Quantitative Analysis of Phytosterols in Cattle Feed, Milk and Fortified Foods

Over recent decades, research has demonstrated a direct correlation between phytosterol consumption and the lowering of low-density lipoprotein cholesterol. The fortification of phytosterols in processed food products has therefore become increasingly popular and as a result, there is a subsequent need for new and improved techniques for quantification of phytosterols in these products. Natural phytosterol fortification of milk by controlled feeding is also becoming a common farming practice although the efficacy of this approach is relatively unknown. Moreover, there are no known reports regarding the resulting phytosterol content in milk under different animal feeding regimes. This study therefore investigated whether different cattle feeds can influence the profile of phytosterols and cholesterol in the milk produced as an alternative to direct fortification. A series of five feeding experiments were performed using common feeds used by Australian dairy farmers and selected formulated rumen protected feeds. In order to achieve this main objective, a new reliable and rapid analytical technique was required which could accurately measure total phytosterols (including the conjugates) at naturally occurring levels in cattle feed and the resulting bovine milk. The analytical method development investigated three hydrolysis techniques to liberate the sterols for extraction. This included acid hydrolysis and enzymatic treatments (for glycosidic bonds) and saponification (for fatty acid ester bonds). The method development also included optimisation of a sample clean-up and instrumentation. The final method parameters were selected based on accuracy, time efficiency, labour intensity and the availability of resources. The optimised analytical method used acid hydrolysis and saponification protocols with simultaneous sterol solvent extraction during the hydrolysis step to avoid the less efficient manual liquid extraction step usually performed at this stage. This was then followed by sample clean-up using an amino propyl phase solid phase extraction for cattle feed samples. All extracts were concentrated to a known volume and derivatised using a silylating reagent to make them thermally stable for analysis. Quantification of sterols was performed using gas chromatography coupled with mass spectrometry and flame ionisation detectors which, allowed for direct quantification and identification of the samples. The total phytosterol determination was based on the sum of the identified plant sterols including brassicasterol, stigmasterol, campesterol, campestanol, β-sitosterol and stigmastanol. A total of twelve different cattle feed types (excluding the rumen protected feed) commonly used in the dairy industry were analysed including lucerne, pasture (rye grass), maize silage, pasture silage, grape marc (dried and wet), wheat, canola, tannin, barley grain, mineral mix, cotton oil and molasses. These feeds were used in various combinations for the controlled feeding trials with the developed analytical method determining that the highest and lowest average phytosterol contents were found in cotton seed oil (256 mg/100 g) and tannin (<35 mg/100 g) respectively. Based on the analytical method developed, the limit of reporting was 35 mg/100 g and 5 mg/100 g for total phytosterols and individual sterols respectively. In addition to these common cattle feeds, a final feeding trial was also conducted with a rumen protected feed with a known high phytosterol content. The results of the feeding trials showed that statistical significances (p < 0.05) were observed for some individual phytosterols and cholesterol in milk under these differing feeding regimes compared to the respective controls. The limit of reporting for the milk was 0.12 mg/100 mL and 0.02 mg/100 mL for total phytosterols and individual sterols respectively. In the case of the phytosterols, where the daily recommended consumption to optimise the health benefits is typically 2 g per day, the levels found in milk were