306 Biosynthesis of Conjugated Linoleic Acid and Its Incorporation into Ruminant’s Products** Man K. Song* and John J. Kennelly1 Department of Animal Science, Chungbuk National University, Cheongju 361-763, Korea ABSTRACT : Bio-hydrogenation of C18-unsaturated fatty acids released from the hydrolysis of dietary lipids in the rumen, in general, occurs rapidly but the range of hydrogenation is quite large, depending on the degree of unsaturation of fatty acids, the configuration of unsaturated fatty acids, microbial type and the experimental condition. Conjugated linoleic acid (CLA) is incompletely hydrogenated products by rumen microorganisms in ruminant animals. It has been shown to have numerous potential benefits for human health and the richest dietary sources of CLA are bovine milk and milk products. The cis-9, trans-11 is the predominant CLA isomer in bovine products and other isomers can be formed with double bonds in positions 8/10, 10/12, or 11/13. The term CLA refers to this whole group of 18 carbon conjugated fatty acids. Alpha-linolenic acid goes through a similar bio-hydrogenation process producing trans-11 C18:1 and C18:0, but may not appear to produce CLA as an intermediate. Although the CLA has been mostly derived from the dietary C18:2 alternative pathway may be existed due to the extreme microbial diversity in the reticulo-rumen. Regardless of the origin of CLA, manipulation of the bio-hydrogenation process remains the key to increasing CLA in milk and beef by dietary means, by increasing rumen production of CLA. Although the effect of oil supplementation on changes in fatty acid composition in milk seems to be clear its effect on beef is still controversial. Thus further studies are required to enrich the CLA in beef under various dietary and feeding conditions. (Asian-Aust. J. Anim. Sci. 2003. Vol. 16, No. 2 : 306-314) Key Words : Bio-hydrogenation, Rumen Bacteria, Unsaturated Fatty Acids, Conjugated Linoleic Acid (CLA), Oil Source, Dietary Manipulation INTRODUCTION BIO-HYDROGENATION IN THE RUMEN Conjugated linoleic acid (CLA) is a component that has An active bio-hydrogenation of unsaturated fatty acids been shown in recent years to have numerous potential by ruminal microbes has been well documented (Wu et al., benefits for human health, including potent cancer-fighting 1991; Wang and Song, 2001; Sasaki et al., 2001, Wang et properties (Ha et al., 1987; Shultz et al., 1992; Ip et al., al., 2002). Earlier studies ostulated that there could be 1999). Other effects include a role in reducing various purposes of hydrogenation in the rumen. The main atherosclerosis (Lee et al., 1994; Nicolosi et al., 1997) and a function of hydrogenation was the disposal of reducing benefit for diabetes treatment (Houseknecht et al., 1998). power which is essential to bacteria living in a reduced CLA is found almost exclusively in animal products. Since environment (Lennarz, 1966). Hydrogenation also has an CLA is a incompletely hydrogenated products by rumen essential role in the utilization of dietary fatty acids by fatty microorganisms in ruminant animals, bovine milk and milk acid-auxotrophic bacteria (Hazlewood and Dawson, 1979). products are among the richest dietary sources. The topic of An alternative suggestion is to detoxify the unsaturated CLA as it relates to ruminant production has been reviewed fatty acids (Kemp and Lander, 1984; Kemp et al., 1984). previously (Griinari and Bauman, 1999; Dhiman, 2000; Diverse bacteria relating to hydrogenation of the Chilliard et al., 2001). The objective of this paper is not to unsaturated fatty acids have been isolated. Kemp et al. provide an extensive review of the literature but rather to (1975) isolated 5 hydrogenating strains, and estimated that provide an overview of the key factors relating to the bio- strains were each present in the rumen at 107-8 per ml. The hydrogenation of unsaturated fatty acids released from the Treponema strain is reported to occur in about the similar dietary lipid hydrolysis by rumen microorganisms and numbers (Yokoyama and Davis, 1971). Verhulst et al. subsequent biosynthesis of CLA in the cow, and to discuss (1985) also isolated 7 hydrogenating strains from the rumen the feasibility and potential of producing CLA enriched fluid. Butyrivibrio fibrisolvens was proved to be major milk and beef. hydrogenating bacteria (Polan et al., 1964; Kepler and Tove, ** This paper was presented at an 2002 International Symposium 1967). Hazlewood et al. (1976) divided the hydrogenating on “Recent Advances in Animal Nutrition” held in New Delhi, bacteria into three groups based on the pattern of end- India (September 22, 2002). products of hydrogenation and on the isomerisations carried * Corresponding Author: Man K. Song. Tel: +82-43-261-2545, out. Bacteria are largely responsible for hydrogenation in Fax: +82-43-269-2549. E-mail: [email protected] the rumen while the protozoa are of only very minor 1 Dairy Research and Technology Centre, Food and Nutritional importance. Science, University of Alberta, Edmonton, AB, Canada T6G 2P5. SONG AND KENNELLY 307 cis-9, cis-12 (Linoleic acid) isomerisation (group A) C18:2 Small intestine cis-9, trans-11 conjugated diene C18:3 CLA hydrogenation (group A) C18:1 (Trans-11) trans-11-octadecenoic acid C18:3 Rumen FEED C18:2 C18:0 C18:2 CLA hydrogenation (group B) C18:1 t-11 C18:3 C18:0 Stearic acid MILK Figure 1. Scheme for the bio-hydrogenation of linoleic acid. Group A or B refer to the two classes of hydrogenating Bacteria Figure 2. Formation of CLA in the cow (Kemp and Lander, 1984). Figure 1 is the typical bio-hydrogenating scheme of those measured in vivo, thus in vitro measurements are linoleic acid (Harfoot and Hazlewood, 1988). reliable to predict the fatty acid metabolism in vivo. The pathway involves an initial isomerisation step resulting in the formation of a conjugated cis-9, trans-11 BIOSYNTHESIS OF CLA IN THE COW acid which then undergoes hydrogenation of its cis double bond leaving trans-11 octadecenoic acid. Finally this is Conjugated linoleic acid (CLA) is formed in the rumen hydrogenated to stearic acid (Dawson and Kemp, 1970; as an intermediate product in the digestion of dietary fat. Verhulst et al., 1985). The conversion of trans-11 C18:1 to Kepler and Tove (1967) in an early study showed that cis-9, C18:0 appears to involve a different group of organisms and trans-11 C18:2, the major isomer of CLA, is the first occurs at a slower rate (Griinari et al., 1997). According to intermediate formed in the biohydrogenation of linoleic Kemp and Lander (1984), group A bacteria mostly acid by the rumen bacteria as shown in Figure 2. Trans-11 hydrogenate linoleic acid to trans-11-octadecenoic acid C18:1 typically accumulates in the rumen. Trans-11 C18:1 while group B bacteria are capable of hydrogenating a wide and cis-9, trans-11 C18:2 account for approximately 50% range of octadecenoic acids, including trans-11 (trans- of the trans fatty acids found in milk fat (Griinari, 1998). vaccenic) acids to stearic acid. Although the cis-9, trans-11 is the predominant CLA In most studies it appeared that bio-hydrogenation in the isomer in bovine milk, other isomers can be formed with rumen occurs rapidly but the range of hydrogenation was double bonds in positions 8/10, 10/12, or 11/13. Each of quite large, depending on the degree of unsaturation of fatty these double bonds can be in a cis or trans configuration, acids, the configuration of unsaturated fatty acids, microbial giving a range of possible CLA isomers. type and the experimental condition. Song and Sohn (1997) The term CLA refers to this whole group of 18 carbon found the rapid bio-hydrogenation of linoleic and linolenic conjugated fatty acids. Alpha-linolenic acid goes through a acid when incubated with oils in vitro as shown in Table 1. similar biohydrogenation process producing trans-11 C18:1 Linolenic acid content in flaxseed oil was higher as 54.9% and C18:0, but does not appear to produce CLA as an than the other 13 fatty acids, but most of the linoleic as well intermediate. Choinard et al. (1998) and Kelly et al. (1998) as linolenic acid was disappeared. Similar results were indicated that the CLA was mostly derived from the dietary observed from the in vitro study by Song and Choi (1998). C18:2. Bessa et al. (2000), however, revealed the possibility Wu et al. (1991) reported that 44-68% of oleic acid, 63-79% of alternative pathway that may be existed in the production of CLA from C18:3 due to the extreme microbial diversity of linoleic acid and 78-90% of linolenic acid were in the reticulo-rumen. Wang et al. (2002) also observed an hydrogenated in the rumen. Fellner et al. (1995) reported increased cis-9, trans-11 CLA proportion from linseed based on in vitro study that bio-hydrogenation of infused incubation compared to that from rapeseed under the two linoleic acid averaged 77%. Palmquist and Jenkins (1980) addition levels of concentrate in incubation solution in vitro suggested that end product from microbial hydrogenation of (Table 2). Linseed is much higher in C18:3 than rapeseed C -unsaturated fatty acids was stearic acid, and this was 18 (Table 1). proved by Jenkins (1993). Because of the extensive bio-hydrogenation of linoleic Wu and Palmquist (1991) and Fellner et al. (1995) and linolenic acid to C18:1 trans-11, several studies have indicated that the extent of hydrogenation was similar to suggested that there may be little accumulation of CLA in 308 BIO-SYNTHESIS AND INCORPORATION OF CLA Table 1. Changes in C18-fatty acids when incubated with oils in vitro for 24 hour Rapeseed oil Linseed oil C18-fatty acids Prior to incubation Post incubation Prior to incubation Post incubation C18:0 1.93 62.20 2.72 61.24 C18:1 53.41 7.96 18.24 6.92 C18:2 25.78 0.31 15.56 0.40 C18:3 9.64 0.30 54.91 0.22 Table 2.