the trisaturated glycerides of bovine milk fat

J . Sci. Food Agric. 1987,39,303-316

The Trisaturated Glycerides of Bovine Milk Fat

William Banks, William W. Christie, John L. Clapperton and Anne K. Girdler

Hannah Research Institute, Ayr KA6 5HL, UK

(Received 9 July 1986; revised version received 26 November 1986; accepted 2 December 1986)

A B S T R A C T

Dietary manipulation has been used to produce a range of milk fats differing substantially in fatty acid composition. These fats have then been fractionated by preparing the mercuric acetate adducts to produce the trisaturated glyceride components. The method produced fractions having a very low unsaturated fatty acid content. Calculations suggest that the trisaturated fractions contained appreciably less 4: 0 than would be predicted, with a compensating increase in the relative proportion of long- chain fatty acids. The triglyceride distributions of the parent fats were shown to be markedly bimodal in nature, whereas those of the trisaturated glycerides were unimodal or exhibited fairly small second peaks. Calcula- tions based on the proportion of unsaturated fatty acids present in the milk fat suggest that the strategy of triglyceride synthesis within the bovine mammary gland is not to reduce the production of trisaturated glycerides to a minimum unless only small amounts of unsaturated fatty acid are available. The melting spectra, determined by differential scanning calorimetry, of the parent milk fats were quite different f rom those of the trisaturated components, with the latter fraction having little material melting below OOC, but complete melting occurred at slightly higher temperatures. The positions of the major endotherms in the melting spectra of the parent fats on the one hand and the trisaturated fractions on the other indicate that extensive solid solution occurred between the unsaturated and trisaturated components of the parent fat. The heat of melting of the trisaturated glycerides was much greater than that of the parent fats.

Key words: Milk fat, fatty acid composition, triglyceride composition, trisaturated glycerides, melting spectra.

303

J. Sci. Food Agric. 0022-5142/87/$03,50 0 Society of Chemical Industry, 1987. Printed in Great Britain

304 W. Banks, W . W. Christie, J . L. Clapperton, A . K . Girdler

1 INTRODUCTION

The fatty acid spectrum of milk fat is dominated by the saturated acids, with a cis- 18 : 1 fatty acid, resulting from enzymic desaturation of 18: 0 in both the intestinal epithelium and the mammary gland, being the only unsaturated fatty acid present in appreciable proportions. These saturated fatty acids contain from 4 to 18 carbon atoms, and consequently the structure of the triglycerides of milk fat is exceedingly complex. This complexity has limited the number of studies carried out on the fine structure of the fat. However, the current decrease in butter consumption is leading to a search for new markets for milk fat. The present limitations in knowledge of structure-property relations must be overcome if, for example, uses for specific fractions of milk fat are to be found.

Because milk fat is so complex, it is necessary to simplify the problem by some kind of fractionation procedure. We have chosen in this instance to examine in detail the properties of the trisaturated glyceride fraction of milk fat. Hawke and co-~orkersl-~ have reported on this aspect, but for technical reasons the triglycerides were first fractionated according to molecular weight and then according to degree of saturation. The fatty acid and triglyceride composition of the total trisaturated glycerides of the parent milk fat could then be constructed using arithmetical procedures, but these are not appropriate to the thermal data. We have developed a technique that allows the trisaturated glyceride fraction to be separated from entire milk fat, and report here on the necessary methodology and the properties of the fractions.

2 EXPERIMENTAL 2.1 Milk fats

Milk was obtained from four groups, each consisting of four cows, i.e. milks from the four cows within the group were bulked. Two groups were housed in the byre, where they were offered typical winter rations consisting of hay and concentrates for 3 weeks. Milk was collected (a.m and p.m.) on the 21st day from each group, and butter was prepared as previously described.5 For the next 3 weeks, the diet of each group was supplemented with either soya oil or palm oil (500 g day-') and butter was prepared from samples taken on the 35th and 42nd days of the trial. The remaining two groups grazed grass pastures, receiving a supplement of molassed sugar-beet pulp for the first 3 weeks, to which soya oil or palm oil (500 g oil cow-' day-') was added for the remaining 3 weeks. Milks were again collected on days 21,35 and 42 and used to make butters. All butters were stored at -20°C until required.

2.2 Fatty acid analysis Milk fat was extracted from butter using chloroform/methanol and analysed as detailed previously.

2.3 Triglyceride analysis

Butter was extracted with n-heptane to give a fat concentration of c. 3 g litre-', and the triglycerides were separated by capillary g.1.c. as described previ~usly.~

The trisaturated glycerides of bovine milk fat 305

2.4 Separation of trisaturated glycerides

Anhydrous milk fat was obtained from butters by melting, washing with hot water and drying in V ~ C U O at 50°C. Mercuric acetate reagent was prepared, consisting of 14 g mercuric acetate in 250 ml methanol, 2-5 ml water and 1 ml glacial acetic acid. A known weight (c. 200 mg) of anhydrous milk fat was transferred to a test tube, mercuric acetate reagent (2.5 ml) and chloroform (2.5 ml) were added and the mixture was refluxed for 8 h before being stored overnight at 50°C. The mixture was evaporated to dryness in a stream of nitrogen. Methanol (2.5 ml) and diethyl ether (6.5 ml) were added, dissolving the lipid and precipitating excess mercury salts. The precipitate was consolidated by centrifugation and the supernatant was carefully decanted. The precipitate was washed (2x 1 ml) with ether and the washings were added to the supernatant, which was then evaporated to dryness. A short column (c. 5 g adsorbent) of Florisil in hexane/diethyl ether (85:15 by volume) was prepared, and the mercury adducts were applied to the column in this solvent. Elution of the non-adducted triglycerides (trisaturated) was carried out with the same solvent (100 ml), the eluate was evaporated to dryness, and the residual trisaturated glyceride was weighed.

2.5 Melting profiles

Melting profiles of the parent milk fats and their constituent trisaturated glyceride fractions were obtained as described previo~sly.~.~

3 RESULTS AND DISCUSSION 3.1 Parent milk fats

The various diets offered to the cows were so chosen as to produce a wide spectrum of milk fats, both in terms of fatty acid composition and the physical properties of the butters. The feeding regimes in the third week provided fats typical of the grazing and indoor feeding situation, corresponding to summer and winter butter, respectively. Within each basic type of diet, the presence of soya oil yielded a more easily spreadable butter, whereas palm oil decreased spreadability. Thus in terms of physical and rheological properties, the milk fats used in this work encompass the range that is available commercially, but they also extend beyond that range, providing greater extremes of hardness and softness.

3.2 Fatty acid compositions of the parent milk fats and of their trisaturated

The fatty acid compositions of the parent milk fats are recorded in Table 1; those of the trisaturated glyceride fractions are shown in Table 2. In each case, the values derived from the control (no added oil) diets are the arithmetic means of the two groups, obtained from milk samples taken on the 21st day of the experi- ment. Similarly each value recorded for a fat-rich dietary treatment is the mean obtained from samples on days 35 and 42.

Comparison of the proportions of unsaturated fatty acids in the parent fats with those of the trisaturated glyceride samples showed that the fractionation tech-

glyceride fractions

306 W. Banks, W. W. Christie, J . L. Clapperton, A . K . Girdler

TABLE 1 Fatty Acid Composition (mmol mol-l) and Number-average Chain Length (P,) of the Milk Fats Obtained from Cows Grazing or Feeding Indoors and Receiving Supplements

Containing No Oil, Soya Oil or Palm Oil

Fatty Grazing Indoor feeding acid Control, Soya Palm Control, Soya

no oil oil oil no oil oil Palm

oil

4: 0 6:O 8 : O

lo:o 12:o 14:O 14: 1 16:O 16: 1 18:O 18: 1 18:2+ P"

132 48 25 46 46

123 22

23 1 12 87

195 32 13.7

120 37 16 25 28 96 14

191 10

121 292 49 14.6

119 32 14 27 29 90 10

300 18 94

246 20 14.4

121 48 26 49 52

130 18

300 15 68

149 24 13-5

139 44 22 33 34

104 14

21 1 8

143 224 23 13.9

132 39 15 24 25 86 10

315 19

104 213

17 14.1

nique had been successful. The most appropriate criterion for the success of the technique is the proportion of 18 : 1 in the two materials. In the parent fats the mean value for 18 : 1 was 220 mmol mol-I, whereas the trisaturated glyceride fractions contained on average only 5 mmol mol-I. When all the unsaturated fatty acids present in the trisaturated fractions are considered, an anomaly

TABLE 2 Fatty Acid Composition (mmol mol-') and Number-average Chain Length (P,) of the Trisaturated Glyceride Fractions of the Milk Fats Obtained from Cows Receiving Various

Diets

Fatty Grazing indoor feeding acid Control, Soya Palm Control, Soya Palm

no oil oil oil no oil oil oil

4:O 135 130 131 122 150 155 6:O 65 52 51 60 59 53 8:O 29 21 20 29 26 20

1o:o 12: 0 14:O 14: 1 16:O 16: 1 18:O 18: l 18:2+ P"

59 64

180 14

322 4

123 4 1

12.8

38 45

158 18

312 5

204 15 2

13.3

35 36

173 12

396 4

138 2 1

13.3

58 64

171 10

390 3

91 1 2

12.9

45 32 48 35

152 120 10 9

300 424 4 3

199 145 4 2 4 3

13.0 13.2


becomes obvious, namely that the 14: 1 accounts for only 5% of all the unsaturated fatty acids in the parent milk fats, but for 50% in the fractions. The anomaly is due to the fact that the g.1.c. technique used in this work does not differentiate between 14:l and 15:O (linear and branched), and the mixture is arbitrarily described as 14 : 1. Hence this group of fatty acids is more saturated than is implied in the designation.

In the parent fats, the lowest values for the number-average carbon-atom content (Pa) was associated with the control diet during both grazing and the indoor feeding situation. However, the highest value was related to the diet supplemented with soya oil during grazing, and to that containing palm oil during the indoor feeding period. The P, values were lower for the trisaturated fractions than for the corresponding parent fat, and although the lowest values were again associated with the control diets, the overall variations was not marked.

3.3 Comparison of calculated and experimental fatty acid compositions for the

If it is assumed that the saturated fatty acids are randomly distributed with respect to the total degree of unsaturation of the triglycerides, the relative distribution of fatty acids in the trisaturated fraction may be calculated by removing the total proportion of unsaturated fatty acids (but excluding 14 : 1) and renormalising the remaining acids. The results, corrected for residual 16 : 1,18 : 1 and 18 : 2+ present in the experimental fractions, of this procedure are shown in Table 3.

On comparing the calculated and experimental values, it was immediately obvious that the actual relative proportion of 4:O was considerably lower than predicted. The fatty acids of intermediate chain length, i.e. 6 : &12 : 0, showed good agreement between theoretical and experimental values. The deficiency in

trisaturated glyceride fractions

TABLE 3 The Calculated Relative Proportions (mmol mol-') of the Fatty Acids Present in the

Trisaturated Glycerides

Fatty Grazing Indoor feeding acid Control, Soya Palm Control, Soya Palm

no oil oil oil no oil oil oil

4:O 171 181 165 148 186 176 6:O 62 57 47 59 59 53 8:O 33 23 20 32 30 20

lo:o 59 37 38 60 44 32 12:o 59 41 40 64 45 33 14:O 161 144 125 159 137 114 14: 1 29 21 14 22 19 13 16:O 303 288 415 367 279 415 18:O 113 182 130 84 189 136

The calculation assumes a proportional distribution of the 4:0, 8:0, 10:0, 12:0, 14:0, 14 : 1, 16: 0 and 18 : 0 present in the parent fats, but the calculated values are corrected for the residual unsaturated fatty acids present in the experimental trisaturated glyceride fractions.

308 W. Banks, W . W. Christie, J . L. Ciapperton, A. K . Girdler

4 : 0 in the experimental fractions was compensated by an increase in the relative proportions of the long-chain fatty acids, i.e. 14 : 0, 16: 0 and 18 : 0. In five of the six samples, there was no bias, inasmuch as there was approximately the same increase for each acid. However, the trisaturated fraction derived from animals grazing grass supplemented with palm oil did show bias, the 14 : 0 being much greater than the calculated value, with a compensatory decrease in the proportion of 16:O.

No explanation can presently be advanced for the anomalous behaviour of this sample. Repeated analysis of the samples confirmed the values shown in Tables 1 and 2, hence the anomaly is real rather than apparent. However, the most significant finding to arise from this comparison of calculated and experimental fatty acid compositions of the trisaturated glycerides is that the proportion of 4 : 0 in the trisaturated fractions was much lower than predicted theoretically, i.e. a definite bias exists against the inclusion of 4 : 0 in trisaturated glycerides.

3.4 Triglyceride distributions for the parent fats and their trisaturated glyceride

The relative proportions (mmol mol-I) of triglycerides of different carbon number (CN), i.e. the number of fatty acid carbon atoms in the triglyceride, are shown in Table 4 for the parent milk fats, and in Table 5 for the trisaturated fractions.

The triglyceride distribution of the control milk fat produced by cows at grass was bimodal in nature, with the low molecular weight region (peak at CN 38) dominating the area of high molecular weight (peak at CN 50). The bimodality was emphasised on feeding fat, with the high molecular weight region becoming more pronounced. Dietary palm oil did not affect the position of the major peak in the low molecular weight component (at CN 38), but soya oil increased it to CN 40; both oils increased the position of the high molecular weight peak, to CN 52 (palm) or to CN 52-54 (soya).

During the indoor feeding period, the control diet produced a milk fat with a triglyceride distribution dominated by the lower molecular weight species (peak at CN 38) and exhibiting only a modest degree of bimodality. Adding fat to the diet increased the degree of bimodality, but the low molecular weight region remained dominant, with the position of the major peak again at CN 38. For the control diet, the peak in the high molecular weight region occurred at CN 50; supplementation with fat broadened this peak so that the maximum occurred at CN 50-52.

The triglyceride distribution of the saturated fraction derived from the grazing control treatment was unimodal in nature, with a maximum at CN 36, and it was fairly symmetrical except for a slight shoulder in the high molecular weight region. The corresponding fractions from the fat-rich grazing treatments retained a major peak at CN 36, but also exhibited bimodality with a second peak occurring in the high molecular weight region at CN 46-48 (dietary soya oil) and CN 48 (palm oil).

The trisaturated fraction derived from the indoor, control diet was similar in its triglyceride distribution pattern to that isolated from the grazing control treatment. Dietary soya oil gave a trisaturated fraction with the low molecular weight peak at CN 36 and the high molecular weight shoulder only slightly more

fractions


TABLE 4 Relative Proportions (mmol mol-I) of Triglycerides of Different Carbon Number (CN) Present in the Parent Milk Fats, and the Number-average value (m,) for Each Fat

Compared with the Theoretical Value (3 X P,) Derived from Fatty Acid Composition

Carbon no. Grazing Indoor feeding

(CN) Control, Soya Palm Control, Soya Palm no oil oil Oil no oil oil oil

26 2 1 1 2 4 1 28 6 2 3 5 5 3 30 13 4 6 13 14 5 32 28 12 12 21 32 10 34 68 37 39 80 72 46 36 126 79 111 149 126 129 38 150 129 160 153 153 179 40 123 138 106 109 112 109 42 77 64 55 84 66 55 44 64 40 42 78 52 46 46 63 52 50 70 55 53 48 72 70 74 75 66 77 50 91 110 132 80 88 126 52 79 130 149 56 90 127 54 37 132 59 14 65 35 CN, 42-1 44.7 43.9 41.5 42.3 43.2 3xP, 41-1 43-8 43-2 40.5 41.7 42.3

-

TABLE 5 Relative Proportions (mmol mol-') of Triglycerides of Different Carbon Number Present in the Trisaturated Glyceride Fractions and the Number-average value (m,) for Each Fraction Compared with the Theoretical Value (3XP,) Derived from Fatty Acid

Composition

Carbon no. Grazing Indoor feeding

Control, Soya Palm Control, Soya Palm no oil oil oil no oil oil oil

(W

26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 mn 3xP,

- 18 19 55

141 218 185 118 90 66 47 33 10 - -

38.1 38.4

- 1 12 6 17 11 42 28

112 104 184 244 163 200 101 99 85 70 66 59 74 58 73 65 53 46 18 9

39.8 39.4 39.9 39.9

- 1

2 10 14 41

131 236 182 117 104 76 53 28 7 - -

38.4 38.7

- 11 20 50

127 215 193 112 71 59 50 48 33 10

38.3 39.0

-

- 3 8

24 96

244 257 91 60 50 53 59 45 10

39.3 39.6

-

310 W. Banks, W. W. Christie, J . L. Clapperton, A . K . Girdler

emphasised. Palm oil increased the position of the main peak to CN 38, and in the high molecular weight region a peak appeared at CN 48.

In each dietary situation, the response to added fat was to emphasise the high molecular weight region of the triglyceride distribution. This outcome is not unexpected, because such supplementation increases the long-chain fatty acid content of the milk fat. Indeed, the value of CN,, the number-average carbon number, derived from the triglyceride distribution was in reasonable agreement with the value calculated from 3xP,, both for the parent fats and for the trisaturated fractions. This good agreement confirms that there are no gross errors in the experimental determinations of the fatty acid and triglyceride compositions.

A marked feature of the triglyceride distributions of the saturated fractions was a virtual absence of CN 54, i.e. tristearin. In the case of the control treatments, CN 52 was absent, but appeared in very limited quantities in response to dietary fat. The main change in the high molecular weight end of the distribution was due to increases in CN 46,48 and 50 in the grazing situation and in CN 48 and 50 during the indoor feeding period. As in the fatty acids, the distribution of molecular species was less sensitive to change in diet in the case of the saturated fractions than in the parent fats.

The single largest determinant of the form of the triglyceride distribution is the placement of 4 : 0 and 6 : 0. It is widely agreed that all 4 : 0 and in excess of 85% of 6:O is found at position 3 in the triacyl-sn-glycerols. Thus, to an excellent approximation, only a single short-chain fatty acid (SCFA), i.e. 4: 0 or 6 : 0, can be found in any triglyceride molecule. The largest triglyceride that contains such an SCFA has CN 42, i.e. CI8,Cl8 and 6:0, but the converse does not apply: triglycerides with CN<42 need not necessarily contain such SCFA. The comparison of the molar proportion of triglycerides containing SCFA, assuming only a

TABLE 6 Molar Proportion of Triglycerides (TG) Containing 4 : 0 and 6 : 0, Assuming All 4 : 0 and 6:O is Found at Position sn 3, in the Parent Milk Fats and in the Trisaturated Glyceride

Fractions, Compared with the Fraction of TG Having a Carbon Number (CN)S42

Grazing Indoor feeding


Parent fats Molar proportion 0.54 0.47 0.45 0.51 0.55 0.51

of TG containing 4:O and 6:O

Molar proportion 0.59 0-47 0.49 0.62 0.58 0.54 of TG with CNS42

Trisaturated glycerides Molar proportion 0.60 0.55 0.55 0.55 0.63 0.62

of TG containing 4:O and 6:O

of TG with CNS42 Molar proportion 0.84 0.71 0.76 0.84 0.80 0.78


single SCFA can be found in any triglyceride, with the molar proportion of triglycerides having CN<42 is shown in Table 6.

In the parent fats, the proportion of triglyceride with CN<42 tended to be greater than that of triglycerides containing SCFA, particularly in the case of the fats derived from the control diets. Triglycerides with CNc42, and not containing SCFA, must contain 8 : 0,lO : 0,12 : 0 or 14 : 0. The proportions of these fatty acids of intermediate chain length decrease when the diet is supplemented with oil. Hence the difference between the total proportion of triglyceride with CNS42 and those containing SCFA would be expected to be greatest in the case of the control diets, which was in fact observed in this work.

In the trisaturated glyceride fractions, it was very obvious that a considerable proportion (at least 20%) of the triglycerides with CNS42 contained neither 4 : 0 nor 6:O. Although, as in the case of the parent fats, this fact was particularly apparent in the two samples derived from control diets, it was still fairly pronounced in the trisaturated fractions obtained from fat-rich diets. As the triglyceride fraction contains a greater proportion of material of CN<42 that has no SCFA content than does the parent fat, the unsaturated glyceride fraction (not examined in this work) must contain correspondingly lower amounts. Position sn 3 of the triglyceride is the last to be filled, so these differences suggest that the bovine mammary gland adopts different strategies in the utilisation of saturated and unsaturated diacylglycerol intermediates in the biosynthesis of triglycerides.

The mammary gland also determines the proportion of trisaturated material in the parent fat, and this work allows comment to be made on the underlying strategy. As shown in Table 7, the major unsaturated fatty acids, i.e. 16 : 1, 18 : 1 and 18: 2+, account for 188-351 mmol mol-l total fatty acid. If it is assumed that the strategy is to minimise the synthesis of trisaturates by ascribing a single unsaturated fatty acid to as many glycerol molecules as possible, the maximum

TABLE 7 Total Proportions (mmol mol-') of Unsaturated Fatty Acids (16:1+18:1+18:2+) in the Parent Milk Fats, the Calculated Maximum Molar Proportion of Triglycerides (TG) Containing Unsaturated Fatty Acids, the Calculated Minimum Molar Proportion of Trisaturated Glycerides, the Experimentally Determined Weight Fraction (w) and

Calculated Molar Fraction (f) of Trisaturated Glycerides

18 : 1+18: 1+18 : 2+ Maximum proportion

of unsaturated TG Minimum proportion

of saturated TG Weight fraction (w)

of saturated glycerides Molar fraction (f)

of saturated glycerides

Grazing

Control, Soya no oil oil

239 351 0-72 1.00

0.28 0

0.29 0.22

0-32 0.25

Palm oil

284 0.85

0.15

0.31

0.35

Indoor feeding

Control, no oil

188 0.56

0.44

0.40

0.44

Soya Palm oil oil

255 249 0-77 0.75

0.23 0.25

0.35 0.34

0.39 0.37

312 W. Banks, W. W. Christie, J . L. Clapperton, A. K . Girdler

proportion of unsaturated triglycerides can be obtained by dividing the sum of the unsaturated fatty acids by 333.3; subtracting the resultant value from 1 gives the minimum proportion of trisaturated glycerides. This figure may then be compared with the experimental value. However, the latter parameter is available as the weight fraction (w), whilst the calculation yields a molar (or number) proportion. The necessary correction can be carried out using:

where the subscripts p, ts and us refer to the parent, trisaturated fraction and unsaturated fraction, respectively, to solve for CN,,,,,. The derived value of CNn,,,, is then used in:

to solve forf, the molar fraction of unsaturated material. The values off obtained in this way are also shown in Table 7. Mechanical losses in the isolation of the trisaturated fractions may lead to values off being slightly underestimated.

Generally, the value o f f , the molar fraction of trisaturated material, was greater than would be expected if the underlying synthetic strategy was to produce the maximum amount of unsaturated triglycerides. In particular, the milk fat of cows receiving grass supplemented with soya oil contained more than enough unsaturated fatty acid to avoid any production of trisaturated material, in spite of which some 25% of the total triglycerides was found in this fraction. However, the sample with the lowest content of unsaturated fatty acids, the milk fat from animals receiving the control, indoor diet, showed coincidence between the f-value and the calculated minimum proportion of trisaturated glyceride. This coincidence suggests that as the proportion of unsaturated fatty acids in milk fat decreases, a point is reached at which the synthetic mechanism minimises the production of trisaturated glycerides.

3.5 Thermal properties

Melting profiles of the milk fats derived from the grazing experiments and from the various indoor feeding treatments are shown in Figs l a and lb , respectively. The control fats exhibited three main melting areas, a low temperature region with a small peak at 5°C (grazing) or 7°C (indoor feeding), a middle melting region with a pronounced peak at 13°C (grazing) or 16°C (indoor feeding), and a broad, high melting, plateau region. Introduction of oil to the diet tended in each case to emphasise the separation of the middle and high melting regions of the resultant butterfats. All the oil-rich diets yielded milk fats with melting peaks at 74°C. In the grazing situation, supplementary soya oil produced a major melting peak at 1"C, whereas palm oil gave a major peak at 14°C. Indoor feeding produced milk fats having peaks melting at 1617°C. Within each of the two systems of feeding, complete melting occurred at the lowest temperature in the case of the control fat, and at the highest temperature when the diet was supplemented with palm oil. Comparing the two feeding systems, complete melting occurred at a lower temperature for a given diet in the grazing situation.


I I I I I I I I -30 -20 -10 0 10 2 0 30 40

Temperature ("C) Fig. 1. Melting profiles of milk fats obtained from various dietary supplements during (a) grazing and (b) indoor feeding period. In each case: - control, no added oil; . . . . added soya oil; ----

added palm oil.

Melting profiles of the trisaturated glyceride components of these butterfats are shown in Fig. 2, samples obtained from grazed animals being shown in Fig. 2a and those from cows fed indoors in Fig. 2b. All six fractions showed minor melting peaks at -3 to +l"C. Trisaturated glycerides derived from the control butterfats gave basically similar melting spectra, except that the major peaks occurred at temperatures 3 4 ° C higher in the case of samples obtained from indoor feeding. The saturated fractions of the milk fats obtained on feeding oils showed pronounced melting gaps, i.e. exothermic peaks due to considerable recrystallisation of the semi-molten material. However, whereas the fractions obtained as a consequence of using dietary soya oil were similar to the control samples in showing two peaks in the middle melting range, those resulting from dietary palm oil exhibited only a single, large endotherm in this region. As in the case of the parent butterfats, complete melting of the trisaturates occurred at the lowest temperature in the case of the control and at the highest temperature when

314 W. Banks, W. W. Christie, J . L. Clapperton, A . K.

1 1 / I / I I I

(a)

(b)

0.5 cal g-'OC-' I

Girdler

I I I I I I -20 -10 0 10 2 0 30 40

Temperature (W

Fig. 2. Melting profiles of the trisaturated glyceride fractions of milk fats obtained from various dietary supplements during (a) grazing and (b) indoor feeding period. In each case: -control, no added oil;

. . . . added soya oil; ---- added palm oil.

they were derived from a diet containing palm oil. Again, when comparing samples from outdoor and indoor feeding, complete melting for a given type of diet occurred at a lower temperature during the outdoor feeding period.

The fact that the trisaturated glycerides exhibited major melting peaks at temperatures at which little melting took place in the parent fat suggests that in the parent fat a considerable degree of solid solution occurs between trisaturated and unsaturated glycerides. Calculations carried out by Taylor et aL4 showed that similar solid solutions occurred on mixing the saturated and unsaturated triglyceride fractions of the high molecular weight component of milk fat. Com- parison of Figs 1 and 2 shows that the trisaturates make only a very small


TABLE 8 Heats of Melting (cal g-l) of the Parent Milk Fats and of their Trisaturated Glyceride

Fractions Grazing Indoor feeding


Parent milk fat 17.4 15.6 16.3 17.9 15.1 19.2 Trisaturated Glyceride 22.5 22.3 24.5 21.2 24.0 30.4

contribution to the total amount of material melting at low temperatures, i.e. below O'C, again in agreement with previous observations. The pronounced exothermic peaks exhibited by the trisaturated glycerides derived from the milk fat of cows receiving oil-rich diets are most probably manifestations of poly- morphic beha~iour .~

The areas under the curves in Figs 1 and 2, excluding the exotherms, represent the heats of melting, AH. Values for this parameter are recorded in Table 8. Consistently, higher values of A H were observed for trisaturated glycerides than for the parent milk fat. For simple triglycerides,* AH increases with melting point (MP), e.g. AH for tricaprylate (MP 8.3"C) is 34.1 cal g-' whereas AH for tristear- ate (MP 73.5'C) is 52.8 cal g-l. A similar tendency has been noted for fractions of milk fat.7,9 However, in our previous even a high melting fraction had a AH of only 22.3 cal g-1, a value surpassed by a number of the trisaturated glyceride fractions. Thus for milk fat, the degree of saturation of the triglycerides appears to be a more important determinant of AH than does the melting point.

4 CONCLUSIONS

The method described herein for separating the trisaturated glyceride fraction of milk fat, and the detailed composition of that fraction, offers an insight into the complex structure of the fat. In that respect, perhaps the most significant point to emerge from the present work is that the trisaturated glycerides contain less 4 : 0 than is calculated from the fatty acid composition of the parent fat.

ACKNOWLEDGEMENTS

J. V. Wilson, C-E. E. Graham and K. Small gave skilled technical assistance.

REFERENCES

1. Morrison, I. M.; Hawke, J. C. Triglyceride composition of bovine milk fat with elevated

2. Morrison, I. M.; Hawke, J. C. Positional distribution of fatty acids in the triglycerides of levels of linoleic acid. Lipids 1977, 12, 994-1004.

bovine milk fat with elevated levels of linoleic acid. Lipids 1977, 12, 1005-1011.

316 W . Banks, W. W. Christie, J . L. Clapperton, A. K . Girdler

3. Morrison, I. M.; Hawke, J. C. Influence of elevated levels of linoleicacid on the thermal properties of bovine milk fat. Lipids 1979, 14, 391-394.

4. Taylor, M. W.; Norris, G. E.; Hawke, J. C. The thermal properties of bovine milk triacylglycerols. N. Z . J. Dairy Sci. Technol. 1978, 13,236-241.

5. Banks, W.; Clapperton, J. L.; Kelly, M. E.; Wilson, A. G. ; Crawford, R. J. M. The yield, fatty acid composition and physical properties of milk fat obtained by feeding soya oil to dairy cows. J. Sci. Food Agric. 1980, 31, 368-374.

6. Banks, W.; Clapperton, J. L.; Ferrie, M. E. The physical properties of milk fats of different chemical compositions. J . SOC. Dairy Technol. 1976, 29(2), 86-90.

7. Banks, W.; Clapperton, J. L.; Girdler, A. K. On the fractional melting of milk fat and the properties of the fractions. J . Sci. Food Agric. 1985,36,421-432.

8. Timms, R. E. Heats of fusion of glycerides. Chem. Phys. Lipids 1978, 21, 113-129. 9. Timms, R. E. The phase behaviour and polymorphism of milk fat, milk fat fractions and

fully hardened milk fat. Aust. J. Dairy Technol. 1980,35,41-53.

the trisaturated glycerides of bovine milk fat

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