Preparation,Properties And Antibacterial Mechanism Of Chitosan Oligosaccharide Modified By Geraniol And Cinnamyl Alcohol

Chitosan oligosaccharide?COS?as a natural non-toxic oligosaccharide,has various biological activities such as antioxidant,anti-inflammatory and antibacterial activity.Using it as a food additive can not only improve the stability of food,but also extend the shelf life of food.Chemical modification by grafting substance with good antibacterial properties is an effective method to improve its antibacterial properties and expands the application scope of COS in practical application.As a natural plant extract,essential oil has attracted public concern and attention in food additives,insecticidal and bacteriostatic.But the complex components,volatile and poor solubility properties of essential oils limit its research and application in practice.Refer to previous research,this study connected the antibacterial cinnamyl alcohol?Cin?and geraniol?Ger?with COS in order to improve the antibacterial property of COS and solve the poor solubility properties of essential oils based on our improved experimental scheme.Another goal was to improve the degree of substitution?DS?and yield of derivatives.Finally,we had explored the antibacterial effect and mechanism of derivatives on E.coli and S.aureus.The main researches and results were as follows:1.Synthesis of COS-M-Ger1-3 and COS-M-Cin1-3.After bromination of Ger and Cin,COS-M-Ger and COS-M-Cin were prepared by reacting Ger-Br and Cin-Br with NH₂ or OH of COS under the catalysis of triethylamine?TEA?.The derivatives were identified by UV-vis spectroscopy,FT-IR spectroscopy,and NMR spectroscopy and three derivatives with different DS were obtained by changing the molar ratio of materials?COS:Ger-Br/Cin-Br=1:1,1:2,1:3?.After characterization,the DS of the derivatives were calculated based on the results of elemental analysis.The results showed that the molar ratio of materials had an effect on the DS of COS-M-Cin,but had a smaller effect on that of COS-M-Ger.These may be due to the benzene ring in Cin has better reactivity than terpenes presented in Ger.The DS of COS-M-Ger1-3 were 0.260,0.278 and 0.283 and their yields were 61%?78%,which were4?7 times higher than that of previous study.The DS of COS-M-Cin1-3 were 0.255,0.328and 0.366,respectively,and the yields were 75%?82%.Compared with previous research,the DS of COS-M-Cin1-3 increased by 1.36?5.72 times,and the yields increased by 50%.2.The properties of COS derivatives.Thermogravimetric analysis?TGA?,X-ray diffraction?XRD?,turbidity methods,plate counting method and agar diffusion method were used to study the thermal stability,crystallinity,solubility and antibacterial of the derivatives.The results showed that the derivatives were slightly unstable than COS,but the thermal decomposition temperature was close to 200°?,so they were still relatively thermally stable.The crystallinity of derivatives was higher than COS and they could be completely dissolved in most organic solvents,acidic and neutral aqueous solutions.The results indicated that COS-M-Cin and COS-M-Ger had good solubility and relatively stable structure.The antibacterial test results showed that COS-M-Cin and COS-M-Ger had obvious inhibitory effects on the growth of E.coli and S.aureus,and their antibacterial effects in neutral solution were better than COS and two commercially available antibacterial agents.The inhibition of S.aureus by both derivatives was stronger than that of E.coli,and the inhibitory effects were dependent on concentration and DS.Finally,COS-M-Ger has a stronger antibacterial effect on S.aureus than COS-M-Cin.3.The antibacterial mechanism of COS derivatives on E.coli and S.aureus.The minimum inhibitory concentration?MIC?of COS-M-Cin1 against E.coli and S.aureus was1.0 mg/m L,and MIC of COS-M-Ger1 against E.coli and S.aureus were 1.0 mg/m L and 0.5mg/mL.Scanning electron microscope?SEM?,conductivity measurement,Zeta potential,hydrophobicity,?-galactosidase leakage and other test methods were used to explore the influence of derivatives on bacterial morphology,cell membrane permeability and surface characteristics.The results showed that the change of cell morphology,leakage of?-galactosidase,the increase of conductivity of the culture fluid,surface potential and hydrophobicity of the bacteria,after treated with derivatives.Detection of intracellular reactive oxygen species?ROS?and proteins to study the effects of derivatives on the internal physiological metabolism of bacteria.The results exhibited that after the derivatives treated the bacteria,the intracellular ROS increased and the types and contents of intracellular proteins changed.The above research showed that the derivatives caused abnormal cell function by changing cell membrane permeability and affecting protein synthesis,leading to cell death.The high-yield and high-substitution COS-M-Cin and COS-M-Ger prepared in this paper have good solubility,thermal stability and obvious antibacterial activity.This study laid a solid theoretical foundation for the efficient preparation of essential oil modified COS derivatives and the application of antibacterial derivatives in the fields of food,agriculture and medicine.