Study On Acylated Regulation Of Ovalbumin/ε-Polylysine Interaction And Properties Of Thymol Nanogel

Thymol(Thy)is a plant essential oil component with good antibacterial activity.However,its application is limited by its poor stability,poor water solubility and low bioavailability.Thy can be encapsulated by nano-carriers constructed with protein matrix,which can improve the solubility of Thy and insulate it from the external environment.Therefore,stable encapsulation and efficient antibacterial effect can be achieved.Ovalbumin(OVA)is the highest proportion of egg white protein.Due to its wide source,low price,safety and non-toxicity,OVA has gradually become an ideal nano-carrier matrix for encapsulating active ingredients,so that it has broad application prospects in food and drug fields.However,natural OVA still has the defects of low emulsification,poor gelation,and poor stability.Many studies have shown that the stability and functional characteristics of OVA encapsulation carriers can be effectively regulated by the strategy of moderate modification of proteins and electrostatic recombination with counterion polymers.On the basis,OVA was used as substrate and succinic anhydride(SA)was used as modifier to prepare succinylated modified OVA(named SOVA)in this paper,then the effect of succinylation modification on the structure and functional characteristics of OVA was explored.Furthermore,SOVA/ε-polylysine(ε-PL)electrostatic complex and SOVA/ε-PL nanogels loaded with Thy were constructed,and the regulation of the interaction between SOVA and ε-PL on the application properties of Thy nanogels was determined by using various techniques such as spectroscopy,electron microscopy and rheology.The main findings are as follows:(1)Succinylated modified OVA was prepared,and the effect of succinylation modification on the structure and function properties of OVA was revealed.With the addition of SA,the succinylation degree of OVA increased gradually and then tended to be stable.When the amount of SA was 9%of OVA mass(named S9OVA),the acylation degree of OVA reached 95%.With the increase of the degree of acylation,the particle size and surface hydrophobicity of SOVA gradually decreased,while the electronegativity,free sulfhydryl content and molecular weight gradually increased.The content of α-helix in the secondary structure gradually decreased by 3.4%-11.9%,while the amorphous structure increased,which means the protein structure continued to expand.Scanning electron microscopy showed that the succinylation modification changed the morphology of OVA from smooth spherical to irregular sheet.The enhancement of intermolecular electrostatic repulsion improved the thermal stability of SOVA,and the maximum peak temperature increased by 19.45℃.Moreover,the amorphous structure of SOVA was enhanced,and the apparent viscosity was reduced and the shear thinning behavior was presented.Meanwhile,the acylation modification strengthened the interaction between OVA and water/oil,which promoted the distribution of protein molecules at the water-oil interface,so that the functional properties of OVA,such as solubility,oil retention,emulsification and emulsion stability(emulsification activity index increased by 1.7-2.7 times)were improved.(2)The SOVA/ε-PL electrostatic composite system was constructed,and the regulatory effects of different acylation degrees and ε-PL:SOVA ratio on the interaction,microstructure,stability and functional properties of the composite system were determined.With the increase ofε-PL content,the particle size of the SOVA/ε-PL complex enlarged until aggregation and precipitation.The higher the acylation degree of SOVA,the smaller the size increase amplitude of the SOVA/ε-PL complex,and the positive charge shift caused by the cation ε-PL is more severely inhibited.With the increase of ε-PL content,the fluorescence intensity of SOVA decreased significantly,and the higher the acylation degree of SOVA,the more ε-PL could be electrostatically synthesized.The main force between SOVA and ε-PL is electrostatic interaction.followed by hydrophobic interaction and hy drogen bond interaction,and the higher the degree of acylation,the stronger the electrostatic interaction and hydrophobic interaction.Transmission electron microscopy(TEM)results showed that SOVA/ε-PL complex presented a compact spherical structure with uniform distribution and the particle size was about 100 nm.The stability test results showed that the higher the degree of acylation,the stronger the thermal stability,ionic stability and pH stability of the complex.In particular,S9OVA/ε-PL could maintain a uniform particle size distribution under the condition of 90℃ heat treatment for 30 min and 100 mM NaCl,and the particle size fluctuation in the range of pH 2-9 was small.The addition of ε-PL has no significant effect on the emulsification of SOVA,but can significantly improve the hardness and springiness of SOVA gel.Meanwhile,the antibacterial effects of S9OVA/ε-PL complex against Escherichia coli,Staphylococcus aureus,Shewanella baltica,and Pseudomonas fluorescens were similar to that of the original ε-PL.(3)The SOVA/ε-PL nanogels loaded with Thy were constructed by heat-induced gel method.The regulatory effects of acylation modification and electrostatic recombination with εPL on the microstructure,environmental stability and antibacterial activity of OVA-based nanogels were analyzed.Based on the analysis of encapsulation efficiency,particle size and polydispersity index(PDI),the optimal core-wall ratio(Thy:S9OVA/ε-PL)was determined to be 1:1.The encapsulation efficiency of the nanogel(named SOPT-NGs)formed under this condition reached 85.49%,and the particle size,PDI and ζ-potential were respectively 167.97 nm,0.21 and-27.10 mV.The internal of SOPT-NGs may be driven by hydrogen bonding,hydrophobic interaction and electrostatic interaction.Succinylation modification can provide a more stable and heat-resistant network structure for protein nanogels.Thy was successfully embedded in the nanogel and existed in an amorphous state.TEM observation showed that the microstructure of SOPT-NGs presented uniformly distributed particles with a particle size of about 60 nm,while the other nanogels were not uniformly distributed and irregularly shaped.SOPT-NGs showed good thermal stability,ionic strength stability and pH stability.The minimum inhibitory concentrations of SOPT-NGs for E.coli,S.aureus,S.baltica and P.fluorescens were 0.076-0.153 mg/mL,which was much lower than free Thy.These results suggested that ε-PL and Thy may have enhanced antibacterial effect in nanogels.The above results showed that the combination strategy of succinylation modification and electrostatic recombination with ε-PL can significantly improve the ability of OVA nanogel to encapsulate and protect Thy,as well as strengthen the antibacterial effect of Thy,which provides a theoretical basis and method reference for the joint application of SOVA,ε-PL and Thy and the development of other multifunctional nanocapsules.