Study On The Preparation,Characterization,and Functional Properties Of Whey Protein-inulin Conjugates

Objectives:In this study,3 kinds of whey protein isolate-inulin conjugates(WPIIn conjugates)with different degree of glycation(DG)were prepared by single factor experiments and orthogonal experiment through the Maillard reaction(MR)between WPI and In.The difference of the microstructure,solubility,emulsibility and other functional properties among the 3 kinds of WPI-In conjugates,as well as the biological activities including antioxidant activity,immune and intestinal microbiota regulation were analyzed,so as to clarify the potential health promotion effect of WPI-In conjugates obtained by MR,provide a theoretical basis for expanding the application of whey protein and inulin which is the characteristic resource of Gansu Province,as well as provide a reference for the safety of food processing.Methods:1.The preparation of WPI-In conjugates:through single-factor experiments,the effects of reaction temperature,reaction time,initial pH of the reaction system,WPI concentration(w/v)and the mass ratio of WPI to In on MR between WPI and In were discussed.According to the results of the single-factor experiments,four factors including reaction time,initial pH of the reaction system,whey protein concentration and the mass ratio of WPI to In were selected for the orthogonal experiment with three levels of each factor,and 3 kinds of WPI-In conjugates with different DG were prepared named whey protein isolate-inulin conjugates with low degree of glycation(WPI-In-LDG),whey protein isolate-inulin conjugates with moderate degree of glycation(WPI-In-MDG)and whey protein isolate-inulin conjugates with high degree of glycation(WPI-In-HDG).2.The characterization of WPI-In conjugates:the Zeta potential and particle size distribution of WPI,WPI-In-LDG,WPI-In-MDG and WPI-In-HDG were determined by Zeta potential and particle size analyzer,and the structure of WPI,WPI-In-LDG,WPI-In-MDG and WPI-In-HDG were observed and analyzed by Fourier transform infrared spectroscopy,circular dichroism and scanning electron microscope.3.The functional properties of WPI-In conjugates:in the in vitro experiment part,taking WPI as a blank control,the solubility,emulsifying activity,emulsification stability,and 1,1-diphenyl-2-picrylhydrazyl(DPPH)free radical scavenging ability of the reaction system were measured before and after the 3 kinds of WPI-In conjugates were prepared.In the animal experiment part,72 adult healthy female Kunming mice were randomly divided into 6 groups named control group(CG),WPI group,In group,WPI-In-LDG group,WPI-In-MDG group and WPI-In-HDG group.The mice in each group were gavaged at 9:00 a.m.every day for 6 weeks.After the end of gavage:(1)The blood of mice in each group was collected from eyeballs and the serums were separated,then the content of immunoglobulins A(Ig A),Ig G,Ig M,and cytokines including interleukin-2(IL-2),IL-4,IL-6,IL-1β,Tumor necrosis factor(TNF-α)and γInterferon(IFN-γ)were measured using the ELISA kits.(2)The spleen,thymus and liver were separated and weighed,then the activities of glutathione peroxidase(GSHPx)and superoxide dismutase(SOD),and the content of malondialdehyde(MDA)in the liver tissue of mice were detected by the ELISA kits.(3)The fresh feces of mice were collected and the community composition of microbiota in the mice feces were analyzed through 16S rRNA high-throughput sequencing.Results:1.The preparation of WPI-In conjugates:With the increase of reaction time and the mass ratio of WPI to In,DG of WPI-In conjugates firstly increased and then decreased.With the increase of initial pH,the glycosylation degree of WPI-In conjugates gradually increased.With the increase of WPI concentration,DG of WPI-In conjugates gradually decreased and then stabilized.The reaction temperature had no significant effect on DG of WPI-In conjugates.(2)Three WPI-In conjugates with different DG were prepared through single factor experiments and orthogonal experiments,respectively:WPI-In HDG(preparation conditions:60℃ of reaction temperature,1 h of the reaction time,initial pH of 8,6.0%of WPI concentration and the mass ratio of WPI to In of 1:4),WPI-In-MDG(preparation conditions:60℃ of reaction temperature,2 h of the reaction time,initial pH of 10,1.5%of WPI concentration and the mass ratio of WPI to In of 1:1)and WPI-In-LDG(preparation conditions:60℃ of reaction temperature,3 h of the reaction time,initial pH of 9,7.5%of WPI concentration and the mass ratio of WPI to In of 1:2),and DG of them were(11.5 8±1.31)%,(6.85±0.51)%and(1.23±0.23)%,respectively.2.The characterization of WPI-In conjugates:(1)Zeta potential and particle size distribution:the particle sizes of WPI-In conjugates were smaller than that of WPI.With the increase of DG,the particle size of WPI-In conjugates gradually decreased.The particle size of WPI-In-HDG was the smallest,with 88.58%distribution in the range of 15～25 nm and 11.42%distribution in the range of 180～250 nm.(2)The Zeta potentials of WPI,WPI-In-LDG,WPI-In-MDG and WPI-In-HDG were(-63.05±0.08)mV,(63.00±0.37)mV,(-58.41±2.09)mV and(-55.30±1.76)mV,respectively.Compared with WPI-In-LDG,the Zeta potentials of WPI-In-MDG and WPI-In-HDG significantly decreased(P<0.05),but still showed good stability.(3)Fourier transform infrared spectroscopy analysis:Compared with WPI,the stretching vibration of C-O were enhanced,and the number of hydroxyls was increased,indicating that In was connected to WPI by covalent bong.(4)Circular dichroism analysis:MR significantly changed the secondary structure of WPI,and the content of α-helix of WPI-In conjugates decreased by 48.24%to 90.44%,the content of β-turn decreased by 73.89%to 99.84%,and the content of β-sheet increased by 85.18%to 119.05%.Compared with WPI-InLDG and WPI-In-MDG,the content of α-helix and β-turn of WPI-In-HDG significantly decreased,and the content of β-sheet significantly increased(P<0.05).(5)Scanning electron microscope observation:Loss of the nearly spherical appearance of WPI was caused by the insert of In.As DG increased,pore structures gradually appeared on the surface of WPI-In conjugates and magnified,which caused the gradually loosened structure of WPI-In conjugates.3.The functional properties of WPI-In conjugates:(1)Solubility:DG affected the solubility of the WPI-In conjugates.As DG increased,the solubility of WPI-In conjugates gradually increased(P<0.05),and WPI-In-HDG showed the highest solubility which was(66.02±3.41)%.(2)Emulsibility:DG affected the emulsibility of WPI-In conjugates.Compared with WPI-In-LDG and WPI-In-MDG,the emulsifying activity and emulsification stability of WPI-In-HDG were significantly higher which were(8.12±0.07)m2/g and 94.27±3.22,respectively(P<0.05).(3)DPPH free radical scavenging ability:DG had no significant effect on the DPPH free radical scavenging ability of WPI-In conjugates(P>0.05).(4)The antioxidant activity of mice liver:WPIIn conjugates improved the GSH-Px and SOD activities,and reduced the MDA content in mice liver(P<0.05).WPI-In-HDG showed the strongest ability to increase the activity of GSH-Px and reduce the content of MDA in mice liver.(5)The immune function of mice:WPI-In conjugates promoted the secretion of Ig M and Ig G,increased the level of anti-inflammatory factors including IL-4 and IL-2,and decreased the level of proinflammatory factor TNF-α in mice serum(P<0.05).WPI-In HDG showed the strongest ability to increase the level of Ig G and IL-4,and WPI-In-MDG showed a stronger ability to increase the level of Ig G.(6)Analysis of intestinal flora in mice:αdiversity analysis showed that the Chao 1 index and ACE index of WPI-In-LDG group and WPI-In-MDG group were significantly higher than that of In group(P<0.05),indicating that WPI-In-LDG and WPI-In-MDG increased the community richness of intestinal microbial communities in mice compared to In.β diversity analysis showed that compared to CG group,WPI group,In group and WPI-In-MDG group,the intestinal microflora structure of mice in WPI-In-HDG group was significantly different(P<0.05),indicating that WPI-In-HDG could alter the intestinal microflora structure of mice.Analysis of species differences showed that WPI-In-LDG increased the relative abundance of beneficial bacteria including Marvinbryantia,Oscillospira and Ruminococcaceae(P<0.05),WPI-In-MDG increased the relative abundance of beneficial bacteria of Adlercreutzia(P<0.05),and WPI-In-HDG increased the relative abundance of beneficial bacteria including Bacteroidetes and Butyricicoccus(P<0.05).Correlation analysis showed that WPI-In conjugates could enhance the antioxidant capacity of mice liver by increasing the abundance of intestinal microbiota Butyricicoccus and Ruminococcaceae,as well as enhance the immune function by increasing the abundance of intestinal microbiota Marvinbryantia.Conclusions:1.WPI-In conjugates showed smaller particle size and more flexible structure than WPI.2.WPI-In conjugates exhibited good solubility,emulsibility,and DPPH free radical scavenging ability,and improved the liver antioxidant capacity and immune function of mice,increased the relative abundance of beneficial bacteria,and improved the composition of intestinal flora in mice.3.The functional properties of WPI-In conjugates are proportional to DG,which are expected to expand the applications of whey protein and inulin in the food systems.