Optimising Microbial Xylanase Hydrolysis of Brewer’s Spent Grain for the Production of Monosaccharides and Oligosaccharides

Abstract

Brewer’s spent grain (BSG) is a major by-product of the brewing industry and contains a considerable amount of carbohydrate, fibre and protein, making it a potential material for valorisation. The carbohydrate fraction of BSG includes hemicellulose, of which arabinoxylan is a major component. However, much of its arabinoxylan is present in an insoluble form and associated with other cell-wall components, limiting its accessibility and conversion into value-added carbohydrate products. This study investigated the xylanase hydrolysis of BSG and optimised the hydrolysis conditions for the production of monosaccharides and xylo-oligosaccharides (XOS). Proximate analysis showed that BSG contained 1.03% moisture, 4.74% ash, 11.8% fat, 48.7% carbohydrate, 21.5% protein and 13.0% crude fibre. Acid hydrolysis further showed the presence of xylose, arabinose, glucose, galactose, mannose, galacturonic acid, glucuronic acid, rhamnose, ribose and fucose, with xylose (17.8%) and arabinose (9.89%) being the major monosaccharides released from the structural carbohydrate fraction. The total monosaccharide content obtained following acid hydrolysis was lower than the carbohydrate content estimated by proximate analysis, potentially due to differences in analytical method, incomplete hydrolysis and sugar degradation during acid treatment. The predominance of xylose and arabinose indicated that BSG is a suitable substrate for xylanase-mediated hydrolysis. To optimise the hydrolysis process, response surface methodology (RSM) using a Box-Behnken design (BBD) was employed to evaluate the effects of pH, incubation time, and enzyme concentration. Xylose and arabinose were evaluated as monosaccharide responses, while xylopentaose (DP5) was selected as the XOS response for optimisation. The responses behaved differently under the tested conditions. Arabinose was mainly influenced by pH, whereas incubation time had the strongest effect on DP5. Enzyme concentration did not have a significant effect on xylose, arabinose, and DP5 within the range studied (50–200 U/g BSG). Validation showed poor agreement for xylose, with an experimental concentration of 94.0 ± 11.8 µg/mL compared with the predicted concentration of 187 µg/mL. Most importantly, xylose showed a different response pattern from arabinose and DP5, indicating that the same hydrolysis conditions could not maximise all three responses. Therefore, the optimisation was refined to focus on arabinose and DP5. The refined optimisation gave a composite desirability of 0.963 at pH 4.0, 50 U/g BSG and an incubation time of approximately 8.8 h. The predicted response for arabinose concentration was 9.30 µg/mL, while the predicted HPLC response assigned to DP5 was 678 µg/mL xylose equivalents. Validation resulted in an arabinose concentration of 12.6 ± 2.12 µg/mL and an HPLC response of assigned to DP5 of 604 ± 13.6 µg/mL xylose equivalents. Mass spectrometric analysis also supported the presence of XOS corresponding to DP2–DP7 in the hydrolysate. Overall, the findings showed that xylanase hydrolysis of BSG can produce both monosaccharides and XOS, and that the most suitable hydrolysis conditions depend on the target product.