Release of Hypoglycin A from Hypoglycin B and Decrease of Hypoglycin A and Methylene Cyclopropyl Glycine...

The transformation of hypoglycin A (HGA), hypoglycin B (HGB), and methylene cyclopropyl glycine (MCPrG) in ruminal fluid batch cultures was investigated, and the effect of these toxins on the batch culture microorganisms using microbial metabolites was measured. An experiment was conducted using ovine ruminal fluid batch cultures and the ANKOM RF Gas Production System over four runs, each with an incubation period of 48 h. The fermenters contained 200 mg of (i) a substrate mixture (80% cellulose, 20% starch; CSM), (ii) CSM and 1.5 mL of a solution of pure toxins (a mixture of 500 ng/mL HGA and MCPrG each; PCM), or (iii) CSM and 100 mg sycamore maple seeds (SMS). Each fermenter contained 30 mL of inoculum (ruminal fluid and buffer, 1:2 v/v). For control, autoclaved ruminal fluid was incubated with CSM, PCM, and SMS, respectively. Samples were taken from the liquid phase of the fermenters and analyzed using liquid chromatography-tandem mass spectrometry (LC/MS-MS) for sycamore maple toxins and metabolites. Microbial activity was assessed using gas production, short chain fatty acids, and NH₃ concentration. Additionally, pH and redox potentials were measured. In PCM, HGA and MCPrG concentrations rapidly decreased (p < 0.05), and were not measurable anymore after a 24 h incubation period. In SMS, the initial concentrations were 4.7 ± 1.4 µg/mL HGA, 19.9 ± 5.41 µg/mL HGB, and 1.2 ± 0.33 µg/mL MCPrG. In SMS, HGA increased in 24 h, coincidently to a decrease in HGB concentration (p < 0.05). We modeled a rapid conversion of HGB to HGA, accompanied by progressive HGA transformation. The concentration of MCPrG was constant until 4 h and decreased afterwards (p < 0.05). In SMS incubations, HGA and MCPrG concentrations of 5.6 ± 1.5 and 0.32 ± 0.090 µg/mL remained after 48 h, respectively. The HGB to HGA conversion and transformation of HGA and MCPrG also occurred in autoclaved ruminal fluid. Gas production and microbial metabolite concentrations were higher in SMS compared to CSM and PCM (p < 0.05), as the seeds were used as an additional substrate by the batch culture microorganisms.