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In this study, carboxymethyl chitosan (CMCS) and N-acetylneuraminic acid (NeuAc) were used to develop C-NeuAc hydrogels to encapsulate Pediococcus pentosaceus QK-1. The mechanical properties, thermal stability, in vitro degradation, and pH sensitivity of the hydrogel were evaluated. The C-NeuAc concentration required for optimal hydrogel performance was 3% (w/v). Hydrogel swelling behaviour was effectively assessed by Fickian diffusion and Schott's second-order kinetic models. The hydrogel demonstrated excellent biocompatibility and low in vitro cytotoxicity. An in vitro assay revealed that the viability of Pediococcus pentosaceus QK-1 in C-NeuAc had decreased by only 1.41 log (CFU/ mL) after exposure to simulated acidic gastric fluid. Moreover, the survival rate of the encapsulated and free Pediococcus pentosaceus QK-1 cells were 80.1% and virtually zero, respectively, after passage through the gastrointestinal tract. It was empirically determined that low temperature and freeze-drying were the ideal condition and method to ensure storage longevity of the hydrogel-encapsulated probiotic. Hence, C-NeuAc hydrogel is highly desirable as a food-grade probiotic delivery vehicle.

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2'-fucosyllactose (2'-FL), one of the simplest but most abundant oligosaccharides in human milk, has been demonstrated to have many positive benefits for the healthy development of newborns. However, the high-cost production and limited availability restrict its widespread use in infant nutrition and further research on its potential functions. In this study, on the basis of previous achievements, we developed a powerful cell factory by using a lacZ-mutant Escherichia coli C41 (DE3)ΔZ to ulteriorly increase 2'-FL production by feeding inexpensive glycerol. Initially, we co-expressed the genes for GDP-L-fucose biosynthesis and heterologous α-1,2-fucosyltransferase in C41(DE3)ΔZ through different plasmid-based expression combinations, functionally constructing a preferred route for 2'-FL biosynthesis. To further boost the carbon flux from GDP-L-fucose toward 2'-FL synthesis, deletion of chromosomal genes (wcaJ, nudD, and nudK) involved in the degradation of the precursors GDP-L-fucose and GDP-mannose were performed. Notably, the co-introduction of two heterologous positive regulators, RcsA and RcsB, was confirmed to be more conducive to GDP-L-fucose formation and thus 2'-FL production. Further a genomic integration of an individual copy of α-1,2-fucosyltransferase gene, as well as the preliminary optimization of fermentation conditions enabled the resulting engineered strain to achieve a high titer and yield. By collectively taking into account the intracellular lactose utilization, GDP-L-fucose availability, and fucosylation activity for 2'-FL production, ultimately a highest titer of 2'-FL in our optimized conditions reached 6.86 g/L with a yield of 0.92 mol/mol from lactose in the batch fermentation. Moreover, the feasibility of mass production was demonstrated in a 50-L fed-batch fermentation system in which a maximum titer of 66.80 g/L 2'-FL was achieved with a yield of 0.89 mol 2'-FL/mol lactose and a productivity of approximately 0.95 g/L/h 2'-FL. As a proof of concept, our preliminary 2'-FL production demonstrated a superior production performance, which will provide a promising candidate process for further industrial production.

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Properties, crystallization behavior and oxidative stabilities of enzymatically catalyzed interesterified fats were investigated in this study. Interesterified fats were catalyzed by Lipase Lipozyme RM IM, through reaction from beef tallow (BT), palm stearin (PS) and camellia oil (CO)under the mass ratio of 7.55: 2.45: 4 (BT: CO: PS) using 3.65% (w/w) of Lipozyme RM IM at 72.6°C for 3.85 h. After reaction, interesterified fats with 36.8°C sliding melting point (SMP) was obtained. Physicochemical properties (fatty acid profile, triacylglycerol profile, solid fat content (SFC), melting and crystallization curve, polymorphic forms) of interesterified fats were characterized. Results proved that interesterified fats blends after interesterification were improved with desirable ß' type crystals and preferable SFC. Triacylglycerol constituent of interesterified fats displayed a decrease in OOO, PSS/SPS, LLL, SSS and increased in PSO/POS/SPO, POO/OPO, POP/PPO, PLO/PLP/PPL by comparison of physical blends without interesterification. Additionally, it is estimated that interesterified fats have a moderate antioxidative stability about 352 days-shelf life at 20°C through the traditional accelerated oxidation test. In conclusion, interesterified fats with desirable properties could be suitable for plastic fats use.

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