Antimethanogenic activity of Monascus metabolites in the rumen revealed by the concentration of statins, their diversity and the presence of acid forms

Monascus-fermented cereals reduce methane production from the rumen. The identification of the metabolites responsible of the antimethanogenic effect is important to assess the potential of this strategy as a mitigation option in ruminant production. This study highlights metabolites from Monascus ruber associated to methane inhibition. An in vitro rumen screening test was used to rank solid-state fermented wheat samples for their ability to inhibit methane. Four active and four less-active samples were selected for metabolomics analysis using liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS) and the identity of discriminant variables responsible for this group distinction was assigned thanks to tandem mass spectrometry (MS/MS) experiments. A total of 28 discriminating metabolites were putatively identified based on their accurate m/z values, fragmentation pathways and information from databases. The chemical structure (identification level 1) was confirmed for 9 of them thanks to the available authentic chemical standards. Most of these metabolites belong to the chemical class of statins and their derivatives (n=13), four of them annotated as statin-like molecules were observed here for the first time. A targeted approach using LC-MS/MS was performed to measure the levels of known metabolites and showed that the lovastatin concentration in active samples was 16-fold greater than in least-active samples. Whereas lovastatin was the major metabolite, up to 40% of the total statins were represented by other statin molecules. Comparison of the functional capability of lovastatin lactone and lovastatin acid demonstrates that the acid form is responsible for the antimethanogenic activity in the rumen environment. This study shows that Monascus-fermented feeds contain a wide variety of statins in both lactone and acid forms. Information from this work provides insight for improving the antimethanogenic efficacy of diets containing bioactive Monascus metabolites in ruminants