The goal of this study was to investigate the composition of milk and whey from the Saanen caprine breed in New Zealand, for the hypothesis; New Zealand Saanen caprine milk and whey have a similar oligosaccharides profile to that previously identified in studies with other caprine breeds. The data shown here is in accordance with the ions and concentrations observed in other caprine breeds [265, 317-320] (Table 1.2), from around the world. The second hypothesis, The method of whey lactose hydrolysis, centrifugation and porous graphitic carbon adsorption chromatography will produce an CMOP on a scale sufficient for subsequent in vitro and in vivo experimentation, was confirmed by the production of a CMOP containing 8% oligosaccharides, 44% monosaccharides, 44% lactose and 4% GOS.
2.5.1 Caprine whey processing
Caprine whey was chosen as the starting material for further enrichment of CMO as most of the fat and protein had already been removed during cheese making, simplifying further oligosaccharide enrichment. After initial centrifugation and ultrafiltration steps, (to decrease the fat and protein concentration of the whey), -galactosidase was used to hydrolyse the lactose to avoid lactose binding to the porous graphitised carbon under the adsorption/desorption conditions used to bind and elute the oligosaccharides. However, the conditions utilised in this study were only sufficient to hydrolase 92% of the lactose to the monosaccharides, glucose and galactose. The inhibitory effect of higher concentrations of galactose in batch systems on lactose hydrolysis have been reported previously [301, 302]. Higher conversion rates may be achieved by continuous systems using immobilised β- galactosidase.
63 After loading the -galactosidase treated sample onto the porous graphitised carbon, the column was washed with three column volumes of deionised water. This partially removed the carbohydrate contaminants (glucose, galactose, GOS and lactose) together with the salts and residual traces of protein and lipids prior to elution with 40% acetonitrile. A final product containing 8% oligosaccharides, 44% monosaccharides, 44% lactose and 4% GOS was obtained. Although acetonitrile is a solvent used in pharmaceutical preparations and it should be avoided in food additives [321], it is expected that complete acetonitrile removal was achieved by evaporation and freeze drying.
2.5.2 Oligosaccharide characterisation and quantification
A recent study has identified 40 different oligosaccharide structures in caprine milk and described the relative abundance of both acidic and neutral oligosaccharides using ultra- performance liquid chromatography [301]. The results of this current study use the relative intensities (peak areas) of extracted mass peaks from the mass spectrometric analysis for the quantification of CMO compared against the specific standards. While it is recognised that the intensity of each ion is dependent on the ability of that particular molecule to ionise in solution, approximate amounts of each putative oligosaccharide were estimated based on the appropriate calibration curves. The seasonality of milk yield, breed and degree of ionisation of the different oligosaccharides could be limitations of the current method and be responsible for variations in CMO concentrations and detection when compared to other studies. Although 40 different oligosaccharide structures have been identified in caprine milk by a recent study [301], the present study has identified and quantified only the most abundant oligosaccharides.
Martinez-Ferez et al. [265] compared the oligosaccharide concentrations among caprine, ovine, bovine and human milk and demonstrated that the CMO concentration was (0.25 to 0.30 g/L ten times higher than BMO (0.03 to 0.06 g/L) and ovine oligosaccharides (0.02 to 0.04 g/L), but less concentrated than HMO (5 to 10 g/L). Our results are similar with CMO
64 concentrations previously described, with the sum of the four most concentrated oligosaccharides (3’ galactosyl-lactose (m/z 503), 3’-sialyl-lactose (m/z 632), 6’-sialyl-lactose (m/z 632), N-glycolylneuraminyl-lactose (m/z 648) and Disialyl-N-lactose (m/z 923)) in mature caprine milk from the Saanen breed in New Zealand, ranging between 0.19 to 0.31 g/L. The variation of oligosaccharide concentrations reported here and by other authors [279, 304] may be due variations of animal breed, feed, lactation stage and/or method of quantification. The approach described here (solid phase extraction with graphitised carbon and the LC-MS), however, could be used as a relatively rapid method to identify whey sources richest in oligosaccharides.
2.5.3 Sialyloligosaccharides prevalence and health effects
The sialyloligosaccharides (N-glycolylneuraminyl-lactose (m/z 648), 3’-sialyl-lactose (m/z
632) and 6’-sialyl-lactose (m/z 632)) are oligosaccharides highly prevalent in New Zealand Saanen caprine colostrum, milk and whey, although there was compositional variation between individual samples (Table 2.4). Over the last two decades, sialyloligosaccharides have been demonstrated to have a range of biological functions in humans. For example, 3’- sialyl-lactose and 6’-sialyl-lactose have been shown to increase the in vitro adhesion of bifidobacteria to intestinal cells [322] and promote, an intestinal microbiota resistant to DSS induced colitis in adult mice [283]. There are also indications that sialyoligosaccharides may reduce the severity of influenza virus infection and ulcers caused by Helicobacter pylori
[284]. Other functions include increased immunity in infants, development of cerebral function, and enhanced proliferation of commensal enteric bacteria [72, 237, 285, 286].
The sialyloligosaccharide N-glycolylneuraminyl-lactose, however, contains the monosaccharide Neu5Gc, found in most mammalian milk (e.g. in the oligosaccharides 3’- sialyl-lactose and 6’-sialyl-lactose present in caprine and bovine milk), but not in human milk which contains Neu5Ac instead. Due to the fact that Neu5Gc differs from the human Neu5Ac, by only one oxygen atom, after the consumption of these oligosaccharides, human
65 cells may take up the Neu5Gc. The immune system does recognise this molecule as foreign, and this may be associated with increasing risk of many diseases, including carcinomas, atherosclerosis, and type-2 diabetes. Although not proven, caution is suggested when using oligosaccharides containing Neu5Gc in a human diet [323].
Sialyloligosaccharides represent 12 out of 23 predominant oligosaccharides found in human milk [271, 278], but only five out of six [261] and four out of seven predominant oligosaccharides described for bovine and caprine milk, respectively [279]. Although there is an equivalent percentage of sialyloligosaccharides in caprine (95%) and bovine milk (91%) compared to neutral oligosaccharides [301], their concentrations are two or three times higher in caprine milk (120-200 mg/L) than in bovine milk (60 mg/L) [261] and bovine infant formulas (15–35 mg/L) [262]. The presence of sialyloligosaccharides in caprine milk and their potential health effects in human milk [282] indicate that CMO may be used in the healthcare and food sectors as ingredients to promote health in both infants and adults.