The Preparation Of Novel Chitosan Esters Andanti Microbial Performance Study

Chitosan,which has prospective applications in a broad range of scientific areas,is a natural linear polysaccharide and attracts significant interest because of its fascinating structural features and good performances. However, chitosan is limited in various application domains for the poor solubility, which it can only be dissolved in low concentration of acid solution due to its compact crystalline structure. Three novel chitosan esters were synthesized and aimed at improving its solubility and antimicrobial activity, which were expected as food additives to be used in the food system for preservation. The effect of reaction factors on the degree of substitution had been investigated and the suitable conditions were achieved. The structures of derivatives were characterized by FTIR and 1H NMR. Biological safety of chitosan derivatives was evaluated bymaximaltolerancedose experimentand micronucleustest. The solubility, foamingpropertyand antimicrobial activity of chitosan derivatives were investigated.Althoughantimicrobial activity of chitosan is widely studied, its antimicrobial mechanismis not clear.Andmostspeculationsassumethat antimicrobial activity of chitosan is closely related to its poly-cationic nature. Therefore,in this study, chitosanmicrospheres were obtained by spraydrying and cross-linkedin different degrees to control the amount of amino, which wereused to study directly the relationship between antimicrobial activity of chitosan and its amino group.The main contents and conclusions are described as follows:1. Preparation and characterization of chitosan esters(1) Preparation of O-acetyl-chitosan. O-acetyl-chitosan (ACS) was prepared by esterification reaction of acetic acid with chitosan in the presence of SOCl2 under high-intensity ultrasound.The substances were collected, filtered, purified anddried to obtain ACS. The optimal conditions were temperature 35℃, reaction time 1 h, the ratio of chitosan to acetic acid,1:4 (g/ml), and the ratio of chitosan to SOCl2, 1:4(mol/mol). The structure of ACS was characterized by FTIR and 1H NMR, certifying that acetyl group was selectively acylated onto the hydroxyl group of chitosan.The maximum degree of substitution was 0.35 under optimum conditions, which was calculated from the peak areas in NMR spectra by integral method.(2) Preparation of N-benzoyl-O-acetyl-chitosan. N-benzoyl-O-acetyl-chitosan (BACS) was prepared by using the selective partial acylation of chitosan, benzoyl chloride and acetic acid under high-intensity ultrasound. The substances were collected, filtered, purified anddried to obtain BACS.The optimal conditions were temperature 35℃, reaction time 2 h, the ratio of chitosan to acetic acid.1:6 (g/ml), and the ratio of chitosan tobenzoyl chloride,1:2 (mol/mol). The structure of BACS was characterized by FTIR and 1H NMR, certifying that the benzyl and acetyl groups were selectively acylated onto the amino and hydroxyl group of chitosan, respectively.The maximum degrees of substitution of benzoyl and acetyl for BACS were 0.26 and 1.15, respectively, which were calculated from the peak areas in NMR spectra by integral method.(3) Preparation of O-acetyl-chitosan-N-2-hydroxypropyl trimethyl ammonium chloride. O-acetyl-chitosan-N-2-hydroxypropyl trimethyl ammonium chloride (AQTS) was prepared by two steps reaction. At first, chitosan-N-2-hydroxypropyl trimethyl ammonium chloride (QTS) was prepared according to the common method. Then, QTS reacted with acetic acid to get O-acetyl-chitosan-N-2-hydroxypropyl trimethyl ammonium chloride (AQTS) in the presence of SOCI2 by mechanical mixing at room temperature for a given time. The reaction product was precipitated in acetone overnight.The desired compound was filtered, dissolvedin distilled water, dialyzed, concentrated, and freeze-dried to obtain the chitosan ester AQTS.The degree of quaternization (DQ) of QTS was 85.5% which was measured bytitration.The structure of AQTS was characterized by FTIR and 1H NMR, certifyingthat esterificationofacetic acid with quaternized chitosan occurred. The degree of acetyl (DA) of AQTS was 2.31, which was calculated from the peak areas in NMR spectra by integral method.2. Performancestudies of chitosan estersBiological safety of chitosan and chitosan derivatives was evaluated by maximaltolerancedose experimentand micronucleus test.According toacute toxicity classification standard of ProceduresandMethodsforToxicolohicalAssessmentonFoodSafety, the results of maximaltolerancedose experimentindicated that chitosan andits derivatives were the actual non-toxic substances. The result of micronucleus test of chitosananditsderivatives was negative. These results indicated that these substances met the standard for safety application of food additives. The solubility, foamingpropertyand antimicrobial activity of chitosanand its derivatives were investigated. The results of solubility test showed that chitosan derivatives had better solubility thanchitosan, for example, BACS was slightlysoluble inethanediol and isopropyl alcohol, QTS wassoluble indistilled water and AQTS exhibited an excellent solubility in both aqueous solution and common organic solvents, such as methanol, ethanol,ethanediol and isopropyl alcohol. The results of foaming test showed that chitosan exhibited weak foam capacity, while ACS, BACS and AQTS showed good foam capacity and foam stability, especially BACS. The results of antimicrobial assays showed that chitosan derivatives exhibited higher antibacterial activity than chitosan against Escherichia coli and Staphylococcus aureus, and inhibition effect was BACS>QTS> ACS>AQTS>chitosan, while only BACS showedsignificantinhibitoryeffect on the growth of Aspergillus niger.These results indicated that chitosan derivatives had better performances than chitosan. Chitosan derivatives had good foaming property and antimicrobial activity and had the potential becoming a foodadditive with a dual role of both foaming agent andfood preservative, which would reduce costs and improve foodsafety.3. The preliminary studyof the antimicrobial mechanism of chitosanChitosan microspheres (CMs) were prepared by spray drying, which dispersed well at 150 rpm in culture. The CMs were rough-surface and spherical shape of diameter of about1-4 μm, which were larger thanStaphylococcus aureus. The spray-dried microspheres were subsequently dispersed in an ethyl acetate solution containing differentconcentrations of glutaraldehyde to cross-link. Then the stability of cross-linked microspheres was evaluated. Three kinds of chitosan microspheres (0.7%CMs,1.0%CMs and 1.2%CMs) were selected for the following experiments, which had higher degreeofcross-linking and stronger stability. Isoelectricpoints of 0.7%CMs,1.0%CMs and 1.2%CMs were measured by titration, which were 5.8,6.8 and 7.1, respectively. The inhibitory effects of the chitosan microspheres with different degreesofcross-linking onthe growth ofStaphylococcus aureuswere investigated under different pH systems. The results showed that the inhibitory effects of 0.7%CMs and 1.0%CMs against Staphylococcus aureus increased with the increasingofCMs concentration at pH 5.5 and 6.5 systems, while 1.2%CMs weaklypromoted the growth of.Staphylococcus aureuswith the increasingofCMs concentration, which indicated that the amount of amino groups of chitosan played an important role in the antimicrobial activity. At pH 7.5 system, three kinds of chitosan microspheres had no inhibitory effects on Staphylococcus aureus, which indicated that the protonationof amino groups on the surface of CMs was a precondition for owning antibacterial activity.