Preparation And Effectiveness Evaluation Of Chitosan Nanoparticles, And Their Biological Properties And Applications As Carrier Of Bioactive Compounds

Chitin, one of the most abundant renewable biopolymer on earth, is a linear chain molecule composed of several hundred units of β-(1→4)-2-acetamido-2-deoxy-β-D-glucan.Based on the different orientations of its microfibrils, chitin can be classified into three forms including α, β and γ. Among them, α-chitin is the most widely used and usually prepared from shrimp and crabs shell. However, β-chitin is mainly extracted from squid pens. Chitosan(CS), the most important derivative of chitin, is prepared by deacetylization of chitin. Chitin and CS have many unique functional properties including biocompatibility, biodegradability, and non-toxicity, and have been widely applied in the field of food, agriculture, medicine, and materials. Chitin and CS are mainly prepared using chemical method in the industrial production, but this prepration method not only results in huge environment pollution, but brings into large threat to human health. Moreover, extraction of chitin using microbial fermentation prevents the uneven deacetylation and relevant molecular weight reduction caused by strong acid and alkali. Furthermore, the remaining fermentation waste contains abundant protein hydrolysate(amino acid and polypeptide), which can be collected as culture medium of other microbes for decreasing the cost of wastewater treatment. Therefore, microbial fermentation is a promising method for the preparation of chitin Hence, microbial fermentation method is key andurgent to prepare the chitin and CS. Moreover, because chitosanase and protease produced by Serratia marcescens B742 fermentation have low enzyme activity, microbial mutation was employed to improve the activity of chitosanase and proteasein this stage of this dissertation research, thus further improving the deprotenization efficacy of shrimp shell powders(SSPs). Meanwhile, CS processes both intramolecular and intermolecular hydrogen bonds, leading to poor water solubility. Hence, the ultrasound-assited(UA) Maillard reaction(MR) was employed to improve its water solubility, antioxidant and antibacterial activity in the second stage of research. Nanothecnology plays significant roles in the fields of biomedicine, phamarcy, biologyand functional foods. CS is a very important material to prepare nanoparticles(NPs). Therefore, α- and β-CS NPs, α- and β-CS Maillard reaction products(MRPs) NPs, α- and β-CS NPsencapsulated tea polyphenol(TP) and TP-Zn complex, α- and β-CS NPs encapsulated catechins(CAT) and CAT-Zn complex were all prepared, and their structural, antioxidant, antibacterial, cell toxicology and cell fluorescence were investigated.(1) To reduce the use of strong acid and alkalin, shrimp shell powders(SSPs) were fermented using successive two-step fermentation of Serratia marcescens B742 and Lactobacillus plantarum ATCC 8014 to extract chitin. Taguchi experimental design with orthogonal array was employed to investigate the most contributing factors on each of the one-step fermentation. The identified optimal fermentation conditions for extracting chitin from SSPs using S.marcescens B742 were 2% SSPs, 2 h of sonication time, 10% incubation level, and 4 d of culture time, while that of using L. plantarum ATCC 8014 fermentation was 2% SSPs, 15% glucose, 10% incubation level, and 2 d of culture time. Successive two-step fermentation using identified optimal fermentation conditions resulted in chitin yield of 18.9% with the final deprotenization(DP) and demineralization(DM) rate of 94.5% and 93.0%, respectively. The obtained chitin was compared with the commercial chitin from SSPs using scanning electron microscopy(SEM), Fourier transform infrared spectrometer(FT-IR) and X-ray diffraction(XRD). Results showed that the chitin prepared by the successive two-step fermentation exhibited similar physicochemical and structural properties to those of the commercial one, while significantly less use of chemical reagents.(2) To improve DP efficacy of SSPs for preparing chitin, S. marcescens B742 mutants were prepared using 2% diethyl diethyl sulfate(DES), UV-irradiation, and/or microwave heating treatments. Both single-stage and multi-stage mutations were investigated for optimizing S. marcescens B742 mutation conditions. Under the optimized mutation conditions(2% DES treatment for 30 min plus successive 20 min UV-irradiation), the protease and chitosanase produced by mutant S. marcescens B742 was 240.15 and 170.6 m U/m L, respectively as compared with 212.58±1.51 and 83.75±6.51 m U/m L, respectively by wild S. marcescens B742. DP(%) efficacy of SSPs by mutant S. marcescens B742 reached 91.43% after 3 d of submerged fermentation instead of 83.37% from the wild S. marcescens B742 after 4 d of submerged fermentation. Molecular mass of protease and chitosanase was 41.20 and 47.10 k Da, respectively based on the sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE), and both enzymes were verified by mass spectrometry analysis. The chitosanase from both wild and mutant S. marcescens B742 was activated by SDS, Tween 20, Tween 40, and Trixon-100, and the protease and chitosanase were strongly inhibited by ethylenediaminetetraacetic acid(EDTA). These results suggested that S. marcescens B742 mutants can be used in the biological production of chitin through deproteinization of SSPs.(3) To improve the production of chitin deacetylase(CDA) for the bioconversion of chitin to CS with desirable functionality, the effect of the nutritional requirement on the CDA production from Rhizopus japonicus M193 fermentation was investigated under submerged conditions. Nutritional elements including glucose(g/L), inoculum level(%), and Mg SO4·7H2O(g/L), as well as culture time(d) were identified as the most critical factors for the CDA production based on the results from Plackett-Burman design(PBD). Taguchi design with orthogonal array was further employed to optimize R. japonicus M193 fermentation conditions based on the results from PBD, in which 2.5% chitin, 5 g/L glucose, 5% inoculumlevel, 0.6 g/L Mg SO4·7H2O, and 5 d culture time were identified as the optimal fermentation condi-tions. Under this condition, the maximum CDA production, deacetylation of degree(DDA) and molecular mass(MM) of produced CS were 547.38 ± 12.06 U/L, 78.85 ± 1.68%, and 125.63 ± 3.74 k Da, respectively. Obtained CS displayed similar physicochemical and structural properties to those of commercial CS extracted using chemical method based on the results from FT-IR, thermogravimetricanalysis(TGA)-differential scanning calorimetry(DSC), and nuclear magnetic resonance(NMR) assays,while the use of chemical reagents was significantly reduced.(4) To improve water solubility of α- and β-CS and their antibacterial and antioxidant activity, α-CS-fructose Millard reaction(MR) was induced by using high intensity ultrasound-assisted(UA) water-bath heating at 80 oC in a model system consisting of 1% chitosan and 0.5, 1 or 1.5% fructose solution(FS). Based on the yield of the Millard reaction products(MRPs), the optimal UA water-bath heating conditions were 7, 5 and 8 h for 0.5, 1 and 1.5% FS, respectively, whereas they were 7, 8, and 7 h for 0.5, 1 and 1.5% FS, respectively by water-bath heating alone. Under the optimal conditions, the solubility of MRPs produced by UA heating reached 8.35-9.65 g/L compared with 5.32-7.37 g/L by water-bath heating alone. The yield of MRPs from UA heating treatments was 12.45, 12.63 and 18.86% for 0.5, 1, and 1.5% FS, respectively verses 5.78, 5.93 and 10.02%, respectively from water-bath heating alone. Reducing power(0.40, 0.47 and 0.65), DPPH free radical scavenging capacity(79.71, 87.10 and 98.70%) and ORAC(533.30, 1218.62 and 841.87 μmol TE/L) of MR solutions from UA heating were significantly higher than those from water-bath heating alone. MRPs showed higher antibacterial activity against Staphylococcus aureus(MIC of 2,500 mg/L) and Escherichia coli(MIC of ~313-625 mg/L) than that of native CS, but no difference(P < 0.05) was found in MRPs from water-bath heating alone. This study suggested that the high intensity UA water-bath heating could produce water soluble α-CS-fructose MRPs with significantly improved functionality.(5) Alpha-and β-CS NPs was prepapred based on the principle of ionic gelation between chitosan and sodium tripolyphosphate(TPP), and their antioxidant and cell toxicology were evaluated. The β-CS was depolymerized to MM)< 40 k Da for obtaining β-CSNPs at < 50 nm. Tea polyphenol(TP)-Zn complex loaded β-CS NPs were further prepared with a TP-Zn complex encapsulation efficacy of 97.33%, average particle size of 84.55 nm, and Zeta-potential of 29.23 m V. TP-Zn complex loaded α-and β-CS NPs exhibited higher antioxidant activity than that of TP loaded β-CS NPs based on reducing power, DPPH radical scavenging activity, and ORAC values. The in vitro release study conducted at p H 4.5 and 7.4 showed that the TP-Zn complex loaded β-CS NPs sustained the release of the TP-Zn complex over 5.5 h. Structural analysis using TEM, atomic force microscopy(AFM), FT-IR, and TGA-DSC indicated that TP-Zn complex was well incorporated into β-CS NPs. FITC-TP-Zn complex loaded β-CS NPs maintained the morphological characteristics of living cells and were well attached to the inside of EBM-2 endothelial cells, corroborating the result of the cytotoxicity test that NPs have hardly any cytotoxicity. This study suggested that TP-Zn complex loaded β-CS NPs can be used as an antioxidants delivery system for food and other applications.(6) β-CS NPs encapsulated catechins(CAT) or CAT-Zn complex at different particle sizes were prepared and their antibacterial activity against E. coli and Listeria innocua was studied. The CAT-Zn complex encapsulated β-CS NPs at different particle sizes were prepared at different ratios of CS and CAT-Zn complex(1:1, 1:3 and 1:5) using ionic gelation technology. Bacterial growth curve, minimum inhibitory concentration(MIC), and minimum bacterial concentration(MBC) of CAT-Zn complex encapsulated β-CS NPs against L. innocua and E. coli were studied. The particle size of CAT-Zn complex encapsulated β-CS NPs at β-CS to CAT-Zn complex ratios of 1:1, 1:3 and 1:5 were 208.0, 479.3 and 590.7 nm, respectively, and had polydispersity index(PDI) of 0.377-0.395 and positive Zeta-potential of 39.17-45.62 m V. The smaller particle size of CAT-Zn complex encapsulated β-CS NPs showed higher antibacterial activity than that of larger particle size ones. MIC and MBC of the smallest particle size of CAT-Zn complex encapsulated β-CS NPs against L. innocua and E. coli were 0.0625 and 0.0313 mg/m L, and 0.125 and 0.0625 mg/m L respectively. This study suggested that encapsulation of CAT-Zn complex in β-CS NPs improved the antibacterial activity of CAT and CAT-Zn complex, and the encapsulate can be used as an antibacterial substance for food and other applications.The main content of this dissertation was to 1) systematically prepare the chitin and chitosas using microbial fermentation method, 2) modify thr chitosan by Maillard reaction for improving the its solubility, antioxidant and antibacterial properties, 3) prepare low particle size of α-and β-CS NPs using inonic gelation technology, 4) investigate their physicochemical, antioxidant, antibacterial, cell toxicology and fluorescene properties. Thus, the research route of this dissertation is from the preparation and modification of chitosan to its utilization for providing helpful references for researchers.