Influence of Linoleic Acid Content of Milk Lipids on Oxidation of Milk and Milk Fat1

Influence of Linoleic Acid Content of Milk Lipids on Oxidation of Milk and Milk Fat1

INFLUENCE OF LINOLE'IC ACID CONTENT OF MILK LIPIDS OXIDATION ON OF MILK AND MILK FAT 1 L. M. SMITH, W. L. DUNKLEY, A~-I)M. RONNING Departments of ...

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INFLUENCE

OF LINOLE'IC ACID CONTENT OF MILK LIPIDS OXIDATION

ON

OF MILK AND MILK FAT 1

L. M. SMITH, W. L. DUNKLEY, A~-I)M. RONNING Departments of Food Science and Technology and Animal Husbandry University of California, Davis SU:~/I~ARY

The concentration of linoleie acid in milk lipids was increased by infusing two cows with a cottonseed oil emulsion, and changes in the oxidative stability of the milk and milk fat were determined. Infusing 150 g of cottonseed oil (as Lipomul I.V.) tripled the linoleic acid content of the milk fat in the next milking. Linoleic acid in the milk phospholipids increased to a smaller extent, and not until the second milking after the infusion. Little change was noted in the concentration of other fatty acids. No significant change occurred in milk production, fat percentage, or the amounts of copper, tocopherols, and carotenoids. A decrease in oxidative stability of the milk fat appeared to be related to the increase in its linoleie acid content. Furthermore, an increase in susceptibility of the milk to copper-induced oxidized flavor, as measured by the thiobarbituric acid test, appeared to be dependent on increased Iinoleic acid content of the phospholipids rather than the milk fat.

5~[ost of the lipids of milk are found in the fat globules and the adsorbed membrane surrounding the globules, but small amounts are also found in the milk serum (5). The triglycerides of the fat globules constitute over 98%, and the phospholipids about t % , of the total weight of the lipids present in milk. The term milk fat, as used in this paper, includes the triglycerides, together with accompanying sterols and trace lipids of the globules, but not the phospholipids, which are found principally in the membrane. Feeding trials are of limited value for studying the role of fatty acid composition of milk lipids in the oxidative deterioration of milk and milk fat. This is because numerous variables associated with feeding influence not only the fatty acids but also other constituents present. For example, different grasses, hays, and concentrate feeds are known to change the fatty acid composition of milk fat. The literature on this subject has been reviewed elsewhere (4, 12). However, the relative effect of a specific feed on the character of the fat may be influenced by the fatty acid composition of the feed, its physical state, and conditions such as the cow's level of nutrition and stage of lactation (1, 3, 4, 11). Furthermore, changes in feed may cause changes in the amounts of

antioxldants and prooxidants in both the lipid and skimmilk fractions of milk (1, 6). The infusion technique described by Tore (12) provides a convenient means of changing the fatty acid composition of milk fat. Further, this method should avoid other changes in milk composition that may be associated with changes in diet. In a previous study from this laboratory (1), alfalfa and oat hays were compared in their influence on the susceptibility to oxidation of milk and milk fat. Significant differences related to the ration were found in both cases. Significant differences were also found in the amounts of natural copper, caxotenoids, tocopherols, and unsaturated fatty acids. However, the relative importance of the change in each of these constituents in determining the susceptibility of milk to oxidation was not assessed. The present study was made to elucidate the relative importance of the linoleie acid content of milk lipids in the oxidative deterioration of milk and milk fat. Data a r e presented showing the influence of the intravenous infusion of a cottonseed oil emulsion on the fatty acid composition of milk fat and phospholipids, and related changes in the oxidative stability of the milk and milk fat. ~F~HODS

Received for publication October 22, 1962. This study was supported in part by funds from the C~alifornia Dairy Council.

Two Jersey cows were fed a concentrate mixture containing 78% milo, 21% cottonseed meal, and 1% oyster shell flour, plus barley

8

L. 1Vf. SMITH, ~V. L. D U N K L E Y , AND ]YI. R O N N I N G

straw free choice. This ration was intentionally low in tocopherol and carotene. A cottonseed oil emulsion, Lipomul I.V. (Upjohn Co., Kalamazoo, Michigan), was infused into the jugular vein of each cow promptly after the ramming milking. The fatty acid composition of the oil, determined by gasliquid chromatography (7), was 0.9% myristic, 23.3% palmitic, 2.5% stearic, 17.5% oleic, and 54.5% linoleic acid. Milk samples were collected at selected milkings before and after the infusions, using stainless-steel equipment and polyethylene bottles. Milk fat samples were prepared by allowing the milk to cream, churning the cream, melting the butter granules, and filtering the clear fat at 50 C. To prepare the phospholipid samples, the butter serum and buttermilk were combined and extracted with ethanol, diethyl ether, and pentane. The lipids were recovered, dried with anhydrous sodium sulfate, and chromatographed on a silicic acid-celite column. The contaminating triglycerides eluted with chloroform were discarded. After the phospholipid fraction was eluted with methanol, it was washed once by the technique of Folch et al. (2), and dried. The procedures used to prepare the milk fat and phospholipid samples have been described in detail (8). Lipid phosphorus was determined by the method of Smith et al. (10). Methyl esters of the fatty acids of the milk fat and phospholipids were prepared, respectively, by alkali- and acid-catalyzed methanol-

ysis. F a t t y acid compositions were determined by gas-liquid chromatography (7, 9). Methods for flavor examination, thiobarbituric acid (TBA) test, fat stability, copper, tocopherol, and total carotenoids were as in a previous study (1). RESULTS AND DISCUSSION

I n the first experiment, infusion of 500 ml of Lipomul (75 g cottonseed oil) into Cow 186 increased the linoleic acid content of the milk fat from 3.1 (before infusion) to 4.3% (the milking following infusion). Since a greater increase seemed desirable fro" this study, the amount of infused emulsion was increased in the second experiment. The chemical and sensory data obtained in the first experiment were consistent with, but not as extensive as, those for the second experiment, presented next. Table I shows the percentages of stearic, oleic, and linoleic acid in the milk fat and in the phospholipids from the milk of Cows 185 and 186 before and after infusion with 1,000 ml of Lipomul (150 g cottonseed oil). The amounts of linoleic acid were roughly tripled in the fat obtained at the milking 12 hr after the infusions. The linoleic acid percentages then declined, by the fourth milking (60 hr after infusions) reaching levels only slightly higher than before the infusions. The increases in linoleic acid were compensated for by relatively minor decreases in the amounts of stearic, oleic, and other fatty acids.

TABLE 1 Influence of Lipomu] infusion ~ on concentration of selected fatty acids in milk fat and ml]k phospholipids

Cow no.

Time before (--) and after (+) infusion

Milk fat Stearic

(br)

185

186

Oleic

Milk phospholipids Linoleic Stearic

(wt v/v)

Ole~e

Linoleic

P

(wt % )

--36 --12 -712 +24 +36 +60

I0.9 ]0.5 9.3 10.1 11.1 8.7

25.6 26.6 22.5 22.1 19.1 31.7

3.0 2.7 8.6 7.7 5.0 4.1

--36 --32 +12 +24 +36 +60

10.5 10.5 8.3 9.3 10.0 8.7

21.7 22.7 19.5 20.8 23.3 21.7

3.6 2.9 10.2 6.9 4.4 4.]

13.3 7.0

34.1 29.3

9.3 10.6

3.2 3.2

40.8 30.3 38.0 37.9

]2.9 10.0 14.8 ]5,.1

3.5 2.8

11.9

9.9 6.8 10.3 lO.O

3.4

* 1,000 ml Lipomul I.V. (Upjoh~ Co., Kalamazoo, Michiga~n) containing ]50 g cottonseed

oil.

OXIDATIVE

INFLUENCE

OF L I N O L E I C

ACID

TABLE 2 Influence of Lipomul infusion ~ on oxidative stability of milk and milk fat Time before (--) and after (+) infusion

TBA increase

Flavor stability b

Fat stability ¢

TBA increase

Flavor stability b

(hr)

(A × iOQ

(hr)

(hr)

(A × iO~)

(hr)

Cow 18g

Cow 186

--36 - -

1

+12 +24 +36 +60 oil.

120 77 ]20 230 2'0'0

i8 30 18 18

70 75 100 135

Fat stability ~

(hr) 14'5

30 94 79 150 80

48 18 36 30 30

145 60 75 95 125

1,000 ml Lipomul I.V. (Upjohn Co., Kalamazoo, Michigan) containing 150 g cottonseed

b Time at which distinct oxidized flavor was first detected in milk samples containing 0.1 ~g/g added copper. ° Induction period at 80 O. I n the phospholipid fractions, the amounts of linoleic acid did not increase until the second milking following the infusions. Also, the increases were relatively much less than in the milk fat. The lipid phosphorus data show that the phospholipid samples were comparable in purity except for the sample isolated from milk obtained from Cow 186 12 hr after infusion. This phospholipid sample was contaminated with about 20% triglycorides and other nonphospholipids, as indicated by its phosphorus content (2.8%, compared to 3.5% in the sample taken before infusion). No attempt was made to correct the percentages of linoleic and other acids to compensate for this impurity. Table 2 shows the influence of the Lipomul infusions on the oxidative stability of the milk and milk fat from the two cows. The TBA increase was greatest for milk produced the third milking after infusion, when the level of linoleic acid in the phospholipids was high (Table 1). Less confidence can be placed in the flavor stability data, because oxidized flavor did not develop during five-day storage in any of the milk samples without added copper (i.e., none of the samples developed the flavor spontaneously). All samples were highly susceptible to copper-induced oxidized flavor, however, because all samples with 0.1 ppm of added copper developed a distinctly oxidized flavor during storage for 48 hr. Hence, the flavor score after a standardized storage time was of little value as a measure of susceptibility to oxidized flavor. As an alternative, samples containing 0.i ppm of added copper were scored twice daily to determine the approxi-

mate time required to develop a distinct oxidized flavor. The results, reported as flavor stability in hours, are an inaccurate measure of stability, because of the long and variable intervals between sensory, examinations. However, they indicate that the infusions increased susceptibility of the milk to the development of oxidized flavor. Tables 1 and 2 show that the oxidative stability of the milk fat was lowest at the first milking after the Lipomul infusion, and the change in stability coincided with the greatest increase in linoleic acid content. I n contrast, the decrease in stability of the milk, in terms of copper-induced oxidized flavor measured by the TBA test, appeared to be dependent on the increase in the linoleic acid content of the phospholipids, because both of these changes were greatest at the second or third milking. Tove (13) found that the infusion of 459 to 900 g of cottonseed oil as Lipomul I.V. reduced milk production immediately after infusion. Such large infusions increased the linoleic acid content of the milk fat to about 25%, but the possible effect on other milk constituents, including prooxidants and antioxidants, was not determined. Table 3 shows that the infusion of 150 g of cottonseed oil in the present study did not influence either milk production or fat percentage. Furthermore, the infusion caused little or no consistent change in the amounts of copper, tocopherol, and total carotenoids. The concentration of linoleic acid in milk fat was increased in the milkings immediately following intravenous infusion of the cow with

L. ~VL SMITH, W. L. DUNKLEY, AND M. RONNING

10

TABLE 3 Miscellaneous analyses of milk and milk f a t before and a f t e r Lipomul infusion i

Cow 185

186

Time before (--) and after ( + ) infusion

Milk Production

Fat

Milk f a t Copper

Carotenoids

Tocopherols

(lb)

(%)

(pRm)

(gg/g)

(gg/g)

--36 --12 +12 +24 +36 +60

11.4 11.3 11.3 10.8 11.0 12.2

4.1

0.026 0.028 0.027

i:2 0.9

23 27

0.4

21

--36 --12 +12 +24 +36

10.4 9.0 9.0 8.4 8.8

0.8 0.6

28 22

0.8

18

+60

9.5

4.1

4.1 5.0 4.1 4.2

0.016 ................

5.6 6.0 5.7 6.9 6.1 4.8

0.031 0.038 0.034 0.028 ................

a 1,000 ml Lipomul X.V. (Upjohn Co., Kalamazoo, Michigan) containing 150 g cottonseed o11. Lipomul. H o w e v e r , a relatively smaller increase was f o u n d in t h e p h o s p h o l i p i d s a n d this increase was n o t o b t a i n e d u n t i l a t least the second milking, 24 h r a f t e r t h e i n f u s i o n . I t h a s n o t been established w h e t h e r a r a t i o n t h a t will increase t h e linoleic acid c o n t e n t of t h e milk fat will p r o d u c e a c o r r e s p o n d i n g increase in the a m o u n t of this acid i n t h e p h o s p h o l i p i d fraction. P r e v i o u s studies (8, 9) a t this l a b o r a t o r y h a v e s h o w n t h a t the m i x t u r e of p h o s p h o l i p i d s f o u n d in milk c o n t a i n s h i g h e r p e r c e n t a g e s of u n s a t u r a t e d f a t t y acids a n d lower p e r c e n t a g e s o f acids below C1o, t h a n t h e triglyeerides. T h e g l y e e r o p h o s p h o l i p i d s largely a c c o u n t f o r these differences. I n t h e p r e s e n t study, i t is notew o r t h y t h a t a l t h o u g h linoleie acid was i n f u s e d into the blood as a triglyceride, t h e a m o u n t s of linoleic aeid were i n c r e a s e d in b o t h the milk triglycerides a n d the p h o s p h o l i p i d s . F u r t h e r more, these increases p e r s i s t e d u n t i l a t least the t h i r d m i l k i n g following i n f u s i o n . These results suggest t h a t t h e i n f u s e d triglycerides were used in the synthesis n o t only of milk p h o s p h o l i p i d s b u t also of m i l k f a t . REFERElffCES (1) DUNKLI~¥, W. L., SMITH, L. M., AND RONNrt¢o, M. Influence of Alfalfa and Oat Hays on Susceptibility of Milk to Oxidized Flavor. J. Dairy Sei., 43:1766. 1960. (2) FOLCH, J., L ~ S , M., Am) STA~-L~V, G. H. S. A Simple Method for the Isolation and Purification of Total IApids from Animal Tissues. J. Biol. Chem., 226: 4,98. 1957.

(3) JACK, E. L, The F a t t y Acids and Glycerides of Cow's Milk Fat. Agr. and Food Chem., 8: 377. 1960. (4) JACk, E. L., AWl) S M I ~ , L. M. Chemistry of Milk F a t : A Review. J. Dairy Sci., 39: 1. 1956. (5) J ~ N ~ s s , R., ANn PA~Yi'ON, S. Principles of Dairy Chemistry. p. 34. J o h n Wiley and Sons, Inc., New York. 1959. (6) KRUKOVSKY,

(7)

(8)

(9)

(i0)

(11)

(12) (13)

V.

•.,

TI~IMBI~F~GE~, G.

W.,

TUI~K, g . L., LOOSLI, J. K., AND HE~WDERSON, C. R. Influence of Roughages on Certain Biochemical Properties of Milk. J. Dairy Sci., 37: 1. 1954. SMITH, L. M. Quantitative F a t t y Acid Analysis of Milk F a t b y Gas-Liquid Chromatography. J. Dairy Sci., 44: 607. 1961. SMITH, L. M., AND JAOK, E. L. Isolagon of Milk Phospholipids and Determination of Their Polyunsaturated F a t t y Acids. J. Dairy Sci., 42: 767. 1959. SM~V~, L. M., AND LOWRY, R. R. F a t t y Acid Composition of the Phospholipids and Other Lipids in Milk. J. Dairy Sci., 45: 581. 1962. SMITH, L. M., LOWI~Y, R. R., AND, JACK, E. L. Determination of Phosphorus in Milk 1Apids. J. Dairy Sci., 42:552. 1959. S M * ~ , L. M., AN]) RONNIN0, M. Comparison of F a t t y Acid Composition of Milk F a t s Produced by Cows Fed Alfalfa, Oat, or Ground, Pelleted Alfalfa l-lay. J. Dairy Sci., 44: 1170. 1961. Tov~, S. B. The Origin of Milk Fat. J. Dairy Sei., 4,3: 1356. 1960. Tow, S. B. Private communication. March, 1961.