No abstract

β-galactosidase (EC 3.2.1.23) are interesting enzymes able to catalyze lactose hydrolysis and transfer reactions to produce lactose-based prebiotics with potential application in the pharmaceutical and food industry. In this work, Aspergillus lacticoffeatus is described, for the first time, as an effective β-galactosidase producer. The extracellular enzyme production was evaluated in synthetic and alternative media containing cheese whey and corn steep liquor. Although β-galactosidase production occurred in all media (expect for the one composed solely by cheese whey), the highest enzymatic activity values (460U/mL) were obtained for the synthetic medium. Ochratoxin A production in synthetic medium was also evaluated and 9days of fermentation was identified as a suitable fermentation time to obtain a crude extract enzyme with mycotoxin concentration below the legal comparable value established for wine and grape juices (2ng/mL). The optimal pH and temperature for the crude extract enzyme was found in the range of 3.5-4.5 and 50-60°C, respectively. The β-galactosidase activity was reduced in the presence of Ba, Fe, Li, K and galactose, while additives (except for ascorbic acid) and detergents exhibited a positive effect on enzymatic activity. This enzyme was able to catalyze the synthesis of prebiotics, namely lactulose (2.5g/L) and a galacto-oligosaccharide (trisaccharide, 6.3g/L), either when whole cells or crude enzyme was used as biocatalyst. The lactulose production using fungal whole cells is herein reported for the first time. Additionally, A. lacticoffeatus was also found to produce an enzyme with fructosyltransferase activity and other prebiotics, namely fructo-oligosaccharide 1-kestose (2.4g/L).

Until now the prebiotic potential of pure trisaccharide raffinose on human health assessed through highthroughput sequencing remains poorly investigated. In this work, an in vitro model using human fecal inocula of two healthy volunteers (D1 and D2) was used to study the prebiotic potential of raffinose and compare it with the well-stablished and commercial prebiotic lactulose. The intestinal microbiota showed preference for raffinose as substrate showing the highest consumption value at 48 h (96.0 ± 0.9% D1 and 95.3 ± 0.7% D2). The fermentation of raffinose decreased the medium pH, the ammonia concentration and the relative amount of Proteobacteria, while increasing the total production of lactate and short chain fatty acids (129.9 ± 2.6 mmol/L D1 and 179.6 ± 0.6 mmol/L D2), CO 2 (10.8 ± 0.8 mmol/L medium D1 and 5.2 ± 0.3 mmol/L medium D2) and the relative amount of Bifidobacterium and Lactobacillus. This study suggests that raffinose holds potential functional properties for human health.

Xylooligosaccharides (XOS) are emergent prebiotics exhibiting high potential as food ingredients. In this work, in vitro studies were performed using human fecal inocula from two healthy donors (D 1 and D2) to evaluate the prebiotic effect of commercial lactulose and XOS produced in a single-step by recombinant Bacillus subtilis 3610. The fermentation of lactulose led to the highest production of lactate (D1: 33.7 ± 0.5 mM; D2:19.7 ± 0.3 mM) and acetate (D1: 77.5 ± 0.6 mM; D2: 81.0 ± 0.7 mM), while XOS led to the highest production of butyrate (D1: 9.0 ± 0.6 mM; D2: 10.5 ± 0.8 mM) and CO 2 (D1: 8.92 ± 0.02 mM; D2: 11.4 ± 0.3 mM). Microbiota analysis showed a significant decrease in the relative abundance of Proteobacteria for both substrates and an increase in Bifidobacterium and Lactobacillus for lactulose, and Bacteroides for XOS.

Zymomonas mobilis is a well-recognized ethanologenic bacterium with outstanding characteristics which make it a promising platform for the biotechnological production of relevant building blocks and fine chemicals compounds. In the last years, research has been focused on the physiological, genetic, and metabolic engineering strategies aiming at expanding Z. mobilis ability to metabolize lignocellulosic substrates toward biofuel production. With the expansion of the Z. mobilis molecular and computational modeling toolbox, the potential of this bacterium as a cell factory has been thoroughly explored. The number of genomic, transcriptomic, proteomic, and fluxomic data that is becoming available for this bacterium has increased. For this reason, in the forthcoming years, systems biology is expected to continue driving the improvement of Z. mobilis for current and emergent biotechnological applications. While the existing molecular toolbox allowed the creation of stable Z. mobilis strains with improved traits for pinpointed biotechnological applications, the development of new and more flexible tools is crucial to boost the engineering capabilities of this bacterium. Novel genetic toolkits based on the CRISPR-Cas9 system and recombineering have been recently used for the metabolic engineering of Z. mobilis. However, they are mostly at the proof-of-concept stage and need to be further improved. Graphical Abstract