Study On The Construction And Properties Of Chitosan-based N-acetylneuraminic Acid Gel Systems

The biological activity of functional nutrients such as Pediococcus pentosaceus and astaxanthin(AST)has been recognised by the public.However,its application in practical products is limited due to the environmental stability of gastrointestinal digestion(e.g.pH and bile salts),processing and storage(e.g.light and temperature).Gel vehicles,with their high systemic stability and unique network structure,are considered as a new strategy to protect the stability of a series of functional nutrients.Through the modification of chitosan(CS)to enhance systemic antibacterial activity and to avoid the shortcomings of traditional antibiotics is also a current research hotspot.Therefore,in this study,gel systems based on CS and N-acetylneuraminic acid(NeuAc)were constructed for the application scenarios of antibacterial materials and encapsulation vehicles,respectively.The binding principle of NeuAc with CS and its derivatives and the mechanism influencing the performance of the gel system were analysed.And the focus was on the antibacterial activity and the encapsulation effect on Pediococcus pentosaceus and AST in different application scenarios.The main research contents and results are as follows:(1)CS-NeuAc hydrogel was constructed by cyclic freeze/thaw.The structure and properties of CS-NeuAc hydrogels with different CS/NeuAc molar ratios were comprehensively investigated by various characterization methods,and their antibacterial properties were qualitatively and quantitatively evaluated.The results showed that CS and NeuAc were crosslinked by forming amide bonds to form hydrogels with an amorphous crystal form,and a distinct three-dimensional network structure with connected pores.The crosslinking degree between CS and NeuAc affected the properties of CS-NeuAc hydrogels.In this case,when the crosslinking degree was 62.8±1.8%at a CS/NeuAc molar ratio of 10:1,and the hydrogel performance was more stable with a swelling rate,water evaporation rate and degradation rate of 710.7 ± 28.6%,78.2±1.6%and 58.6 ± 1.5%,respectively.CS-NeuAc hydrogel exhibited excellent biocompatibility.Compared to CS,CS-NeuAc hydrogels exhibited superior antibacterial activity,with antibacterial rates over 95%and 92%against E.coli and S.aureus,respectively.(2)Carboxymethyl chitosan/N-acetylneuraminic acid(C-NeuAc)hydrogels were constructed by a simple mixing method.The effects of different polymer concentrations on the hydrogel properties and the protective effect of C-NeuAc hydrogel on Pediococcus pentosaceus QK-1 were systematically analyzed.The results showed that the mechanical properties,thermal stability,in vitro degradability and pH sensitivity of the C-NeuAc hydrogels were regulated by the C-NeuAc polymer concentration.For specific requirements of the encapsulated vehicle,C-NeuAc(3%)hydrogel was chosen for probiotics encapsulation.The swelling behaviour of hydrogels and their encapsulation systems were effectively assessed by Fickian diffusion and Schott’s second-order kinetic models.An in vitro digestion assay revealed that the viability of Pediococcus pentosaceus QK-1 in C-NeuAc hydrogel was decreased by only 1.41 log.Compared to free cells(virtually inactivated),C-NeuAc hydrogel exerted a significant protective effect on Pediococcus pentosaceus QK-1 with survival rate of 80.1%.Furthermore,storage stability assay revealed that low temperature and freeze-drying were ideal storage methods for the hydrogel-encapsulated probiotic.(3)Carboxymethyl chitosan/N-acetylneuraminic acid/oxidized hydroxyethyl cellulose(C-NeuAc/OHEC)hydrogels were constructed under the principle of Schiff base reaction.The effects of the degree of Schiff base reaction on the hydrogel performance were comprehensively investigated,and the synergistic effects were analysed by co-encapsulation of Pediococcus pentosaceus RQ-1 and oligomeric isomaltose(IMO)in the hydrogel.The results showed that the C-NeuAc/OHEC hydrogel surfaces are smooth,the interiors shows a continuous three-dimensional network structure,and crystalline in the amorphous state.The performance of hydrogels such as thermal stability,rheology,swelling behaviour,porosity,pH sensitivity and degradability can be modulated by the volume ratio of the C-NeuAc polymer and OHEC.The hydrogels were relatively stable and suitable as a vehicle for co-encapsulation of Pediococcus pentosaceus RQ-1 and IMO when C-NeuAc:OHEC=5:4.The encapsulation efficiency of the hydrogel was more advantageous in the presence of co-encapsulation with IMO(91.02-94.63%),and there was IMO concentration dependence.An in vitro assay revealed that C-NeuAc/OHEC hydrogels provided good protection for Pediococcus pentosaceus RQ-1 against harsh simulated gastrointestinal conditions.The survival rate of Pediococcus pentosaceus RQ-1 was only 77.0%in the absence of IMO.In comparison with free IMO,the survival rate of Pediococcus pentosaceus RQ-1 co-encapsulated with 0.5%,1.0%and 1.5%IMO were 84.4%,90.8%and 90.3%,respectively.Thus,co-encapsulation with C-NeuAc/OHEC plus IMO significantly improved probiotic survival.Furthermore,storage stability assay revealed that 4℃and 1.0%IMO co-encapsulation were the best storage method for the hydrogel-encapsulated probiotic.(4)The construction of Emulsion gels were based on amphiphilic gel factors(C-NeuAc or CMCS),gelatin and camellia oil.The physicochemical properties of the novel emulsion gels were analysed to further evaluate the effect of the systems on the bioaccessibility and storage stability of encapsulated AST.The results showed that the formation of these emulsion gels mainly involved inter-and intramolecular hydrogen bonding.The gel systems were filled with distinct lipid droplets,which were regular spherical in shape and smooth in texture.These emulsion gels are mainly elastic and shear thinning materials,with gel strength,hardness,viscosity and rheological behaviour varying with gelatine concentration.Emulsion gels that exhibited lower swelling behaviour in simulated gastric fluid(SGF)and simulated intestinal fluid(SIF)were used as encapsulation vehicles for AST.The CN-G3 vehicle containing 3%gelatine was more favourable for the slow release of AST during digestion,and the bioaccessibility of the encapsulated AST was 31.53±2.98%,significantly lower than that of CMCS-G3(37.70±3.42%).Storage stability assay revealed that AST-encapsulated emulsion gels could be stored away from light and at low temperature.