Grafting Mechanism And Application Of Phenolic Acid Grafted Chitosan By Laccase-catalysed Oxidation

Chitosan is a common natural polysaccharide biological macromolecules, having many distinctive properties, of which the antimicrobial and antioxidant properties are two very important ones. As a kind of wide-used natural antibacterial agent, chitosan has some bacteriostasis ability against bacteria and fungi. However due to the lack of hydrogen atom donor, the chitosan is not able to be a good chain breaking antioxidant, which makes its antioxidant activity too weak. Normally, chitosan is only to be used as an auxiliary antioxidant, which limits its application in many areas.This paper started from the phenolic-grafted chitosan obtained by laccase-catalyzed oxidation to improve its antioxidation and antibacterial properties of chitosan. In the conditions that pH was value 6.5, reaction time was 4h and reaction temperature was 40℃, gallic acid (GA),3,4-Dihydroxybenzoic acid (DA) and p-hydroxybenzoic acid (PHA) were selected because of the simple structure to modify chitosan by using laccase as a biological catalyst. Phenolic acid would be turned into active quinone intermediate under laccase-catalyzed oxidation before being grafted onto chitosan. Then the changes of andtioxidation and antibacterial properties of functional chitosans were studied, as well as the changes of structures. Based on those, the preliminary mechanism of grafting was explored and the value of application of this method was evaluated.In the first part, the main mechanism of grafting reaction was investigated. DA was selected as the model compound to modify chitosan. The change of concentration of components in the reaction system was real-time monitored using ReactIR. The results showed that DA was adsorbed onto chitosan before further reaction. GA, DA and PHA were selected as the model compounds to modify chitosan to explore the further reaction of grafting after phenolic being adsorbed onto chitosan through methods of Py-GC/MS, State-solid 13C NMR, FTIR and linear potentiometric titration experiments. Analysis by Py-GC/MS showed that the aromatic structure which was produced by the phenolic acid grafted onto chitosan appeared in the results. That indicated that the phenolic acids were connected to chitosan by chemical bonds after grafting, rather than by physical adsorption. Further study on grafting reaction was carried on by FTIR. The results showed the attenuating of the modified chitosans’ absorption peak near the wavenumber 3440cm-1, and the emergence of the acromion in the wavenumber 1650cm-1. Those changes meant the decrease of N-H and the emergence of the C=N key, showing that the phenolic acids were connected to chitosan by chemical bonds after grafting. So the Schiff base reaction and Michael addition reaction occurred between amino in chitosan and phenolic hydroxyl groups could be deduced. The results detected by Solid-state 13C NMR, showed that near the PPM which was 80, the signal of chitosan derivatives occourred a new peak compared with the original grafted chitosan. That represented the tertiary carbon amine, illustrated new chemical bond in amino of chitosan after grafting reaction, and the more hydroxyl numbers of the phenolic acid, the more obvious the changes of this signal. Grafting rate was measured by linear potentiometric titration experiments. The results showed that GA which contained the most hydroxyl groups had the highest grafting rate, and the PHA which contained the least hydroxyl groups had the lowest grafting rate. We could see that the grafting ratio and hydroxyl number was positively related, which was in accordance with results of previous study and could be further confirmed by each other.The main mechanism could be obtained by comprehensive analysis of results above. In the process of the grafting reaction, phenolic acid firstly contacted with chitosan by physical adsorption before being turned into active quinone intermediate under laccase-catalyzed oxidation and then grafted onto chitosan through Micheal addition reaction and Schiff base reaction under the catalyzed oxidation by laccase, and the more hydroxyl groups, the effect of grafting was better.The second part accessed the application value of modified chitosan.The test about antioxidation properties was carried out by ABTS+method, using modified chitosan derivatives powder treated by laccase/phenolics. Experimental results showed that the laccase/gallic acid modified chitosan derivatives obtained the strongest antioxidation power. The sequence of antioxidation properties from strong to weak was:GA-chitosan, DA-chitosan, PHA-chitosan and original chitosan. To characterize the antioxidant properties of products from another point of view, potassium ferricyanide method was used to determine the reduction ability of chitosan derivatives, the results showed that the reduction power sequence was the same with that measured by ABTS+method: GA-chitosan, DA-chitosan, PHA-chitosan and original chitosan. The reducing capacity could be increased by 86.98% at most.The products were also applied in wet paper towels, and the antibacterial ability of chitosan and derivatives were determined by Oxford cup method and shaking flask method to evaluate the value of the application as additives in wet tissue paper. The results showed that the antibacterial properties of chitosan derivatives after grafting had also been improved, and the degree of increasing was also related to the phenolic acids, of which the sequence from high to low was:GA-chitosan, DA-chitosan, PHA-chitosan and original chitosan. The results of the study indicated that the antibacterial ability could be increased by 44.57% at most.