# Dietary Compounds Influencing the Sensorial, Volatile and Phytochemical Properties of Bovine Milk
## 2 Results and Discussion
### Phytochemical Analyses  
Isoflavones are important as they have the ability to be directly transferred from feed to milk, and subsequently to be reduced to compounds that can potentially impact the sensory properties of milk and other dairy products. In particular, formononetin has been linked to the production of p-cesol [33]. At day three, p-Cresol was not detected in the milk samples, but was detected in all samples at days nine and 14 of storage. It is possible that it was present in the milk samples at day three in sulfonated form or below levels of detection, and was subsequently released by enzymatic action, specifically by arylsulfatase during the storage period [34]. The concentrations of formononetin were found to be significantly correlated to white clover (CLV) feed samples (Figure 1a). Levels of apigenin, daidzein, and genistein were found to be significantly different between the raw (r) and pasteurized (p) GRS, CLV, and TMR milk samples. Daidzein and genistein were highly correlated to rCLV milk and formononetin was more closely correlated with rGRS milk (Figure 1b).  
The concentration of formononetin was found to be highest in rGRS milk, as previously mentioned, and formononetin is likely degraded to p-cesol, a compound that has been associated with barnyard aroma in dairy products. Both r and p GRS milk had the highest levels of p-cresol at days nine and 14. Further, pGRS milk was also more correlated with barnyard aroma than the pCLV and pTMR. The significant correlation of formononetin to CLV feed samples (Figure 1) is expected as leguminous plants such as clover are naturally rich in phytoestrogens [35]. The difference in levels between r and p mills suggests an effect of pasteurization on the compounds, but it is possible that some or all of the formononetin present in the samples was demethylated to daidzein, which is highest in rCLV milk and not detected in the corresponding CLV feed samples (Figure S1). It is also possible that daidzein was further reduced via hydrogenation and ring scission to equal (a microbial metabolite of isoflavure with high estrogenic activity) [36] or metabolized to O-desmethylangolensin [37]. The composition of the individual bovine gut microflora impacts largely on the metabolism of daidzein and subsequently on the rate of equal excretion [35]. Although formononetin is closely correlated to rGRS milk, the TMR feeding system implemented in this study is partly soya-based, in addition to containing grass silage and maize silage, which could explain its proximity to pTMR milk (Figure 1). The other three isoflavones (daidzein, genistein, and apigenin) are significantly correlated to rCLV milk. Numerous isoflavures are readily reduced or converted to other phytoestrogens. As previously mentioned,formononetin can be converted to other isoflavones such as daidzein and subsequently equal [38]. Genistein and daidzein both require degradation of the active compound by gut microflora in order to become bioavailable. Note that S(-)-equol is the active metabolite of daidzein. Genistein, a metabolite of biochanin A, is generally metabolized to glucuronides and sulfate conjugates [39]. Genistein can also be degraded to the higher homolog of p-cresol, 4-ethyl-phenol, by gut microflora [40]. It is known thay 4-Ethyl-phenol is an inactive metabolite with no estrogenic activity. Further, p-Cresol-sulfate has been shown to be a gut-mediated metabolite of genistein. Apigenin has been reported to be metabolized to luteolin, mediated by the enzyme cytochrome P450 [41]. Isoflavone metabolites are also known to be excreted in the urine of ruminants [42] and so losses occur. Moreover, [PERSON] [43] has postulated that the epithelial cells in the mammary gland may only be semi-permeable to estrogenic compounds resulting in a limited transfer from the blood to the milk. Thus, it seems likely that any isoflavones that are present through ingestion can be metabolized to odor-active compounds that potentially impact the sensory properties of bovine milk.