Comparison of enzymatic activities of the reactions of linoleic and linolenic acids ruminal biohydrogenation


Introduction: Biohydrogenation (BH) is a microbial hydrogenation of dietary unsaturated fatty acids occurring in the rumen. BH is of interest because it directly affects the fatty acids composition of milk and meat. The linoleic acid (C18:2) BH is divided into three reactions: isomerisation into conjugated linoleic acids (CLA), reduction to trans-C18:1 and then to stearic acid (C18:0); that of alpha-linolenic acid (C18:3) into four reactions: isomerisation to conjugated linolenic acid (CLnA), reduction to trans11,cis15-C18:2, then to trans-C18:1 and finally to C18:0. The aim of this study was to compare enzymatic activities of the reactions of C18:2 BH to those of C18:3 BH. Materials and methods: Rumen fluid was collected from a dry dairy cow and strained on a metal sieve (1,6mm). Then, it was mixed with Chloramphenicol (Cm), an inhibitor of protein synthesis in prokaryotes, at a dose of 1mg/mL . Incubations were prepared by adding 1mL of rumen fluid + Cm, with 1mL of bicarbonate buffer and 1mg of C18:2 or C18:3 (purity ≥ 99%, Sigma), and were conducted in a waterbath at 39°C, with 3h agitation, in 3 replicates. Fatty acids were quantified by gas chromatography. Then rate (v, mg/L/h) and efficiency (E, %) of the reactions were calculated. Results and Discussion: The isomerisation of C18:3 was quicker and more efficient than that of C18:2, which was probably saturated2 (v = 129.6 vs. 94.4 mg/L/h; E = 80.2 vs. 52.7%, respectively. The reductions of conjugated isomers were rapid and efficient, mainly for CLnA (v = 123.7 mg/L/h; E= 95.5%) compared to CLA (v = 78.1 mg/L/h; E= 82.0%). However, for C18:2 BH, cis9,trans11-CLA disappeared quicker than trans10,cis12-CLA so that their respective production after 3h incubation was +0.016mg vs. +0.073mg. The last reduction of C18:2 BH was the slowest (v = 63.8 mg/L/h; E= 68.9%), and constituted the limiting step, resulting in trans-C18:1 accumulation. The second reduction of C18:3 BH was very slow and poorly efficient (v = 48.8 mg/L/h; E= 38.2%), so that trans11,cis15-C18:2 highly accumulated (+0.450mg produced). The last reduction of C18:3 BH was also a slow and poorly efficient reaction (v = 38.8 mg/L/h; E= 41.9%), so that trans-C18:1 would probably have accumulated with a longer incubation. Conclusion: The BH of C18:2 and C18:3 were not exactly similar. C18:2 BH was slower, its isomerisation seemed to be rapidly saturable and the limiting step was the final reduction inducing an accumulation of trans-C18:1. For C18:3 BH, first and second reactions were rapid, so that few CLnA was present in the media. Contrarily, the third and fourth reactions were slow so that trans11,cis15-C18:2 firstly accumulated. Such an evolution was previously reported in vitro with live mixed ruminal bacteria2, indicating that the evaluation of BH does not require live bacteria, and confirming the validity and interest of this enzymatic approach

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