Fatty acids and triglycerides have a multiplicity of applications in the food industry, animal nutrition, cosmetics and in the pharmaceutical sector. Depending on whether they are free saturated or unsaturated fatty acids or triglycerides with an increased content of saturated or unsaturated fatty acids, they are suitable for a very wide range of applications; thus, for example, polyunsaturated fatty acids are added to baby formula to increase the nutritional value. The various fatty acids and triglycerides are obtained mainly from microorganisms such as Mortierella or from oil-producing plants such as soya, oilseed rape, sunflowers and others, where they are usually obtained in the form of their triacyl glycerides. Alternatively, they are obtained advantageously from animals, such as fish. The free fatty acids are prepared advantageously by hydrolysis.
Whether oils with unsaturated or with saturated fatty acids are preferred depends on the intended purpose; thus, for example, lipids with unsaturated fatty acids, specifically polyunsaturated fatty acids, are preferred in human nutrition since they have a positive effect on the cholesterol level in the blood and thus on the possibility of heart disease. They are used in a variety of dietetic foodstuffs or medicaments.
Especially valuable and sought-after unsaturated fatty acids are the so-called conjugated unsaturated fatty acids, such as conjugated linoleic acid. A series of positive effects have been found for conjugated fatty acids; thus, the administration of conjugated linoleic acid reduces body fat in humans and animals, and increases the conversion of feed into body weight in the case of animals (WO 94/16690, WO 96/06605, WO 97/46230, WO 97/46118). By administering conjugated linoleic acid, it is also possible to positively affect, for example, allergies (WO 97/32008) or cancer (Banni et al., Carcinogenesis, Vol. 20, 1999: 1019–1024, Thompson et al., Cancer, Res., Vol. 57, 1997: 5067–5072).
Conjugated linoleic acid (=CLA) is an intermediate of linoleic acid metabolism in ruminants. CLA refers to a mixture of positional and geometric isomers of linoleic acid, involving double bonds at positions 9 and 11, 10 and 12 or 11 and 13, and has gained considerable attention in recent years because of the many beneficial effects attributed to the cis-9, trans-11 and trans-10, cis-12 isomers, in particular. These include anti-carcinogenic activity, antiatherogenic activity, the ability to reduce the catabolic effects of immune stimulation, the ability to enhance growth promotion and the ability to reduce body fat (Martin and Banni, 1998 for review, and references therein). The isomers can differ positionally (mainly at positions 7 and 9, 9 and 11; or 10 and 12) (Ha et al., 1987) and geometrically (cis-cis, cis-trans, trans-cis, trans-trans). Of the individual isomers of CLA, cis-9, trans-11-octadecadienoic acid has been implicated as the most biologically active because it is the predominant isomer incorporated into the phospholipids of cell membranes, liver phospholipids and triglycerides (Kramer et al., 1998). This is the only isomer incorporated into the phospholipid fraction of cell membranes of animals fed a mixture of CLA isomers (Ha et al., 1990; Ip et al., 1991). This isomer is also the predominant dietary form of CLA, obtained from fats derived from ruminant animals, including milk, dairy products and meat (Chin et al., 1992, O'Shea et al., 2000).
Studies have shown that CLA may have potential in the prevention of a wide range of human medical conditions, and a number of potential health benefits have been described for CLA, including as mentioned above anticarcinogenic activity, antiatherogenic activity, potential in the prevention of diabetes, obesity and bone disorders. Given that dietary CLA has the potential to beneficially affect human health, it is important to identify effective strategies to enrich the natural form of CLA in food products. Currently, the effective level of dietary CLA for disease prevention in humans is not known. Furthermore, the long-term health implications of a low dietary CLA intake at critical stages throughout life are unknown, as for example in formula-fed infants, compared with breast-fed infants, the latter group receiving a relatively higher CLA intake. In the rodent model, dietary CLA was more effective as an anticarcinogen when consumed during periods of active mammary gland development (Ip et al., Nutr. Cancer, 1995, 24: 241–247), which may indicate that increased CLA intake during adolescence might preferentially decrease the risk of cancer in women.
Few data concerning the CLA intake are available. In Germany the daily CLA intake has been estimated to be 0.36 g/day for women and 0.44 g/day for men (Fritsche et al., 1998). CLA in human tissue is predominantly the isomere cis-9, trans-11-octa-decadienoic acid (>95%), three minor isomers have also been identified. Trans-9, trans-11–18:2, cis-9, cis-11–18:2 and trans9, cis-11–18:2, [Fritsche et al., Zeitschrift Lebensmittel. Untersuchung Forschung A-Food Research & Technology 205:415–418 (1997)]. The origin is thought to be dietary and the consumption of cheddar cheese, a good source of CLA, has been shown to enhance plasma CLA levels in men, [Huang et al. Nutr. Res. 14:373–386 (1994)]. In another study it was found that the relationship between milk fat intake and the occurence of cis-9, trans-11-octadecadienoic acid in human tissue was significantly correlated, [Jiang et al., Am. J. Clin. Nutr. 70:21–29 (1999)]. Safflower oil, a rich source of linoleic acid, did not increase plasma CLA levels suggesting that the intestinal flora of humans do not possess the ability to convert linoleic acid to conjugated linoleic acid, however a CLA increase was observed in some subjects [Herbel et al., Am. J. Clin. Nutr. 67: 332–337 (1998)]. Dietary trans fatty acid has been shown to increase serum CLA, [Salminen et al., Nutritional Biochemistry 9: 93–98 (1998)]. It was in this study concluded that CLA may be formed by desaturation of trans fatty acid possibly by a liver enzyme as has been described for rats.
The principial dietary sources of CLA are milk, dairy products and meat from ruminants, but as a result of differences in environmental conditions and diet of the ruminant species, the CLA content of milk and beef fat vary substantially [Michelle et al., Advances in Conjugated Linoleic Acid Research, Volume 1 (1999)]. Among the richest dietary sources of CLA are milk, dairy products, beef and lamb (Chin et al., J. Food Comp. and Anal., 1992, 5: 185–197; Fritsche and Steinhart, Z. Lebensm. Unters. Forsch., 1998, A206: 77–82 and Lipid, 1998, 6S: 190–210).
In fat from ruminant meats and dairy products, the cis-9, trans-11 CLA isomer is present at 80–90% of the total CLA isomers (Chin et al., J. Food Comp. and Anal., 1992, 5: 185–197).
As mentioned above the origin of CLA in foods is mainly due to the biohydrogenation of dietary linoleic acid by anaerobic rumen bacteria. Accordingly the main dietary sources of CLA are meat from ruminant animals and dairy products, and the main CLA isomer found is cis-9, trans-11-C18:2, (80–90%). In uncooked meats, lamb and beef answer for the highest CLA levels 5.6 mg/g of fat in lamb and 4.3 mg/g of fat in beef (Chin et al., 1992; Fritsche and Steinhart, 1998 ). CLA levels in milk varies with season, highest values occuring when pastures are lush and rich in PUFAs, hence levels of CLA in dairy products such as cheese also varies. In vegetable oils CLA is present in low amounts (0.2–0.7 mg/g fat) and contain higher levels of the isomer trans-10, cis-12-C18:2 (˜40%) (Chin et al., 1992). Since fatty acids with conjugated double bonds are a well-known phenomena in plants, specific enzyme systems are belived to be involved. Meats from non-ruminant animals can contain CLA in lower amounts and it may occur from dietary sources such as feeding meat meal. It could also be explained by formation of CLA by intestinal flora as has been shown for rats (Chin et al., J. Nutr., 1993, 124: 694–701). CLA can also be produced by free radical-based double bond shifting during autooxidation and during partial hydrogenation performed industrially.
CLA can be manufactured synthetically from alkaline isomerization of linoleic and linolenic acids, or vegetable oils containing linoleic or linolenic acids. Two reactions are catalyzed when heating oil at 180° C. under alkaline conditions; hydrolysis of the fatty acid ester bond from the triglyceride lipid backbone, which produces free fatty acids, and conjugation of unconjugated unsaturated fatty acids with two or more approproiate double bonds (WO 99/32604). This method produces about 20–35% cis-9, trans-11 CLA and about the same amount of trans-10, cis-12 CLA, but enrichment of either of the isomers relative to the other is possible by using a fractional crystallization procedure.
In addition other isomers are produced mainly trans, trans isomers.
The chemical preparation of conjugated fatty acids, for example conjugated linoleic acid, is also described in U.S. Pat. Nos. 3,356,699 and 4,164,505.
The presence of conjugated unsaturated fatty acids in milk fat was first established by Booth et al. Bioch. J. 29, 133–137 (1935), who also showed that these fatty acids increased in milk fat when cows were turned out to pasture after winter, by demonstrating an increased absorption of the fatty acids in the ultra-violet (UV) region at 230 nm. CLA is formed in the rumen during microbial biohydrogenation of dietary linoleic acid (Kepler and Tove, 1969 Methods in enzymology Vol XIV, p105; J. Biol. Chem., Vol 246, No. 14, 1970: 3612–3620 and J. Biol. Chem., Vol 246, No. 9, 2765–2771). The complete biohydrogenation of linoleic acid in the rumen is a three step process, yielding C 18:0, stearic acid, as an end product. The first reaction, the conversion of linoleic acid to cis-9, trans-11 CLA by linoleic acid isomerase of rumen bacteria occurs very rapidly, followed by slower conversion to trans-11-C 18:1 (FIG. 1). Some of the CLA formed in the rumen is absorbed into blood and incorporated into milk fat. In addition to the biohydrogenation reaction leading to CLA synthesis, CLA can also be produced endogenously from trans-11-C 18:1 in mammary tissues. Trans-11-C 18:1 accumulates in the rumen due to the slower conversion step to stearic acid, and following absorption from the digestive tract is utilized by different tissues e.g. the mammary gland as a substrate for CLA synthesis by the action of Δ-9-desaturase. Indeed, this may be the major pathway of CLA synthesis in lactating cows, accounting for about 64% of the CLA in milk fat.
In addition member of strains of propionibacteria were previously identified to synthesise CLA from linoleic acid by Jiang et al. (1998).
WO 99/29886 describes the use of certain bacterial strains found among food grade bacteria, particularly among dairy starter cultures, which have the ability to produce CLA in vitro by fermentation. Furthermore WO 99/29886 decribes that said bacteria may be used to provide food or feed products enriched in CLA, and also pharmaceutical products containing CLA as active ingredients.
Isomerases are enzymes which bring about an isomerisation of substrate. Linoleic acid isomerase catalyzes the isomerisation reaction of lionoleic acid to cis-9, trans-11-octadecadienoic acid. This membrane bound enzyme was isolated and characterised by Kepler & Tove (1969). The isomerisation reaction occurs in the middle of a long hydrocarbon chain remote from any functional group and requires no cofactors. The enzyme exhibits max activity with substrates linoleic and linolenic acid within a narrow concentration range. Three parameters are involved in the binding of substrate to linoleic acid isomerase: 1.) the π system of a substrate double bond, 2.) hydrophobic interaction and 3.) hydrogen bonding of the substrate carboxyl group. A proposed model for the isomerization of linoleic acid by linoleic acid isomerase is illustrated in FIG. 2. Pictured at the active site are an electrophile (E) that interacts with one of the substrate double bonds, and two basic centers, one of wich (B) is hydrogen bonded to the carboxyl group and the other (B—H) which serves as a donor for the hydrogen added at C-13 (FIG. 2).
WO 99/32604 describes a linoleate isomerase from Lactobacillus reuteri. The enzyme activity leads to the conversion of linoleic acid to six different CLA species which are as follows: (cis,trans)-9,11-CLA, (trans,cis)-10,12-CLA, (cis,cis)-9,11-CLA, (cis,cis)-10,12-CLA, (trans,trans)-9,11-CLA and (trans,trans)-10,12-CLA.
The disadvantages of the abovementioned process is that the yield of the reaction is very low, the purity of the CLA produced is for an industrial process not sufficient and that the process takes place with only low space-time yields. This leads to economically unattractive processes.
Thus, there is still a great need for a single, economic birthdenological industrial process for the production of CLA which does not have the abovementioned disadvantages and therefore for new genes which encode enzymes which participate in the biosynthesis of conjugated linoleic acid and which allow to synthesize and produce it on an industrial scale.