Study On Covalently Grafting Lactoferrin And Chitooligosaccharides And Its Antioxidant,emulsification And Digestion/fermentation Properties

Lactoferrin(LF),as a food-source protein with good nutritional and functional properties,has broad potential application in food industry.However,the shortcomings of thermal stability,oxidation resistance and emulsification had limited its application in food processing.The preparation of protein-sugar covalent complexes through Maillard reaction was an effective pathway to improve the functional properties of proteins.Chitooligosaccharides(COS)was the only natural basic amino oligosaccharide with positive charge in nature,which had antioxidant and intestinal microbiota regulation properties.However,the mechanism of preparing LF-COS covalent complexes through Maillard reaction and the improvement of the functional properties of LF-COS covalent complexes were still not clear.In this study,the covalent grafting mechanism of LF-COS covalent complexes were clearly verified by biomass spectrum firstly.Then the structure of LF-COS covalent complexes was characterized at multiple scales,and the properties of antioxidant,emulsification,digestion and fermentation properties were also investigated.The main results were listed as follows:(1)LF-COS covalent complexes were prepared through Maillard reaction,and the effect of reaction time,reaction temperature and protein/sugar weight ratio on emulsifying activity(EAI)and emulsifying stability(ES)were studied.The optimized reaction conditions were as follows:temperature of 70 ~oC,LF/COS ratio of 1:2(w/w)and reaction time of 4 h.Under this reaction condition,the EAI and ES of LF-COS were 34.42 m~2/g and 82.48 min,which were 8.8%and 27.64%higher than that of LF,respectively.The structure of LF-COS covalent complex was also characterized.The new high molecular weight polymer in SDS-PAGE electrophoresis and the increase of C-N bond structure in FT-IR electrophoresis preliminarily verified the successful covalent grafting of LF and COS.Differential calorimetry scanning(DSC)analysis showed that covalently grafted with COS could effectively improved the thermal stability of LF.(2)Matrix assisted laser desorption ionization time of flight mass spectrometry(MALDI-TOF-MS)was applied to analyze the molecular weight distribution of LF-COS covalent complexes.Compared with LF,the peak molecular weight distribution increased from 82669.93 Da to 84243.88 Da,which further verified the successfully covalent grafting between LF and COS.Liquid chromatography high resolution tandem mass spectrometry(LC-MS/MS)was used to identify the glycosylation sites of LF-COS covalent complexes,and 11 glycosylation sites(5lysine K:K118,K358,K423,K541,K563 and 6 arginine R:R66,R94,R131,R152,R277,R622)of LF were identified to participate in covalent binding with COS.DPPH and ABTS free radical scavenging experiments and Oxidative free radical absorption experiments were applied to evaluate the antioxidant capacity of LF-COS covalent complexes,and the results indicated that the antioxidant capacity of LF-COS covalent complex was significantly improved by comparing with LF.Combined with endogenous fluorescence spectrum and sequence analysis,it was found that all the peptides involved in glycosylation reaction sites contained at least one aromatic amino acid(phenylalanine,histidine,tryptophan and tyrosine)with potential antioxidant activity.Covalently binding with COS changed the structure of glycosylated peptides in LF and exposed more of these amino acid residues with antioxidant potential,thus improving the antioxidant activity of LF.(3)By controlling the reaction time(2-8 h),LF-COS covalent complexes with different grafting degree(1.28%-6.82%)were obtained.The second structure,tertiary structure and surface hydrophobicity of LF-COS covalent complexes were analyzed by circular dichroism,endogenous fluorescence and surface hydrophobicity index,respectively.O/W type emulsions were fabricated by high-speed shear method in one step,and the storage stability test results showed that the emulsion storage stability of LF-COS covalent complexes were significantly improved by comparing with that of LF,especially for the LF-COS covalent complexes under reaction time of 4 h.At the protein concentration of 2%(w/v),the creaming index(CI)of the emulsions after 30days of storage decreased from 38.89%of LF to 10.34%of LF-COS covalent complex under 4 h.Confocal laser scanning microscopy(CLSM)showed that the LF-COS covalent complexes could form tighter membrane structure around droplets than LF.By comparing with LF,the protein adsorption capacity at the emulsion interface showed that LF-COS 4 h covalent complex increased 35.31%.Rheological analysis also displayed the viscosity of the emulsions stabilized by the LF-COS covalent complexes were significantly higher than that of LF.It could be seen that COS covalently grafted with LF improved its emulsification mainly due to two reasons:COS covalently grafting could improve the hydrophilic/hydrophobic balance and conformational compliance of LF,so that LF could expand in the emulsion interface more quickly and adsorb on the surface of oil droplets;LF-COS covalent complexes had larger particle size,higher interfacial adsorption rate,and could form a viscoelastic gel network structure in the emulsion,thus could reduce the droplet flow rate and droplet contact probability,and improve the emulsion storage stability.(4)The digestive properties of LF-COS covalent complex in stomach and small intestine and its glycolysis properties in large intestine were investigated by constructing in vitro simulated digestion and glycolysis model.SDS-PAGE was used to analyze the molecular weight changes of protein samples after digestion.It was found that LF-COS covalent complex was more easily decomposed by pepsin and trypsin than LF,and the degree of digestion was improved.The total amount of short-chain fatty acids produced by LF-COS covalent complex in the fermentation stage was significantly increased,which was 2623.10μg/m L after 48 h of fermentation,and increased 9.41%by comparing with LF.The diversity analysis of intestinal flora after the fermentation of 48 h showed that the number of microbial species in LF-COS covalent complex group was 598,which was 5.81%higher than that of LF.In addition,the content of Firmicutes in LF-COS covalent complex group decreased,while that of Bacteroidetes increased,and the results indicated that LF-COS covalent complex had a certain regulation effect on the diversity of intestinal flora,and had a potential promotion effect on intestinal health.This study provided a theoretical basis for the improvement of functional properties and nutritional value of lactoferrin,and provided a reference for the expanded application of protein-saccharides covalent complexes in the field of functional foods.