The Bio-preparation Of Neokestose And Enzymatic Synthesis Of Neokestose Laurate

Neokestose, a novel fructooligosaccharide (FOS), was reported to possess superiorprebiotic activity to commercial FOS. Such favorable functionalities may boost the marketdemand of neo-FOS. However, no commercial neo-FOS have been produced until now,mainly due to the high cost for their trace amount extraction from vegetables and fruits ortheir low microbial productivity via~6G-fructofuranosidase (~6G-FFase). The whole-cellbiotransformation process, and simultaneous production of~6G-FFase and astaxanthin byXanthophyllomyces dendrorhous were firstly developed to make neokestose practicallyfeasible for commercial production in the present study. Moreover, the properties for foodprocessing were investigated to provide theoretical guidance for the application in foodproduct. Finally, the neokestose was acylated to enlarge the application of this functionalfactor.The effects of production parameters on the biotransformation of sucrose wereinvestigated to enhance the yield of neokestose by Xanthophyllomyces dendrorhous. Cellsshowed optimal~6G-FFase activity at neutral pH and the yield of neokestose showed nosignificant differences between buffer and buffer-free systems. Cell concentration (>80g/L)negatively affected the maximum neokestose yield. Sucrose concentration positivelyincreased the maximum yield of neokestose. Elevating the reaction temperature to30℃, theneokestose productivity increased1.85-fold compared with that at20℃. Meanwhile, cell ageof32h enabled the biotransformation of sucrose more efficiently. In addition, free cellsexhibited a higher productivity over immobilized cells. The maximum neokestoseconcentration finally reached227.72g/L from400g/L sucrose under the optimal conditions.The effects of medium composition and culture conditions on the simultaneousproduction of~6G-FFase and astaxanthin were investigated to reduce the capital cost ofneokestose production by Xanthophyllomyces dendrorhous. The sucrose and corn steep liquor(CSL) were found to be the optimal carbon source and nitrogen source, respectively. CSL andinitial pH were selected as the critical factors using Plackett-Burman design. Maximum~6G-FFase242.57U/mL with5.23mg/L astaxanthin was obtained at CSL52.5mL/L and pH7.9by central composite design. Neokestose yield could reach238.12g/L under the optimizedmedium conditions. Cost analysis suggested66.3%of substrate cost was reduced comparedwith that before optimization. These results demonstrated that the optimized medium andculture conditions could significantly enhance the production of~6G-FFase and astaxanthinand remarkably decrease the substrate cost, which opened up possibilities to produceneokestose industrially. The heat and pH stability of neokestose was evaluated compared with commercial FOS bysimulating different thermo sterilization. The results showed that neokestose at neutral andalkaline pH displayed high heat and pH stability during pasteurization (85℃,30min), hightemperature for short time sterilization (100℃,30min and121℃,20min) and ultra hightemperature sterilization (135℃,30s). The neokestose at pH2and pH3retained42.04%and78.99%during pasteurization (85℃,30min), indicating a significant degradation. Neokestoseat pH4retained97.08%and89.83%during high temperature short time sterilization (100℃,30min and121℃,20min), suggesting a relatively high stability compared to that of FOS. Onthe other hand, the effects of neokestose on the gelatinization and retrogradation of rice starchwere studied compared with FOS, IMO and sucrose using a differential scanning calorimetry(DSC), and the results suggested that the addition of neokestose could increase thegelatinization temperature of rice starch in the order of neokestose>FOS>IMO>sucrose, andreduce the gelatinization enthalpy which showed negatively correlation with the concentrationof neokestose. Moreover, the effect of neokestose on the retrogradation of rice starch wasevaluated. The results indicated that10%of neokestose could significantly retard theretrogradation of rice starch when stored at4℃for short term (7days) and long term (28days);30%of neokestose could inhibit the retrogradation of rice starch when stored at4℃for longterm (28days);10%of neokestose could retard the retrogradation of rice starch when stored at25℃for long term (28days),30%of neokestose had no significant effect on the retragradationof rice starch when stored at25℃. Furthermore, neokestose showed better inhibition effect onthe retrogradation of rice starch compared with that of FOS and IMO at the tested conditions.An acylation modification of neokestose using Candida antarctica lipase B asbiocatalyst was investigated aiming to further broaden the potential application of neokestose.The process conditions were optimized, and the highest conversion rate of82.38%wasachieved under the optimized conditions:20%DMSO in2-methyl-2-butanol (v/v), molarratio of12(vinyl laurate to neokestose), temperature of50℃, molecular sieves of100g/L.Finally, a neokestose feeding strategy was developed based on the kinetics of neokestoseacylation, driving neokestose laurate yield up to61.35mg/mL. The process proposed couldmake it economically and environmentally feasible for large scale production. The neokestoselaurate was purified using C18solid phase extraction column. The structure of neokestoselaurate was determined by1H and13C NMR，COSY and HMQC, and the results showed thatthe novel chemical was6-O-laurylneokestose. Finally, CMC of6-O-laurylneokestose wasdetermined to be352μmol/L using Du Nouy plate method.