殼聚糖納米粒子的制備和功效評價及其在生物活性物質(zhì)載體中的應(yīng)用
發(fā)布時間：2018-09-09 10:36
【摘要】：甲殼素(Chitin)是目前自然界中僅次于纖維素的第二大可再生有機化合物資源,其化學結(jié)構(gòu)式是β-(1→4)-2-乙酰氨基-2脫氧-D-葡萄糖。殼聚糖(Chitosan,CS)是甲殼素N-脫乙�；漠a(chǎn)物,其生物活性比甲殼素高,已廣泛應(yīng)用于食品、醫(yī)藥、污水處理和材料等領(lǐng)域中�；诩讱に睾蜌ぞ厶俏⒗w維排列方向的不同,可分為α、β和γ型甲殼素和殼聚糖,與許多現(xiàn)存的聚合物相比,殼聚糖及其衍生物具有無毒、吸附性強、生物可降解和生物相溶性等特性。在工業(yè)生產(chǎn)中,甲殼素和殼聚糖的制備主要采用強酸和強堿等化學方法提取,該方法對環(huán)境造成極大的污染和人體健康造成較大威脅。微生物發(fā)酵具有環(huán)境友好、成本低且發(fā)酵產(chǎn)品特性均一等優(yōu)點,從而使得微生物發(fā)酵制備甲殼素和殼聚糖的迫切性和必要性。但鑒于微生物發(fā)酵產(chǎn)生蛋白酶和殼聚糖酶酶活較低,因此采用微生物突變的方法提高殼聚糖酶和蛋白酶的酶活,以提高微生物發(fā)酵提取甲殼素的去蛋白質(zhì)效率。此外,殼聚糖由于其分子量較大且分子間和分子內(nèi)存在大量的氫鍵,導致其水溶性較差,因此其應(yīng)用也受到大大的限制。因此,采用超聲波輔助制備殼聚糖衍生產(chǎn)物,以提高殼聚糖的水溶性和進一步提高其抗氧化和抗菌活性。隨著納米技術(shù)的快速發(fā)展,納米粒子在醫(yī)藥、生物、醫(yī)學和保健食品等領(lǐng)域發(fā)揮著巨大的應(yīng)用價值,而殼聚糖是制備納米粒子的重要素材之一,接下來制備了α-和β-殼聚糖納米粒子,并研究了物化特性、以及在抗氧化、抗菌、細胞毒性和細胞熒光中的應(yīng)用。本文可分為六個部分:(1)目前工業(yè)中主要采用化學法生產(chǎn)甲殼素和殼聚糖,但卻對環(huán)境造成了較大的污染。首先研究了粘質(zhì)沙雷氏菌b742(serratiamarcescensb742)和植物乳酸桿菌atcc8014(lactobacillusplantarumatcc8014)對蝦殼粉進行連續(xù)兩步發(fā)酵提取甲殼素。首先根據(jù)粘質(zhì)沙雷氏菌b742的去蛋白質(zhì)效率和植物乳酸桿菌atcc8014的去灰分效率,通過正交實驗優(yōu)化發(fā)酵實驗條件,確定了粘質(zhì)沙雷氏菌b742的發(fā)酵優(yōu)化條件是2.0%的蝦殼粉(w/v)、超聲波1.5h、10%的接種量(w/v)和4d的發(fā)酵時間。植物乳酸桿菌atcc8014的發(fā)酵優(yōu)化條件是2.0%蝦殼粉(w/v)、15%葡萄糖(w/v)、10%的接種量(w/v)和2d的發(fā)酵時間。發(fā)酵后提取的甲殼素通過掃描電鏡(scanningelectronmicroscopy,sem)、傅里葉拉曼光譜(fouriertransforminfraredspectrometer,ft-ir)和x-射線衍射(x-raydiffraction,xrd)進行結(jié)構(gòu)表征,結(jié)果表明蛋白和灰分的去除率分別為94.48和92.99%,甲殼素的產(chǎn)率為18.9%。(2)鑒于粘質(zhì)沙雷氏菌b742發(fā)酵蝦殼粉去蛋白質(zhì)效率較低,本階段使用化學和物理相兩階段相結(jié)合進行粘質(zhì)沙雷氏菌b742突變以提高殼聚糖酶和蛋白酶的酶活。首先用2%硫酸二乙酯(des)、紫外輻射和微波加熱進行單因素突變實驗,以殼聚糖酶和蛋白酶酶活為指標確定出最佳的突變條件分別為2%des處理30min、紫外輻照20min和微波加熱30s。接著進行組合突變以進一步提高突變效率,最佳的突變組合條件是2%des處理30min再進行紫外輻照20min。結(jié)果表明粘質(zhì)沙雷氏菌b742突變后殼聚糖酶和蛋白酶的酶活分別為240.15和170.6mu/ml,而野生型粘質(zhì)沙雷氏菌b742產(chǎn)生的殼聚糖酶和蛋白酶的酶活分別為212.58和83.75mu/ml。突變型粘質(zhì)沙雷氏菌b742發(fā)酵去蝦殼粉中蛋白去除率為91.43%(野生型粘質(zhì)沙雷氏菌b742為83.37%)。通過質(zhì)譜和十二烷基硫酸鈉-聚丙烯酰胺凝膠電泳(sds-page)分析表明突變前后殼聚糖酶蛋白酶的分子量無變化,分別為41.20和47.10kda。此外,還研究了sds、吐溫-20、吐溫-40和一曲拉通x-100干擾條件對殼聚糖酶和蛋白酶酶活的變化。(3)基于前階段粘質(zhì)沙雷氏菌b742和植物乳酸桿菌atcc8014連續(xù)兩階段發(fā)酵提取甲殼素的基礎(chǔ)上,本階段采用優(yōu)化日本根霉菌m193(rhizopusjaponicusm193)發(fā)酵培養(yǎng)基以提高甲殼素脫乙酰酶(cda)酶活而提取殼聚糖。首先以cda為主要指標,通過響應(yīng)面法(plackett-burman設(shè)計)篩選發(fā)酵的實驗條件(即發(fā)酵因素和水平),接著通過正交試驗設(shè)計確定優(yōu)化條件(2.5%的甲殼素、5g/l的葡萄糖、5%的接種量、0.6g/l的mgso4·7h2o和5d的培養(yǎng)時間)。然后通過sem、ft-ir和核磁共振分析(nuclearmagneticresonance,nmr)對甲殼素和殼聚糖的結(jié)構(gòu)進行分析,并與化學法提取的殼聚糖進行比較。結(jié)果表明在優(yōu)化的發(fā)酵條件下,cda、殼聚糖的脫乙酰度和殼聚糖的分子量分別為547.38±12.06mu/l、78.85±1.68%和125.63±3.74kda。與化學提取方法相比,微生物發(fā)酵的殼聚糖在分子量和化學結(jié)構(gòu)上都具有一定的優(yōu)越性。(4)殼聚糖因其分子量較大,分子間和分子內(nèi)有較強的氫鍵,導致其水溶性較差,因此為了提高殼聚糖的水溶性、抗菌和抗氧能力,本階段使用高強度超聲波輔助水浴加熱提高α-殼聚糖-果糖的美拉德反應(yīng)程度。基于美拉德反應(yīng)產(chǎn)物的產(chǎn)率,優(yōu)化出高強度超聲波輔助水浴加熱的條件,在0.5、1和1.5%的果糖下的加熱時間分別是7、5和8h。在優(yōu)化的條件下,美拉德反應(yīng)產(chǎn)物的溶解度為8.35-9.65g/l(水浴加熱的為5.32-7.37g/l)。在0.5、1和1.5%的果糖下,美拉德反應(yīng)產(chǎn)物的產(chǎn)率分別為12.45、12.63和18.86%(水浴加熱的為5.78、5.93和10.02%)。在優(yōu)化條件下,α-殼聚糖-果糖美拉德反應(yīng)產(chǎn)物的還原力分別為0.40、0.47和0.65,dpph清除能力分別為79.71、87.10和98.70%,氧自由基吸附能力(oxygenradicalabsorptioncapacity,orac)分別為533.30、1218.62和841.87μmolte/l。此外,美拉德反應(yīng)產(chǎn)物有較強的抗菌能力,其抗金黃色葡萄球菌的最小抑菌濃度(minimuminhibitionconcentration,mic)為2,500mg/l,大腸桿菌的最小抑菌濃度范圍為313-625mg/l,且水浴加熱對不同比率果糖下α-殼聚糖-果糖美拉德產(chǎn)物的抗菌能力沒有顯著性差異(p0.05)。結(jié)果表明高強度超聲波輔助水浴加熱能顯著提高美拉德反應(yīng)程度、α-殼聚糖的水溶性、抗菌和抗氧化特性。(5)納米材料因具有小尺寸效應(yīng)、較高的包裹效率、高靈敏度等特性,已廣泛用于材料、醫(yī)藥、生物和食品等領(lǐng)域中。在優(yōu)化的條件下,基于殼聚糖與多聚磷酸鈉之間的離子交聯(lián)制備了α-和β-殼聚糖納米粒子、茶多酚和茶多酚-zn復合物裝載的α-和β-殼聚糖納米粒子,以及其包裹抗氧化物質(zhì)的緩釋及抗氧化特性。解聚后的β-殼聚糖的分子量和粒徑分別小于40kda和50nm。茶多酚-zn復合物裝載的β-殼聚糖納米粒子的包裹效率、粒徑和zeta-電位依次為97.33%、84.55nm和29.23mv。此外,相比于茶多酚裝載的β-殼聚糖納米粒子,茶多酚-zn復合物裝載的β-殼聚糖納米粒子有著較高的抗氧化能力(還原力、dpph清除能力和orac)。體外緩釋實驗表明,在ph4.5和7.4下,茶多酚或茶多酚-zn復合物裝載的β-殼聚糖納米粒子在5.5h內(nèi)可持續(xù)釋放茶多酚或茶多酚-zn復合物。sem、tem、原子力顯微鏡(atomicforcemicroscopy,afm)熱重分析和差失熱量掃描結(jié)果表明β-殼聚糖納米粒子已包裹了茶多酚-zn復合物。熒光顯微鏡表明異硫氰酸熒光素(fluoresceinisothiocyanate,fitc)標記的β-殼聚糖納米粒子已吸附于ebm-2內(nèi)皮細胞(ebm-2endothelialcells)上。此外,茶多酚-zn復合物裝載的β-殼聚糖納米粒子在一定濃度范圍內(nèi)對ebm-2內(nèi)皮細胞有著較高的細胞活力。結(jié)果表明茶多酚-zn復合物裝載的β-殼聚糖納米粒子可作為抗氧化物質(zhì)的遞送載體用于食品和其它領(lǐng)域中。(6)在研究殼聚糖納米粒子作為載體在抗氧化應(yīng)用的基礎(chǔ)上,進一步研究了不同納米粒徑包裹的兒茶素-zn復合物的制備和抗菌特性�；陔x子交聯(lián)技術(shù)制備了不同比率的β-殼聚糖和兒茶素-zn復合物(1:1、1:3和1:5)裝載的β-殼聚糖納米粒子。研究了兒茶素-zn復合物裝載的β-殼聚糖納米粒子在抑菌生長、最小抑菌濃度、最小細菌濃度的抗菌特性(單核李斯特增生無害菌和大腸桿菌)。結(jié)果表明不同β-殼聚糖和兒茶素-zn復合物比率(1:1、1:3和1:5)裝載的β-殼聚糖納米粒子的粒徑分別為208.0、479.3和590.7nm,顯示出較好的分散度和zeta-電位。此外,不同比率的β-殼聚糖和兒茶素-zn復合物(1:1、1:3和1:5)裝載的β-殼聚糖納米粒子的粒徑越小,其抗菌特性越強。所有的納米粒子其抗單核李斯特增生無害菌的抗菌活性要高于大腸桿菌。兒茶素-zn復合物裝載的最小粒徑的β-殼聚糖納米粒子對單核李斯特增生無害菌和大腸桿菌的最小抑菌濃度分別為0.0625和0.03125mg/ml,最小細菌濃度分別為0.125和0.0625mg/ml。結(jié)果表明兒茶素-zn復合物裝載的β-殼聚糖納米粒子可作為抗菌劑用于食品和其它領(lǐng)域中。本篇論文的主要內(nèi)容是(1)系統(tǒng)的采用微生物發(fā)酵法制備α-甲殼素和殼聚糖;(2)將制備的殼聚糖通過美拉德反應(yīng)改性提高α-殼聚糖的水溶性、抗氧化和抗菌特性;(3)通過離子交聯(lián)技術(shù)制備小粒徑的α-和β-殼聚糖納米粒子;(4)研究了它們的物化特性、抗氧化、抗菌、細胞毒性和熒光等特性。本篇論文的研究路線是從殼聚糖的制備、改性、殼聚糖納米粒子的制備以及其物化和功能特性,旨在為今后殼聚糖及其應(yīng)用的研究工作者們提供有益參考。
[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.
【學位授予單位】：上海交通大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R318
，
本文編號：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.
【學位授予單位】：上海交通大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R318
，
本文編號：2232137
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