Method for increasing the concentration of conjugated...

Drug – bio-affecting and body treating compositions – Preparations characterized by special physical form – Food or edible as carrier for pharmaceutical

Reexamination Certificate

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C424S442000, C426S002000, C426S623000, C426S630000, C426S635000

Reexamination Certificate

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06635271

ABSTRACT:

The invention relates to a method for increasing the concentration of conjugated linoleic acid, i.e. chemically precisely expressed the cis-9,trans-11 isomer of octadecadienoic acid (CLA, cis-9,trans-11-C18:2) in the milk fat and/or the tissue fat of a ruminant by feeding to the ruminant, either as such or mixed with other feed, separately or together with other fatty acids, vaccenic acid, i.e. chemically precisely expressed the trans-11 isomer of octadecenoic acid (trans-11-C18:1).
Recently it has been observed that conjugated linoleic acid, i.e. CLA, provides quite effective protection against several forms of cancer (Ha Y. L. et al., Cancer Res. 1990, 50:1097; Ip C. et al., Cancer 1994, 74:1050). It has been observed to be effective against skin and stomach cancers of the mouse and against cancer of the mammary gland of the rat (Scimeca J. A. et al., Diet and Breast Cancer. American Inst. Of Cancer Res. 1994, Plenum Press, London). The growth of human cancer cells has also successfully been inhibited in cell cultures (Shultz T. D. et al., Canc. lett. 1992, 63:125). It has also been observed to have other metabolic effects, some of which clearly suggest effects on health (Banni, S. and J.-C. Martin. 1998. Trans fatty acids in human nutrition. Ed. Sebedio and Christie, The Oily Press, Dundee).
In nature, CLA is present mainly as a component in the tissue fat and the milk fat of ruminants, cis-9,trans-11 being the main isomer (approx. 90%). Small amounts of other isomers are also present in milk, but in the present context, CLA denotes specifically the cis-9,trans-11 isomer.
It is thus an object of the present invention to provide a method suitable for animal husbandry by which the concentration of CLA in the milk fat and/or the tissue fat of a ruminant can be increased. This has been achieved according to the invention in the manner disclosed in the accompanying patent claims.
The method according to the present invention is suitable for all ruminants, in particular for lactating ruminants, such as the cow, whereby it is possible to produce for consumers milk and milk products with a modified fatty acid composition. The change in the fatty acid composition of milk and of milk products is advantageous for the health of consumers. Usually CLA, i.e. cis-9,trans-11-C18:2 fatty acid, constitutes 0.3-0.7% by weight of all of the fatty acids in cow's milk. Even at this concentration it may already protect people consuming milk products from cancer, if the intake of milk fat is sufficiently high (Knekt P. R. et al., Brit. J. of Cancer 1996, 73:687). Habits in the consumption of food fats have changed in recent years. The consumption of fats, and in particular milk fat, has decreased, and as a consequence of this also the intake of CLA in food has decreased. By increasing CLA concentration in milk by amount corresponding to the decrease in the consumption of milk fat it would be possible to return the intake of CLA in food to the level of previous years without needing to increase the consumption of fats. The increasing of the concentration of CLA in milk may thus have a great significance for public health. Increasing the concentration of CLA in milk to a level higher than previously could have an especially positive effect, since in experiments on animals it has been observed that the protective effect of CLA has a clear dose response (Ip C. et al., Cancer 1994, 74:1050). CLA in food may also lower the amount of bad cholesterol (LDL) in the blood (Lee et al. 1994. Atherosclerosis. 108:19). The natural CLA isomer of milk fat has been ingested by people for thousands of years without any observed disadvantages.
The formation of conjugated linoleic acid (cis-9,trans-11-C18:2) during the isomerization of linoleic acid (cis-9,cis-12-C18:2) in the rumen has been described as follows:
The concentration of CLA in milk has successfully been increased by feeding cows vegetable oils which contain linoleic acid (Griinari J. M. et al., J. Dairy Sci. 1998, 81:1251). Kelly et al. (Kelly M. L. et al., J. Nutr. 1998, 128:881) compared three vegetable oil additions to feed and their effect on milk CLA concentration and observed that the oil addition with the highest concentration of linoleic acid (sunflower oil) gave the best milk CLA response. They noted that the concentration of CLA in milk can be increased by feeding to animals oily fats specifically containing linoleic acid.
It has been believed that the CLA which has passed into milk fat is derived solely from the linoleic acid present in animal feed, i.e. from octadecadienoic acid cis-9,cis-12 isomer, which the microbes of the rumen isomerize to cis-9,trans-11-C18:2 before reducing it to vaccenic acid (trans-11-C18:1) and further to stearic acid (C18:0). CLA is not formed in notable amounts in the hydrogenation of linolenic acid, i.e. the cis-9,cis-12,cis-15-C18:3 isomer of octadecatrienoic acid, but vaccenic acid is also an important biohydrogenation product of linolenic acid (Harfoot and Hazlewood 1988, The rumen microbial ecosystem. Ed. Hobson, P. N. Elsevier Science Publishers B. V., Amsterdam, The Netherlands). According to the prevailing theory, some of the CLA formed as an intermediate product in the biohydrogenation of linoleic acid escapes from the rumen along with the continuous outflow of the rumen fluid before it becomes reduced to vaccenic acid. The CLA which has escaped from the rumen is absorbed mainly in the small intestine, is transferred by the blood circulation to the mammary gland and is bound there as part of the fatty acid composition of milk fat.
The authors of the present invention observed that fish oil, which contains low levels of linoleic acid (composition shown in Table 1), increased the CLA concentration in milk (Table 2).
Table 1 Fatty acid composition of fish oil
Myristic acid (14:0)
8.6
Palmitic acid (16:0)
18.6
Palmitoleic acid (cis-9-16:1)
11.9
Stearic acid (18:0)
3.6
Oleic acid (cis-9-18:1)
9.4
Other cis-acids
0.9
Cis-Vaccenic acid (cis-11-18:1)
5.2
Elaidic acid (trans-9-18:1)
0.2
Linoleic acid (18:2, n-6)
2.5
Linolenic acid (18:3, n-3)
1.7
EPA (20:5, n-3)
10.2
DHA (22:6, n-3)
7.6
TABLE 2
Effect of the feeding of fish oil on
the fatty acid composition of milk
(% of total fatty acids)
No oil
Fish oil
addition
addition
C12:0
3.4
1.9
C14:0
12.8
9.3
C16:0
38.1
34.3
C18:0
9.7
3.8
C18:1
26.1
28.6
trans-11-C18:1
1.1
3.2
C18:2
2.4
2.6
C18:3
0.2
0.5
CLA
0.7
1.9
The generally approved mechanism of CLA formation described above (Harfoot and Hazlewood 1988) also does not explain the increase of the concentration of CLA in the milk when cows receive pasture grass as their only feed (Griinari et al. 1998 J. Dairy Sci. 81 (Suppl. 1):300 (Abst.)), since the principal fatty acid in the fat present in pasture grass is linolenic acid and the proportion of linoleic acid is typically quite low.
Based on observations cited above, the present inventors drew the conclusion that the CLA in milk may also be derived from a source other than the linoleic acid isomerization taking place in the rumen. Furthermore, since in the rumen of a cow feeding on nothing but pasture grass certain CLA isomers (cis-9,trans-11-C18:2 and trans-9,trans-11-C:18:2) were present at an average ratio of 1:1, but in milk the concentration of the cis-9,trans-11-C18:2 isomer was 10 times higher than that of trans-9,trans-11-C:18:2 isomer (Griinari and Numeral, unpublished), the present inventors noted that the CLA of milk fat may in a certain feeding situation be formed to a large extent elsewhere than in the rumen. It has been observed that the above-mentioned CLA isomers transfer from the digestive tract to the milk at almost the same rate, so that what is involved in this situation is not the discrimination of one of the fatty acid isomers (Chouinard et al. 1998. J. Dairy Sci. 81 (Suppl. 1):353 (Abst.)).
Thus the present invention is based on the surprising observation that an oil addition to feed containing low levels of linoleic acid caused a notable increase in the concentration of CLA in the milk. The initial substance of CLA must, however, be form

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