# Dietary Compounds Influencing the Sensorial, Volatile and Phytochemical Properties of Bovine Milk
## 2 Results and Discussion
### Volatile Analyses (Feed, Raw and Pasteurized Milk)  
Volatile profile analysis by headspace solid-phase microextraction gas-chromatography mass spectrometry (HS-SPME GCMS) was performed on the GRS, CLV, and TMR feed samples, and on the r and p GRS, CLV, and TMR milk samples on days 3, 9, and 14 of refrigerated storage. The transfer of VOCs from feed to bovine milk is well documented, and studies have shown that volatile compounds in forage and feed can enter milk by two mechanisms, namely absorption via the digestive tract (rumen or intestine) before diffusing into the blood and subsequently the mammary gland, and/or through the pulmonary system wherein volatiles present in the air are inhaled, absorbed through the lungs, enter the blood steam, and diffuse into the mammary gland [1; 44]. Respectively, 90 and 104 compounds were identified in GRS and CLV feed samples, consisting mainly of aldehydes, ketones, esters, alcohols, and hydrocarbons. A further 94 compounds were identified in TMR samples consisting mainly of aldehydes, ketones, esters, alcohols, acids, and hydrocarbons (Supplementary Figures S2 and S3). Figure 1: **(a)** Principal component analysis (PCA) biplot showing the correlations between the isoflavones (apigenin, daidzein, formononetin, and genistein) identified in feed samples (grass, grass/clover and TMR) samples and the corresponding raw (r) and pasteurized (p) (grass (GRS), clover (CLV) and total mixed ration (TMR) milk samples as determined by liquid chromatography tandem mass spectrometry (LC-MSMS); **(b)** PCA biplot showing the correlation of the isoflavones (apigenin, daidzein, formononetin, and genistein) to raw (r) and pasteurized (p) GRS, CLV and TMR milk samples as determined by LC-MSMS. Eleven aldehydes, 10 ketones, 30 esters, 10 alcohols, seven acids, two fatty acid esters, one terpene, four furrans, five hydrocarbons, two phenols, two sulphur compounds, two lactones, four pyrazines, and one ether compound varied significantly (\(p<0.001\)) between feed types (Table 1). As well as the direct transfer from feed, alterations in VOCs in milk can occur during pasteurization (thermal) or storage (oxidative, microbial and enzymatic). Of the 32 volatile compounds identified in the feed samples and the corresponding raw milk samples, 20 were identified across all samples (decanal, heptanal, hexanal, nonanal, 2-heptanone, 2-pentanone, acetone, acetophenone, 2-methyl-1-butanol, 3-methyl-1-butanol, cumene, mestilbene, 2,4-dimethylfuran, 1,3-bis(1,1-dimethylethyl)-benzene, 2,4-dimethyl-benzaldehyde, p-xylene, tert-butylbenzene, toluene, dimethyl sulphide and vinylisopentyl ether). It is probable that some of these compounds were transferred directly from the feed to the milk. Decanal (sweet aldehyde), heptanal (green, fatty, herbal), hexanal (green, fatty), nonanal (waxy, orange-peel, fatty), octanal (aldehyde, waxy, fatty), and pentanal (fermented, cardboard-like, bready, nutty) are lipid oxidation products resulting from fatty acid degradation. Further, [32; 45] 2-heptanone (fruity, spicy, sweet) and 2-pentanone (sweet, fruity, ethereal) are secondary oxidation products. Acetone (hay, earthy, wood pulp) has previously been reported to originate from the diet of cows [19], while acetophenone (floral) is a product of phenylalanine metabolism [46] and is also a product of the Maillard reaction, which has been attributed to the heated or sterilized flavor of ultra-heat treated milk [47]. Moreover, 2-Methyl-1-butanol (roasted, wine, onion, fruity) and 3-methyl-1-butanol (fermented) may have originated from the degradation of isoleucine and leucine, respectively, by _Saccharomyces cerevisiae_, as well as other yeasts [48]. They may have also been produced from their corresponding methylketones by reductase activity or by lactic acid bacteria [49]. Further, 1-Pentanol (fermented, bready, yeasty) is derived from the primary aldehyde pentanal by oxidation [1] and \(\alpha\)-Pinene (herbal) is most likely derived from plant diet, and was highest in GRS feed, rGRS, and pGRS milk samples.