Use of headspace solid-phase microextraction for the analysis and characterisation of volatile compounds in rumen contents : a thesis presented in partial fulfillment of the requirements for the degree of Masterate of Science in Chemistry at Massey University

Abstract

Appendix 2&3 removed due to copyright restrictions. Please consult print copy in Library.Volatile fatty acids (VFAs), alkyl phenols and indolic compounds are produced by rumen microbes during the fermentation of forages in ruminants. In this study, ruminal fluid obtained from sheep was examined by headspace solid-phase microextraction (SPME) sampling followed by GC-MS analysis. This technique provides a non-invasive, clean and selective method to characterize the volatiles in ruminal fluid from an in vitro fermentation system. The factors which can influence the extraction efficiency were studied and include the SPME fibre, sample volume, pH of sample matrix (rumen fluid) and extraction time by the fibre in the headspace. The optimum experimental conditions for the analytes in question included: polyacrylate fibre to perform the headspace SPME above 20 mL of rumen fluid in a 68 mL vial for 5 min, followed by immediate GC-MS analysis. The pH of the rumen fluid sample greatly influenced VFA extraction efficiency. Quantitative analysis of p-cresol, m-cresol, indole and skatolc with SPME were compared with steam distillation simultaneous extraction. This comparison showed that the HS-SPME method was semi-quantitative. The optimum in vitro system (16 mL of rumen fluid and 4 mL of artificial saliva in a 68 mL vial incubated at 39°C) was utilised to study production of indole, skatolc and p-cresol from the anaerobic fermentation of tryptophan and tyrosine. Spirulina is an abundant source of dietary protein. Therefore, ¹³C labelled spirulina was used to study the metabolism of protein and formation of analytes derived from ruminal metabolism of protein. A series of labelled end products, including toluene, acetic acid, propanoic acid, iso-butyric acid, n-butyric acid, iso-valeric acid, n-valeric acid, p-cresol, indole, skatole, dimethyldisulfide and dimethyltrisulfide were detected by GC-MS. This result indicates that these compounds are the products of ruminal metabolism of spirulina. When applied to the in vitro rumen system the headspace SPME technique provides a fast approach to study metabolism of target compounds and allows the researcher to follow proposed pathways with labelled substrate