壳聚糖纳米粒子的制备和功效评价及其在生物活性物质载体中的应用
发布时间：2018-09-09 10:36
【摘要】：甲壳素(Chitin)是目前自然界中仅次于纤维素的第二大可再生有机化合物资源,其化学结构式是β-(1→4)-2-乙酰氨基-2脱氧-D-葡萄糖。壳聚糖(Chitosan,CS)是甲壳素N-脱乙酰基的产物,其生物活性比甲壳素高,已广泛应用于食品、医药、污水处理和材料等领域中。基于甲壳素和壳聚糖微纤维排列方向的不同,可分为α、β和γ型甲壳素和壳聚糖,与许多现存的聚合物相比,壳聚糖及其衍生物具有无毒、吸附性强、生物可降解和生物相溶性等特性。在工业生产中,甲壳素和壳聚糖的制备主要采用强酸和强碱等化学方法提取,该方法对环境造成极大的污染和人体健康造成较大威胁。微生物发酵具有环境友好、成本低且发酵产品特性均一等优点,从而使得微生物发酵制备甲壳素和壳聚糖的迫切性和必要性。但鉴于微生物发酵产生蛋白酶和壳聚糖酶酶活较低,因此采用微生物突变的方法提高壳聚糖酶和蛋白酶的酶活,以提高微生物发酵提取甲壳素的去蛋白质效率。此外,壳聚糖由于其分子量较大且分子间和分子内存在大量的氢键,导致其水溶性较差,因此其应用也受到大大的限制。因此,采用超声波辅助制备壳聚糖衍生产物,以提高壳聚糖的水溶性和进一步提高其抗氧化和抗菌活性。随着纳米技术的快速发展,纳米粒子在医药、生物、医学和保健食品等领域发挥着巨大的应用价值,而壳聚糖是制备纳米粒子的重要素材之一,接下来制备了α-和β-壳聚糖纳米粒子,并研究了物化特性、以及在抗氧化、抗菌、细胞毒性和细胞荧光中的应用。本文可分为六个部分:(1)目前工业中主要采用化学法生产甲壳素和壳聚糖,但却对环境造成了较大的污染。首先研究了粘质沙雷氏菌b742(serratiamarcescensb742)和植物乳酸杆菌atcc8014(lactobacillusplantarumatcc8014)对虾壳粉进行连续两步发酵提取甲壳素。首先根据粘质沙雷氏菌b742的去蛋白质效率和植物乳酸杆菌atcc8014的去灰分效率,通过正交实验优化发酵实验条件,确定了粘质沙雷氏菌b742的发酵优化条件是2.0%的虾壳粉(w/v)、超声波1.5h、10%的接种量(w/v)和4d的发酵时间。植物乳酸杆菌atcc8014的发酵优化条件是2.0%虾壳粉(w/v)、15%葡萄糖(w/v)、10%的接种量(w/v)和2d的发酵时间。发酵后提取的甲壳素通过扫描电镜(scanningelectronmicroscopy,sem)、傅里叶拉曼光谱(fouriertransforminfraredspectrometer,ft-ir)和x-射线衍射(x-raydiffraction,xrd)进行结构表征,结果表明蛋白和灰分的去除率分别为94.48和92.99%,甲壳素的产率为18.9%。(2)鉴于粘质沙雷氏菌b742发酵虾壳粉去蛋白质效率较低,本阶段使用化学和物理相两阶段相结合进行粘质沙雷氏菌b742突变以提高壳聚糖酶和蛋白酶的酶活。首先用2%硫酸二乙酯(des)、紫外辐射和微波加热进行单因素突变实验,以壳聚糖酶和蛋白酶酶活为指标确定出最佳的突变条件分别为2%des处理30min、紫外辐照20min和微波加热30s。接着进行组合突变以进一步提高突变效率,最佳的突变组合条件是2%des处理30min再进行紫外辐照20min。结果表明粘质沙雷氏菌b742突变后壳聚糖酶和蛋白酶的酶活分别为240.15和170.6mu/ml,而野生型粘质沙雷氏菌b742产生的壳聚糖酶和蛋白酶的酶活分别为212.58和83.75mu/ml。突变型粘质沙雷氏菌b742发酵去虾壳粉中蛋白去除率为91.43%(野生型粘质沙雷氏菌b742为83.37%)。通过质谱和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sds-page)分析表明突变前后壳聚糖酶蛋白酶的分子量无变化,分别为41.20和47.10kda。此外,还研究了sds、吐温-20、吐温-40和一曲拉通x-100干扰条件对壳聚糖酶和蛋白酶酶活的变化。(3)基于前阶段粘质沙雷氏菌b742和植物乳酸杆菌atcc8014连续两阶段发酵提取甲壳素的基础上,本阶段采用优化日本根霉菌m193(rhizopusjaponicusm193)发酵培养基以提高甲壳素脱乙酰酶(cda)酶活而提取壳聚糖。首先以cda为主要指标,通过响应面法(plackett-burman设计)筛选发酵的实验条件(即发酵因素和水平),接着通过正交试验设计确定优化条件(2.5%的甲壳素、5g/l的葡萄糖、5%的接种量、0.6g/l的mgso4·7h2o和5d的培养时间)。然后通过sem、ft-ir和核磁共振分析(nuclearmagneticresonance,nmr)对甲壳素和壳聚糖的结构进行分析,并与化学法提取的壳聚糖进行比较。结果表明在优化的发酵条件下,cda、壳聚糖的脱乙酰度和壳聚糖的分子量分别为547.38±12.06mu/l、78.85±1.68%和125.63±3.74kda。与化学提取方法相比,微生物发酵的壳聚糖在分子量和化学结构上都具有一定的优越性。(4)壳聚糖因其分子量较大,分子间和分子内有较强的氢键,导致其水溶性较差,因此为了提高壳聚糖的水溶性、抗菌和抗氧能力,本阶段使用高强度超声波辅助水浴加热提高α-壳聚糖-果糖的美拉德反应程度。基于美拉德反应产物的产率,优化出高强度超声波辅助水浴加热的条件,在0.5、1和1.5%的果糖下的加热时间分别是7、5和8h。在优化的条件下,美拉德反应产物的溶解度为8.35-9.65g/l(水浴加热的为5.32-7.37g/l)。在0.5、1和1.5%的果糖下,美拉德反应产物的产率分别为12.45、12.63和18.86%(水浴加热的为5.78、5.93和10.02%)。在优化条件下,α-壳聚糖-果糖美拉德反应产物的还原力分别为0.40、0.47和0.65,dpph清除能力分别为79.71、87.10和98.70%,氧自由基吸附能力(oxygenradicalabsorptioncapacity,orac)分别为533.30、1218.62和841.87μmolte/l。此外,美拉德反应产物有较强的抗菌能力,其抗金黄色葡萄球菌的最小抑菌浓度(minimuminhibitionconcentration,mic)为2,500mg/l,大肠杆菌的最小抑菌浓度范围为313-625mg/l,且水浴加热对不同比率果糖下α-壳聚糖-果糖美拉德产物的抗菌能力没有显著性差异(p0.05)。结果表明高强度超声波辅助水浴加热能显著提高美拉德反应程度、α-壳聚糖的水溶性、抗菌和抗氧化特性。(5)纳米材料因具有小尺寸效应、较高的包裹效率、高灵敏度等特性,已广泛用于材料、医药、生物和食品等领域中。在优化的条件下,基于壳聚糖与多聚磷酸钠之间的离子交联制备了α-和β-壳聚糖纳米粒子、茶多酚和茶多酚-zn复合物装载的α-和β-壳聚糖纳米粒子,以及其包裹抗氧化物质的缓释及抗氧化特性。解聚后的β-壳聚糖的分子量和粒径分别小于40kda和50nm。茶多酚-zn复合物装载的β-壳聚糖纳米粒子的包裹效率、粒径和zeta-电位依次为97.33%、84.55nm和29.23mv。此外,相比于茶多酚装载的β-壳聚糖纳米粒子,茶多酚-zn复合物装载的β-壳聚糖纳米粒子有着较高的抗氧化能力(还原力、dpph清除能力和orac)。体外缓释实验表明,在ph4.5和7.4下,茶多酚或茶多酚-zn复合物装载的β-壳聚糖纳米粒子在5.5h内可持续释放茶多酚或茶多酚-zn复合物。sem、tem、原子力显微镜(atomicforcemicroscopy,afm)热重分析和差失热量扫描结果表明β-壳聚糖纳米粒子已包裹了茶多酚-zn复合物。荧光显微镜表明异硫氰酸荧光素(fluoresceinisothiocyanate,fitc)标记的β-壳聚糖纳米粒子已吸附于ebm-2内皮细胞(ebm-2endothelialcells)上。此外,茶多酚-zn复合物装载的β-壳聚糖纳米粒子在一定浓度范围内对ebm-2内皮细胞有着较高的细胞活力。结果表明茶多酚-zn复合物装载的β-壳聚糖纳米粒子可作为抗氧化物质的递送载体用于食品和其它领域中。(6)在研究壳聚糖纳米粒子作为载体在抗氧化应用的基础上,进一步研究了不同纳米粒径包裹的儿茶素-zn复合物的制备和抗菌特性。基于离子交联技术制备了不同比率的β-壳聚糖和儿茶素-zn复合物(1:1、1:3和1:5)装载的β-壳聚糖纳米粒子。研究了儿茶素-zn复合物装载的β-壳聚糖纳米粒子在抑菌生长、最小抑菌浓度、最小细菌浓度的抗菌特性(单核李斯特增生无害菌和大肠杆菌)。结果表明不同β-壳聚糖和儿茶素-zn复合物比率(1:1、1:3和1:5)装载的β-壳聚糖纳米粒子的粒径分别为208.0、479.3和590.7nm,显示出较好的分散度和zeta-电位。此外,不同比率的β-壳聚糖和儿茶素-zn复合物(1:1、1:3和1:5)装载的β-壳聚糖纳米粒子的粒径越小,其抗菌特性越强。所有的纳米粒子其抗单核李斯特增生无害菌的抗菌活性要高于大肠杆菌。儿茶素-zn复合物装载的最小粒径的β-壳聚糖纳米粒子对单核李斯特增生无害菌和大肠杆菌的最小抑菌浓度分别为0.0625和0.03125mg/ml,最小细菌浓度分别为0.125和0.0625mg/ml。结果表明儿茶素-zn复合物装载的β-壳聚糖纳米粒子可作为抗菌剂用于食品和其它领域中。本篇论文的主要内容是(1)系统的采用微生物发酵法制备α-甲壳素和壳聚糖;(2)将制备的壳聚糖通过美拉德反应改性提高α-壳聚糖的水溶性、抗氧化和抗菌特性;(3)通过离子交联技术制备小粒径的α-和β-壳聚糖纳米粒子;(4)研究了它们的物化特性、抗氧化、抗菌、细胞毒性和荧光等特性。本篇论文的研究路线是从壳聚糖的制备、改性、壳聚糖纳米粒子的制备以及其物化和功能特性,旨在为今后壳聚糖及其应用的研究工作者们提供有益参考。
[Abstract]:Chitin is the second largest renewable organic compound after cellulose in nature. Its chemical structure is beta-(1_4)-2-acetylamino-2-deoxy-D-glucose. Chitosan (CS) is the product of N-deacetylation of chitin. Its biological activity is higher than chitin. It has been widely used in food, medicine and sewage treatment. Chitosan and its derivatives are nontoxic, highly adsorptive, biodegradable and biocompatible compared with many existing polymers. In industrial production, chitin and chitosan are prepared. Microbial fermentation is an environmentally friendly, low-cost and homogeneous fermentation product, which makes it urgent and necessary to prepare chitin and chitosan by microbial fermentation. The enzyme activity of protease and chitosanase produced by fermentation is low, so the enzyme activity of chitosanase and protease is improved by microbial mutation to improve the protein removal efficiency of chitin extracted by microbial fermentation. Therefore, the preparation of chitosan derivatives assisted by ultrasound can improve the water solubility of chitosan and further enhance its antioxidant and antimicrobial activities.With the rapid development of nanotechnology, nanoparticles play an important role in the fields of medicine, biology, medicine and health food. Chitosan is one of the important materials for the preparation of nanoparticles. Then we prepared alpha-and beta-chitosan nanoparticles and studied their physicochemical properties as well as their applications in antioxidation, antibacterial, cytotoxicity and cell fluorescence. This paper can be divided into six parts: (1) Chitin and chitosan are mainly produced by chemical methods in industry at present, but not by chemical methods. Chitin was extracted from shrimp shell powder by continuous two-step fermentation of Serratia marcescens b742 and Lactobacillus plantarum atcc8014. The protein removal efficiency of Serratia marcescens b742 and the ash removal of Lactobacillus plantarum atcc8014 were studied. The optimal fermentation conditions of Serratia marcescens b742 were 2.0% shrimp shell powder (w / v), 1.5 h, 10% inoculation amount (w / v) and 4 d fermentation time. the optimal fermentation conditions of Lactobacillus plantarum atcc8014 were 2.0% shrimp shell powder (w / v), 15% glucose (w / v), 10% inoculation amount (w / v) and 2 d. The structure of chitin was characterized by scanning electron microscopy (sem), Fourier Raman spectroscopy (ft-ir) and X-ray diffraction (xrd). the results showed that the removal rates of protein and ash were 94.48 and 92.99%, respectively. (2) In view of the low protein removal efficiency of Shrimp Shell Powder fermented by Serratia marcescens B742, a chemical and physical two-stage method was used to mutate Serratia marcescens B742 to improve the enzyme activity of chitosanase and protease. Chitosanase and protease activity were used as the index to determine the optimal mutation conditions for 30 minutes, 20 minutes of ultraviolet irradiation and 30 seconds of microwave irradiation, respectively. Then combined mutation was carried out to further improve the mutation efficiency. The optimal mutation combination conditions were 2% des treatment for 30 minutes and then ultraviolet irradiation for 20 minutes. The enzyme activities of chitosanase and protease were 240.15 mu/ml and 170.6 mu/ml respectively, while those of wild-type Serratia marcescens b742 were 212.58 mu/ml and 83.75 mu/ml, respectively. The protein removal rate of shrimp chitosan powder fermented by mutant Serratia marcescens b742 was 91.43% (wild-type Serratia marcescens b742 was 83.37%). The molecular weight of chitosanase protease was 41.20 kDa and 47.10 kda, respectively, before and after the mutation. In addition, the effects of sds, tween-20, Tween-40 and trantone X-100 on the activity of chitosanase and protease were studied. (3) Based on the pre-stage mucin Chitosan was extracted by continuous two-stage fermentation of Serratia b742 and Lactobacillus plantarum atcc8014. Rhizopus japonicus m193 (rhizopus japonicus m193) was used to improve the activity of chitin deacetylase (cda). first, chitosan was extracted by response surface methodology (plackett-burman). The optimum conditions (2.5% chitin, 5 g / L glucose, 5% inoculation, 0.6 g / L MgSO 4.7 H 2O and 5 d culture time) were determined by orthogonal design. then the chitin and chitosan were analyzed by sem, FT-IR and nuclear magnetic resonance (nmr). The results showed that under the optimized fermentation conditions, the degree of deacetylation and the molecular weight of chitosan were 547.38 (+ 12.06mu/l), 78.85 (+ 1.68%) and 125.63 (+ 3.74 kda), respectively. (4) Chitosan is poor in water solubility because of its high molecular weight and strong hydrogen bonds between and within molecules. In order to improve the water solubility, antibacterial and antioxidant properties of chitosan, high intensity ultrasound-assisted water bath heating was used to improve the Maillard reaction of alpha-chitosan-fructose. The yield of Maillard reaction products was optimized. The heating time of Maillard reaction products was 7,5 and 8 h under 0.5,1 and 1.5% fructose respectively. Under the optimized conditions, the solubility of Maillard reaction products was 8.35-9.65 g/l (5.32-7.37 g/l heated in water bath). The products of Maillard reaction were obtained under 0.5,1 and 1.5% fructose. The yields were 12.45, 12.63 and 18.86% (heated by water bath, 5.78, 5.93 and 10.02%) respectively. Under the optimum conditions, the reducing power of the products of Maillard reaction of alpha-chitosan-fructose was 0.40, 0.47 and 0.65, the scavenging power of DPPH was 79.71, 87.10 and 98.70%, and the oxygen radical absorption capacity (orac) was 533.30, 1. 218.62 and 841.87 um olte/l. In addition, Maillard reaction products had strong antibacterial activity. The minimum inhibitory concentration (mic) of Maillard reaction products against Staphylococcus aureus was 2,500 mg/l, and the minimum inhibitory concentration of Escherichia coli was 313-625 mg/l. Maillard products were produced under different ratios of fructose by water bath heating. The results showed that high intensity ultrasound-assisted water bath heating could significantly improve Maillard reaction degree, water solubility, antibacterial and antioxidant properties of alpha-chitosan. (5) Nano-materials have been widely used in materials, medicine, raw materials because of their small size effect, high encapsulation efficiency and high sensitivity. In the fields of food and food, under optimized conditions, alpha-and beta-chitosan nanoparticles, alpha-and beta-chitosan nanoparticles loaded with tea polyphenols and tea polyphenols-zn complexes, and their antioxidant properties encapsulated in antioxidants were prepared by ionic crosslinking between chitosan and sodium polyphosphate. The molecular weight and particle size of the nanoparticles were less than 40 kDa and 50 nm, respectively. the encapsulation efficiency of the nanoparticles was 97.33%, 84.55 nm and 29.23 mv, respectively. Oxidative capacity (reductivity, DPPH scavenging capacity and orac). in vitro slow-release experiments showed that at pH 4.5 and 7.4, the sustained release of tea polyphenols or tea polyphenols-zn complex loaded beta-chitosan nanoparticles within 5.5 H. sem, tem, atomic force microscopy (afm) thermogravimetric analysis and differential scanning calorimetry Fluorescence microscopy showed that fluorescein isothiocyanate (fitc) labeled beta-chitosan nanoparticles were adsorbed on ebm-2 endothelial cells (ebm-2 endothelial cells). in addition, the beta-chitosan nanoparticles loaded on the tea polyphenol-zn complex were also observed. The results showed that the beta-chitosan nanoparticles loaded with tea polyphenol-zn complex could be used as delivery carriers for antioxidants in food and other fields. (6) Based on the study of antioxidant applications of chitosan nanoparticles as carriers, further studies were carried out. The preparation and antibacterial properties of catechin-zn nanoparticles encapsulated with different nano-particles were studied. The beta-chitosan nanoparticles loaded with different ratios of beta-chitosan and catechin-zn (1:1,1:3 and 1:5) were prepared by ion-crosslinking technique. The antibacterial growth and minimal inhibition of beta-chitosan nanoparticles loaded with catechin-zn complex were studied. The results showed that the diameters of the nanoparticles loaded with different ratios of beta-chitosan to catechin-zn (1:1,1:3 and 1:5) were 208.0,479.3 and 590.7 nm, respectively, showing good dispersion and zeta-potential. The smaller the particle size of the beta-chitosan nanoparticles loaded with the ratio of beta-chitosan and catechin-zn (1:1,1:3 and 1:5), the stronger their antibacterial properties. The antibacterial activities of all the nanoparticles against Listeria monocytogenes were higher than those of E. The minimal inhibitory concentrations of nucleostatin-zn complex loaded beta-chitosan nanoparticles were 0.0625 mg/ml and 0.03125 mg/ml, respectively. The results showed that beta-chitosan nanoparticles loaded with catechin-zn complex could be used as antimicrobial agents in food and other fields. Alpha-chitosan and chitosan were prepared by microbial fermentation; (2) Chitosan was modified by Maillard reaction to improve the water solubility, antioxidant and antibacterial properties of alpha-chitosan; (3) preparation of small size alpha-and beta-chitosan nanoparticles by ion cross-linking technology; (4) their physicochemical properties, antioxidant, antibacterial and cytotoxic properties were studied. The research route of this paper is the preparation and modification of chitosan, the preparation of chitosan nanoparticles and their physicochemical and functional properties. The purpose of this paper is to provide a useful reference for the future research of chitosan and its application.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R318
，
本文编号：2232137
[Abstract]:Chitin is the second largest renewable organic compound after cellulose in nature. Its chemical structure is beta-(1_4)-2-acetylamino-2-deoxy-D-glucose. Chitosan (CS) is the product of N-deacetylation of chitin. Its biological activity is higher than chitin. It has been widely used in food, medicine and sewage treatment. Chitosan and its derivatives are nontoxic, highly adsorptive, biodegradable and biocompatible compared with many existing polymers. In industrial production, chitin and chitosan are prepared. Microbial fermentation is an environmentally friendly, low-cost and homogeneous fermentation product, which makes it urgent and necessary to prepare chitin and chitosan by microbial fermentation. The enzyme activity of protease and chitosanase produced by fermentation is low, so the enzyme activity of chitosanase and protease is improved by microbial mutation to improve the protein removal efficiency of chitin extracted by microbial fermentation. Therefore, the preparation of chitosan derivatives assisted by ultrasound can improve the water solubility of chitosan and further enhance its antioxidant and antimicrobial activities.With the rapid development of nanotechnology, nanoparticles play an important role in the fields of medicine, biology, medicine and health food. Chitosan is one of the important materials for the preparation of nanoparticles. Then we prepared alpha-and beta-chitosan nanoparticles and studied their physicochemical properties as well as their applications in antioxidation, antibacterial, cytotoxicity and cell fluorescence. This paper can be divided into six parts: (1) Chitin and chitosan are mainly produced by chemical methods in industry at present, but not by chemical methods. Chitin was extracted from shrimp shell powder by continuous two-step fermentation of Serratia marcescens b742 and Lactobacillus plantarum atcc8014. The protein removal efficiency of Serratia marcescens b742 and the ash removal of Lactobacillus plantarum atcc8014 were studied. The optimal fermentation conditions of Serratia marcescens b742 were 2.0% shrimp shell powder (w / v), 1.5 h, 10% inoculation amount (w / v) and 4 d fermentation time. the optimal fermentation conditions of Lactobacillus plantarum atcc8014 were 2.0% shrimp shell powder (w / v), 15% glucose (w / v), 10% inoculation amount (w / v) and 2 d. The structure of chitin was characterized by scanning electron microscopy (sem), Fourier Raman spectroscopy (ft-ir) and X-ray diffraction (xrd). the results showed that the removal rates of protein and ash were 94.48 and 92.99%, respectively. (2) In view of the low protein removal efficiency of Shrimp Shell Powder fermented by Serratia marcescens B742, a chemical and physical two-stage method was used to mutate Serratia marcescens B742 to improve the enzyme activity of chitosanase and protease. Chitosanase and protease activity were used as the index to determine the optimal mutation conditions for 30 minutes, 20 minutes of ultraviolet irradiation and 30 seconds of microwave irradiation, respectively. Then combined mutation was carried out to further improve the mutation efficiency. The optimal mutation combination conditions were 2% des treatment for 30 minutes and then ultraviolet irradiation for 20 minutes. The enzyme activities of chitosanase and protease were 240.15 mu/ml and 170.6 mu/ml respectively, while those of wild-type Serratia marcescens b742 were 212.58 mu/ml and 83.75 mu/ml, respectively. The protein removal rate of shrimp chitosan powder fermented by mutant Serratia marcescens b742 was 91.43% (wild-type Serratia marcescens b742 was 83.37%). The molecular weight of chitosanase protease was 41.20 kDa and 47.10 kda, respectively, before and after the mutation. In addition, the effects of sds, tween-20, Tween-40 and trantone X-100 on the activity of chitosanase and protease were studied. (3) Based on the pre-stage mucin Chitosan was extracted by continuous two-stage fermentation of Serratia b742 and Lactobacillus plantarum atcc8014. Rhizopus japonicus m193 (rhizopus japonicus m193) was used to improve the activity of chitin deacetylase (cda). first, chitosan was extracted by response surface methodology (plackett-burman). The optimum conditions (2.5% chitin, 5 g / L glucose, 5% inoculation, 0.6 g / L MgSO 4.7 H 2O and 5 d culture time) were determined by orthogonal design. then the chitin and chitosan were analyzed by sem, FT-IR and nuclear magnetic resonance (nmr). The results showed that under the optimized fermentation conditions, the degree of deacetylation and the molecular weight of chitosan were 547.38 (+ 12.06mu/l), 78.85 (+ 1.68%) and 125.63 (+ 3.74 kda), respectively. (4) Chitosan is poor in water solubility because of its high molecular weight and strong hydrogen bonds between and within molecules. In order to improve the water solubility, antibacterial and antioxidant properties of chitosan, high intensity ultrasound-assisted water bath heating was used to improve the Maillard reaction of alpha-chitosan-fructose. The yield of Maillard reaction products was optimized. The heating time of Maillard reaction products was 7,5 and 8 h under 0.5,1 and 1.5% fructose respectively. Under the optimized conditions, the solubility of Maillard reaction products was 8.35-9.65 g/l (5.32-7.37 g/l heated in water bath). The products of Maillard reaction were obtained under 0.5,1 and 1.5% fructose. The yields were 12.45, 12.63 and 18.86% (heated by water bath, 5.78, 5.93 and 10.02%) respectively. Under the optimum conditions, the reducing power of the products of Maillard reaction of alpha-chitosan-fructose was 0.40, 0.47 and 0.65, the scavenging power of DPPH was 79.71, 87.10 and 98.70%, and the oxygen radical absorption capacity (orac) was 533.30, 1. 218.62 and 841.87 um olte/l. In addition, Maillard reaction products had strong antibacterial activity. The minimum inhibitory concentration (mic) of Maillard reaction products against Staphylococcus aureus was 2,500 mg/l, and the minimum inhibitory concentration of Escherichia coli was 313-625 mg/l. Maillard products were produced under different ratios of fructose by water bath heating. The results showed that high intensity ultrasound-assisted water bath heating could significantly improve Maillard reaction degree, water solubility, antibacterial and antioxidant properties of alpha-chitosan. (5) Nano-materials have been widely used in materials, medicine, raw materials because of their small size effect, high encapsulation efficiency and high sensitivity. In the fields of food and food, under optimized conditions, alpha-and beta-chitosan nanoparticles, alpha-and beta-chitosan nanoparticles loaded with tea polyphenols and tea polyphenols-zn complexes, and their antioxidant properties encapsulated in antioxidants were prepared by ionic crosslinking between chitosan and sodium polyphosphate. The molecular weight and particle size of the nanoparticles were less than 40 kDa and 50 nm, respectively. the encapsulation efficiency of the nanoparticles was 97.33%, 84.55 nm and 29.23 mv, respectively. Oxidative capacity (reductivity, DPPH scavenging capacity and orac). in vitro slow-release experiments showed that at pH 4.5 and 7.4, the sustained release of tea polyphenols or tea polyphenols-zn complex loaded beta-chitosan nanoparticles within 5.5 H. sem, tem, atomic force microscopy (afm) thermogravimetric analysis and differential scanning calorimetry Fluorescence microscopy showed that fluorescein isothiocyanate (fitc) labeled beta-chitosan nanoparticles were adsorbed on ebm-2 endothelial cells (ebm-2 endothelial cells). in addition, the beta-chitosan nanoparticles loaded on the tea polyphenol-zn complex were also observed. The results showed that the beta-chitosan nanoparticles loaded with tea polyphenol-zn complex could be used as delivery carriers for antioxidants in food and other fields. (6) Based on the study of antioxidant applications of chitosan nanoparticles as carriers, further studies were carried out. The preparation and antibacterial properties of catechin-zn nanoparticles encapsulated with different nano-particles were studied. The beta-chitosan nanoparticles loaded with different ratios of beta-chitosan and catechin-zn (1:1,1:3 and 1:5) were prepared by ion-crosslinking technique. The antibacterial growth and minimal inhibition of beta-chitosan nanoparticles loaded with catechin-zn complex were studied. The results showed that the diameters of the nanoparticles loaded with different ratios of beta-chitosan to catechin-zn (1:1,1:3 and 1:5) were 208.0,479.3 and 590.7 nm, respectively, showing good dispersion and zeta-potential. The smaller the particle size of the beta-chitosan nanoparticles loaded with the ratio of beta-chitosan and catechin-zn (1:1,1:3 and 1:5), the stronger their antibacterial properties. The antibacterial activities of all the nanoparticles against Listeria monocytogenes were higher than those of E. The minimal inhibitory concentrations of nucleostatin-zn complex loaded beta-chitosan nanoparticles were 0.0625 mg/ml and 0.03125 mg/ml, respectively. The results showed that beta-chitosan nanoparticles loaded with catechin-zn complex could be used as antimicrobial agents in food and other fields. Alpha-chitosan and chitosan were prepared by microbial fermentation; (2) Chitosan was modified by Maillard reaction to improve the water solubility, antioxidant and antibacterial properties of alpha-chitosan; (3) preparation of small size alpha-and beta-chitosan nanoparticles by ion cross-linking technology; (4) their physicochemical properties, antioxidant, antibacterial and cytotoxic properties were studied. The research route of this paper is the preparation and modification of chitosan, the preparation of chitosan nanoparticles and their physicochemical and functional properties. The purpose of this paper is to provide a useful reference for the future research of chitosan and its application.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R318
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本文编号：2232137
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