Galacto-oligosaccharides (GOS) are used as prebiotic ingredients in various food and pharmaceutical formulations. Currently, production of GOS involves the enzymatic conversion of lactose by transgalactosylation using β-galactosidase. The purity of the resulting product is low, typically limited to up to 55% GOS on total carbohydrate basis due to the presence of non-reacted lactose, and the formation of by-products glucose and galactose. In industrial practice high-purity GOS is manufactured by removing the unwanted mono- and disaccharides from raw GOS with simulated moving bed (SMB) chromatography. This purification step is associated with high processing cost that increases the price of pure GOS and limits its marketability. The last decades have witnessed a growing interest in developing competitive biotechnological processes that could replace chromatography. This paper presents a comprehensive review on the recent advancements of microbial GOS purification, a process commonly referred to as selective fermentation or selective metabolism. Purification strategies include: (i) removal of glucose alone or together with galactose by lactose negative yeast species, that typically results in purity values below 60% due to remaining lactose; (ii) removal of both mono- and disaccharides by combining the fast monosaccharide metabolizing capacity of some yeast species with efficient lactose consumption by certain lactose positive microbes, reaching GOS purity in the range of 60–95%; and (iii) the application of selected strains of Kluyveromyces species with high lactose metabolizing activity to achieve high-purity GOS that is practically free from lactose and monosaccharides. Graphical abstract

Galacto-oligosaccharides (GOS) are prebiotic compounds widely used for their health-promoting effects. Conventionally, GOS is produced by the enzymatic conversion of lactose in stirred tank reactors (STR). The high operational costs associated with enzyme inactivation and removal might be reduced by the application of enzyme membrane reactors (EMR). In this study, we aimed to assess the potential of continuous GOS production by EMR using soluble Biolacta N5, a Bacillus circulans-derived commercial enzyme preparation. The steady-state performance of the EMR equipped with an ultrafiltration module was investigated as function of residence time (1.1–2.8 h) and enzyme load (17–190 U·g⁻¹) under fixed operational settings of temperature (50 °C), pH (6.0), lactose feed concentration (300 g·kg⁻¹), and recirculation flow-rate (0.18 m³·h⁻¹). Results indicate that the yield of oligosaccharides with higher degree of polymerization (DP3-6) in STR (approx. 38% on total carbohydrate basis) exceeds that measured in EMR (ranging from 24% to 33%). However, a stable catalytic performance without a significant deterioration in product quality was observed when operating the EMR for an extended period of time (>120 h). Approx. 1.4 kg of DP3-6 was produced per one gram of crude enzyme preparation over the long-term campaigns, indicating that EMR efficiently recovers enzyme activity.

Research background. Several loop-mediated isothermal amplification (LAMP) assays with good performance characteristics have been developed for the detection of Listeria monocytogenes in food; however, there are only a few cases in which DNA extraction, amplification and sensing have been performed in a single-tube system. Experimental approach. The efficiency of DNA extraction by lysis buffers was tested using LAMP. New primer sets for LAMP assays were designed using PrimerExplorer V5 software. The sensitivity and specificity of the LAMP inner primers were determined by optimised PCR. The end-point detection involved gel electrophoresis, turbidity and eriochrome black T (EBT) colour reaction. The sensitivity, specificity and limit of detection (LOD) of the developed LAMP assays were then characterised using L. monocytogenes, non-monocytogenes Listeria and non-Listeria bacterial strains. Results and conclusions. Both the alkaline cell lysis-based sodium hydroxide and Tris-HCl (HotSHOT)+Tween buffer and the Triton X-100 and sodium azide-based TZ buffer generated amplifiable DNA templates under isothermal conditions for LAMP. However, the TZ buffer produced a significantly higher DNA yield than the HotSHOT+Tween buffer. LAMP primers were designed to target the hlyA and inlA virulence genes of L. monocytogenes. The sensitivity and specificity of the LAMP inner primers were 100 % for both genes; however, the PCR reaction targeting the inlA gene generated fewer non-specific PCR products than the hlyA-targeting PCR. The sensitivity of the InlA LAMP assay was 100 %, while its specificity was 96 %. The LOD was 500 fg per reaction, which corresponds to 157 genome copy numbers. The combination of DNA extraction, LAMP amplification, and colorimetric endpoint detection in a single tube resulted in a LAMP assay suitable for the detection of L. monocytogenes in food under laboratory conditions, with potential for further development for on-site detection with microfluidic platforms. Novelty and scientific contribution. To the best of our knowledge, this is the first report of a LAMP assay targeting the inlA gene of L. monocytogenes. The developed single-tube LAMP assay is well-suited for integration with microfluidic systems.