A major challenge in isolating oligosaccharides from dairy streams is to enrich oligosaccharides while simultaneously reducing the content of simple sugars (mono- and disaccharides) that do not possess the desired prebiotic functions. An integrated approach based on optimized conditions that favor maximum lactose hydrolysis, monosaccharide fermentation and oligosaccharides recovery by nanofiltration was developed. Upon complete lactose hydrolysis and fermentation of the monosaccharides by yeast, nanofiltration of fermented whey permeate from colostrum enabled the recovery of 95% of the oligosaccharides at high purity. While the number of commercially available standards has limited the quantification of only a few sialylated oligosaccharides, the application of both high performance anion-exchange chromatography with pulsed amperometric detection and mass spectrometry provided a complete profile of the final product. Approximately 85% of the oligosaccharides in the final concentrate were sialylated, with the remainder being neutral.

The purification of caprine milk oligosaccharides (COS) by membrane filtration has been hampered by the low concentration of target COS and high concentration of lactose. In addition, their molecular weight proximity hinders the recovery of a COS fraction with high degree of purity and recovery yield. In this work, the recovery of a high purity COS concentrate was obtained by the optimization of an integrated approach including complete lactose hydrolysis, fermentation of the resulting monosaccharides and nanofiltration. All carbohydrates were quantified using High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC PAD). Defatted goat whey was ultrafiltered with discontinuous diafiltrations to increase the recovery of COS in the whey permeate which was then subsequently concentrated by nanofiltration. COS recovery yields of 75% with negligible amounts of monosaccharides (0.3% of the initial amount of lactose in the whey permeate) were achieved. A final retentate containing 67.6 and 34.4% of acidic and neutral oligosaccharides respectively was obtained from caprine milk.

Enzymatic hydrolysis of lactose has been shown to improve the efficiency and selectivity of membrane-based separations toward the recovery of bioactive oligosaccharides. Achieving maximum lactose hydrolysis requires intrinsic process optimization for each specific substrate, but the effects of those processing conditions on the target oligosaccharides are not well understood. Response surface methodology was used to investigate the effects of pH (3.25-8.25), temperature (35-55°C), reaction time (6 to 58 min), and amount of enzyme (0.05-0.25%) on the efficiency of lactose hydrolysis by β-galactosidase and on the preservation of biologically important sialyloligosaccharides (3'-siallylactose, 6'-siallylactose, and 6'-sialyl-N-acetyllactosamine) naturally present in bovine colostrum whey permeate. A central composite rotatable design was used. In general, β-galactosidase activity was favored at pH values ranging from 3.25 to 5.75, with other operational parameters having a less pronounced effect. A pH of 4.5 allowed for the use of a shorter reaction time (19 min), lower temperature (40°C), and reduced amount of enzyme (0.1%), but complete hydrolysis at a higher pH (5.75) required greater values for these operational parameters. The total amount of sialyloligosaccharides was not significantly altered by the reaction parameters evaluated, suggesting specificity of β-galactosidase from Aspergillus oryzae toward lactose as well as the stability of the oligosaccharides at pH, temperature, and reaction time evaluated.

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