本文選題：膠質(zhì)芽孢桿菌胞外多糖 + 提取　； 參考：《江南大學(xué)》2017年博士論文

【摘要】：細(xì)菌胞外多糖是一類具有多元結(jié)構(gòu)和獨(dú)特性能的生物大分子,因其優(yōu)良的理化性質(zhì)和生物活性而被廣泛地應(yīng)用于食品、醫(yī)藥、化工等領(lǐng)域。盡管目前已發(fā)現(xiàn)的細(xì)菌胞外多糖種類很多,但真正具有實(shí)際應(yīng)用價值和實(shí)現(xiàn)工業(yè)化生產(chǎn)的卻很少,多糖產(chǎn)量、生產(chǎn)成本以及對構(gòu)效關(guān)系研究的欠缺是制約多糖生產(chǎn)和應(yīng)用的主要因素,因此尋找具有獨(dú)特性能的新型高產(chǎn)多糖是當(dāng)前研究的熱點(diǎn)。膠質(zhì)芽孢桿菌(Bacillus mucilaginosus)是土壤中一類特殊的硅酸鹽菌,能夠利用有限資源產(chǎn)生大量胞外多糖。目前對于B.mucilaginosus的研究主要集中在其解鉀溶磷固氮功能以及污水處理上,B.mucilaginosus能夠降解土壤中的礦物質(zhì)(硅酸鹽、磷灰石、磷鈣石),使其轉(zhuǎn)化為可供植物吸收的營養(yǎng)物質(zhì);同時還可以作為絮凝劑和吸附劑除去污水中的各種污染物質(zhì),B.mucilaginosus這些功能被認(rèn)為和其產(chǎn)生的胞外多糖密切相關(guān)。此外,研究發(fā)現(xiàn)B.mucilaginosus胞外多糖還具有一定的生物活性,能夠促進(jìn)小鼠胃潰瘍部位的修復(fù)以及黃仔雞免疫器官的發(fā)育。然而對于B.mucilaginosu 胞外多糖基本信息的研究非常少,其化學(xué)結(jié)構(gòu)還不為人所知,極大地限制了其應(yīng)用發(fā)展。因此對B.mucilaginosus胞外多糖化學(xué)結(jié)構(gòu)和理化性質(zhì)進(jìn)行基礎(chǔ)性研究將有助于了解其構(gòu)效關(guān)系,拓寬其潛在應(yīng)用領(lǐng)域。本研究通過對B. mucilaginosus SM-01發(fā)酵液進(jìn)行提取優(yōu)化,得到酸性雜多糖BMPS,通過各種表征方法對BMPS的一級結(jié)構(gòu)和高級結(jié)構(gòu)進(jìn)行了研究,為開發(fā)B.mucilaginosus胞外多糖提供了理論依據(jù),同時基于生物活性對其作為傷口修復(fù)材料進(jìn)行了初步探討,拓寬了B.mucilaginosus胞外多糖的應(yīng)用前景,主要結(jié)果如下:(1)通過考察稀釋、升溫、改變pH、加入鹽的量對發(fā)酵液粘度的影響,選擇稀釋與加鹽協(xié)同使用的方法降低發(fā)酵液粘度。在發(fā)酵液中加入3% (w/v) NaCl,比較不同稀釋倍數(shù)下,離心和硅藻土抽濾對發(fā)酵液除菌和除蛋白的影響以及對抽濾通量的影響,發(fā)現(xiàn)抽濾去除菌體和雜蛋白的效果遠(yuǎn)高于離心,在稀釋倍數(shù)為3倍的情況下抽濾,能保持較好的抽濾通量,除菌率為92.6%,除蛋白率為69.7%。以硅藻土作為吸附劑,比較不同添加量對除菌、除蛋白以及多糖回收率的影響,發(fā)現(xiàn)添加10 g/L硅藻土后經(jīng)重復(fù)抽濾三次,鏡檢無菌體存在,蛋白含量接近于零,多糖回收率達(dá)76.4%�？疾觳煌亓舴肿恿康某瑸V膜以及操作壓力對超濾濃縮的影響,發(fā)現(xiàn)采用200 kDa截留分子量的超濾膜在0.1 MPa操作壓力下超濾基本能截留所有多糖組分,經(jīng)過超濾濃縮除鹽,多糖最終總回收率為73.2%,內(nèi)毒素含量低于0.25 EU/mL,此提取工藝簡單易行且適合工業(yè)生產(chǎn)。對濃縮得到的粗多糖經(jīng)離子交換色譜分離純化,再利用體積排阻色譜和光散射聯(lián)用進(jìn)行純度鑒定和分子量測定,發(fā)現(xiàn)B.mucilaginosus SM-01胞外多糖為均一的酸性多糖,命名為BMPS,其重均分子量Mw為2.67×106Da。(2)通過對BMPS進(jìn)行化學(xué)組成分析發(fā)現(xiàn)BMPS不含磷酸基團(tuán)和硫酸基團(tuán),糖醛酸含量為14.3%,O-乙�；繛�4.99%。通過單糖分析得到其單糖組成為葡萄糖(Glc)、甘露糖(Man)、葡萄糖醛酸(GlcA)和一個未知糖醛酸(AM),摩爾比為3.2: 2: 0.5: 0.3。對BMPS先進(jìn)行超聲降解再進(jìn)行溫和酸水解得到一個中性寡糖片段BMPS-H,通過基質(zhì)輔助激光解吸飛行時間質(zhì)譜得到其Mw為1771.43 Da,單糖組成為Glc和Man,摩爾比為1.5: 1。通過甲基化分析發(fā)現(xiàn)BMPS-H分子為線性結(jié)構(gòu),主鏈由1,4-Man和1,4-Glc以及少量1,3-Glc構(gòu)成,通過核磁共振得到其糖基連接順序以及乙�；倪B接位點(diǎn),進(jìn)而推斷出BMPS-H的化學(xué)結(jié)構(gòu)為:β-D-Glc (1→[4)-β-D-Man (1→4)-β-D-Glc (1→]→3)-β-D-Glc (1→[4)-β-D-Man (1→4)-β-D-Glc (l→]n-β-D-Glc 2 2 OAc OAc其中m和n代表不同的重復(fù)單元數(shù)。(3)通過粘度測定發(fā)現(xiàn)BMPS在純水中顯示典型的聚電解質(zhì)行為,加入NaNO3能有效抑制其聚電解質(zhì)效應(yīng),同時通過經(jīng)驗(yàn)B值法(B = 0.018)推斷BMPS在溶液中以半剛性鏈存在。通過超聲降解得到不同分子量的BMPS級分,利用粘度法和光散射法測得各級分在0.1mol/LNaNO3溶液中的Mw、數(shù)均分子量Mn、均方根旋轉(zhuǎn)半徑s2z1/2、第二維利系數(shù)A2、流體力學(xué)半徑Rh以及特性粘數(shù)[η]等參數(shù)。BMPS所有級分的A2均為正值,說明0.1 mol/LNaNO3為其良溶劑,在該體系中所有級分均無聚集產(chǎn)生,適合進(jìn)行溶液性質(zhì)分析。通過[η]和Mw的指數(shù)關(guān)系(α= 0.82)、[η]和ss2z1/2的指數(shù)關(guān)系(α' = 0.64)、s2z1/2與Rh的比值(ρ= 1.82)得出分子量范圍為37.4×104-267×104Da的BMPS在0.1 mol/L NaN03溶液中以半剛性鏈構(gòu)象存在。通過無擾蠕蟲狀圓筒模型進(jìn)行鏈構(gòu)象參數(shù)計算,得到單位圍長摩爾質(zhì)量ML、持續(xù)長度q以及分子鏈直徑d分別為663.5 nm-1、9.5 nm、0.6 nm。通過AFM對其形貌進(jìn)行表征,發(fā)現(xiàn)分子鏈以直鏈或無規(guī)彎曲的形態(tài)存在,測得平均鏈直徑為0.68 nm,與理論模型計算結(jié)果相符合。(4)通過對BMPS的穩(wěn)態(tài)流變行為研究發(fā)現(xiàn)BMPS溶液為典型的假塑性流體,即使在較低的濃度下(0.01%)也具有剪切變稀的行為,且其剪切變稀行為具有濃度依賴性。通過Williamson模型獲得BMPS在濃度區(qū)間為0.0101%-1.2%的零剪切粘度,由零剪切粘度與濃度的關(guān)系得到BMPS的臨界交疊濃度為0.6%,說明BMPS分子鏈比一般半剛性鏈高分子更易發(fā)生纏結(jié)。通過動態(tài)流變研究發(fā)現(xiàn),BMPS溶液在低頻區(qū)以粘性為主的粘彈性體存在,而在高頻區(qū)隨著振蕩頻率的增大則會形成瞬態(tài)的網(wǎng)絡(luò)結(jié)構(gòu),且隨著濃度的增大,其形成瞬態(tài)網(wǎng)絡(luò)結(jié)構(gòu)的能力越強(qiáng)。(5)通過考察聚乙烯醇(polyvinyl alcohol,PVA)濃度和紡絲電壓對PVA納米纖維的影響,選擇8%的PVA溶液作為助紡劑,紡絲電壓為14 kV;將BMPS與PVA以質(zhì)量比為3/100、5/100、10/100、20/100比例制成混合溶液進(jìn)行靜電紡絲。隨著BMPS比例的增大,混合溶液的電導(dǎo)率和粘度相應(yīng)增大,納米纖維的直徑逐漸變小,紡絲過程逐漸困難,由紅外圖譜可以發(fā)現(xiàn)BMPS和PVA之間形成了微弱的氫鍵。將不同比例的BMPS/PVA混紡膜通過戊二醛交聯(lián)10min,能夠改變其水溶性同時保持纖維形態(tài)。通過細(xì)胞相容性實(shí)驗(yàn)發(fā)現(xiàn)PVA纖維膜和BMPS/PVA混紡膜均能促進(jìn)NIH3T3細(xì)胞的增殖,且相比較PVA纖維膜,BMPS/PVA混紡膜更適合細(xì)胞的生長與黏附,因此通過BMPS與PVA混紡制得的納米纖維膜有望成為一種新型傷口修復(fù)材料。
[Abstract]:Bacterial extracellular polysaccharide is a kind of biological macromolecule with multiple structure and unique properties. Because of its excellent physical and chemical properties and biological activity, it has been widely used in the fields of food, medicine, chemical industry and so on. Although there are many kinds of bacterial extracellular polysaccharide found at present, the real application value and industrialization production are very few. The production of polysaccharides, the cost of production and the lack of research on structure-activity relationship are the main factors that restrict the production and application of polysaccharides. Therefore, it is a hot spot to find a new high yield polysaccharide with unique properties. Bacillus mucilaginosus is a special kind of silicate bacteria in the soil, which can make large use of limited resources. At present, the research on B.mucilaginosus mainly focuses on its potassium solution and nitrogen fixing function and sewage treatment. B.mucilaginosus can degrade minerals (silicate, apatite, phosphonite) in the soil and convert it into nutrient substance for plant absorption; and it can also be used as flocculant and adsorbent to remove sewage. In addition, the study found that B.mucilaginosus extracellular polysaccharide also has a certain biological activity, which can promote the repair of gastric ulcer in mice and the development of the immune organs of Yellow Broilers in mice. However, the B.mucilaginosus extracellular polysaccharide group (B.mucilaginosu) The study of this information is very small, its chemical structure is not well known, and its application development is greatly limited. Therefore, the basic research on the chemical structure and physicochemical properties of B.mucilaginosus extracellular polysaccharide will help to understand its structure-activity relationship and broaden its potential application field. This research is carried out through the B. mucilaginosus SM-01 fermentation broth. The acid heteropoly BMPS was extracted and optimized. The primary structure and advanced structure of BMPS were studied by various characterization methods. It provided a theoretical basis for the development of B.mucilaginosus extracellular polysaccharide. At the same time, based on biological activity, it was discussed as a wound repair material, which widened the application of B.mucilaginosus extracellular polysaccharide. The main results are as follows: (1) by investigating the influence of dilution, heating up, changing pH, adding salt content to the viscosity of the fermentation broth, selecting the method to reduce the viscosity of the fermentation liquid with the synergistic use of dilution and salt, and adding 3% (w/v) NaCl in the fermentation liquid to compare the effect of centrifugation and diatomite filtration on the deproteinizing and deproteinizing of the fermentation liquid under different dilution times. As well as the effect on the filtration flux, it was found that the effect of the filtration and removal of the bacteria and the heteroprotein was much higher than that of the centrifuge. In the case of the dilution multiple of 3 times, the extraction filtration rate was better, the rate of bacteria removal was 92.6%, the protein rate was 69.7%. and the diatomite was used as the adsorbent, and the effects of different addition amounts on the removal of bacteria, protein and polysaccharide recovery were compared. After 10 g/L diatomite was added, it was repeatedly pumped three times, and the protein content was close to zero, and the recovery rate of polysaccharide was 76.4%. to investigate the effect of ultrafiltration membrane with different interception molecular weight and the effect of operating pressure on ultrafiltration concentration. It was found that ultrafiltration with 200 kDa intercepting molecular weight could be basically intercepted under the operating pressure of 0.1 MPa. The total total recovery rate of polysaccharide was 73.2% and the content of endotoxin was less than 0.25 EU/mL. The extraction process was simple and suitable for industrial production by ultrafiltration concentration and desalination. The extraction process was simple and suitable for industrial production. The purified polysaccharide was purified by ion exchange chromatography, and then the purity identification and molecular measurement were used by volume exclusion chromatography and light scattering. It is found that B.mucilaginosus SM-01 extracellular polysaccharide is a homogeneous acid polysaccharide named BMPS, and its weight average molecular weight Mw is 2.67 x 106Da. (2). By chemical composition analysis of BMPS, it is found that BMPS does not contain phosphoric acid group and sulphuric acid group, glucuronic acid content is 14.3%, O- acetyl content is 4.99%. through monosaccharide analysis to become grapes. Carbohydrate (Glc), mannose (Man), glucuronic acid (GlcA) and an unknown glucuronic acid (AM), the mole ratio is 3.2: 2: 0.5: 0.3., and a neutral oligosaccharide fragment BMPS-H is obtained by ultrasonic degradation and mild acid hydrolysis of BMPS, and a Mw 1771.43 Da is obtained by the matrix assisted laser desorption flight time mass spectrometry, and the monosaccharide composition is the molar ratio. A linear structure of BMPS-H molecules was found by methylation analysis for 1.5: 1.. The main chain was composed of 1,4-Man and 1,4-Glc and a small amount of 1,3-Glc. The connection sequence of glycosyl groups and the junction sites of acetyl groups were obtained by nuclear magnetic resonance. The chemical structure of BMPS-H was deduced to be: beta -D-Glc (1 to [4) - beta -D-Man (1 to 4) - beta -D-Glc (1 to] 3) - beta -D-Glc ( 1 - [4) - beta -D-Man (1 to 4) - beta -D-Glc (L to]n- beta -D-Glc 22 OAc OAc in which m and N represent the number of different repeating units. (3) it is found that BMPS in pure water shows typical polyelectrolyte behavior through viscosity measurement, and the addition of NaNO3 can effectively inhibit its polyelectrolyte effect, and the empirical values method (0.018) is used to deduce the semi rigidity of the solution in the solution. The BMPS grade of different molecular weights is obtained by ultrasonic degradation. The Mw of different levels in 0.1mol/LNaNO3 solution is measured by viscosity method and light scattering method. The number of molecular weights Mn, the mean square root rotation radius s2z1/2, the second VL coefficient A2, the Rh of the hydrodynamic radius Rh and the characteristic viscosity number [ETA] are all positive, indicating that 0 .1 mol/LNaNO3 is a good solvent in which all the fractions of the system are not aggregated and are suitable for the analysis of the properties of the solution. Through the exponential relationship between [ETA] and Mw (alpha = 0.82), the exponential relationship between [ETA] and ss2z1/2 (alpha '= 0.64), the ratio of s2z1/2 to Rh (P = 1.82), the molecular weight range of 37.4 * 104-267 * 104Da BMPS in 0.1 mol/L NaN03 solution The existence of a semi rigid chain conformation. Through the calculation of the chain conformation parameters by the unperturbed vermicular cylinder model, the unit length mole mass ML, the length Q and the molecular chain diameter D are 663.5 nm-1,9.5 nm respectively, and the 0.6 nm. is characterized by AFM, and the molecular chain is found to be in the form of straight chain or random bending, and the average chain straight is measured. The diameter is 0.68 nm, which is in accordance with the theoretical model calculation results. (4) through the study of the steady rheological behavior of BMPS, it is found that BMPS solution is a typical pseudoplastic fluid, even at lower concentration (0.01%) has shear thinning behavior, and its shear thinning behavior is concentration dependent. The concentration range of BMPS is obtained by Williamson model. The zero shear viscosity of 0.0101%-1.2% is derived from the relationship between the zero shear viscosity and the concentration of the BMPS to get the critical overlapping concentration of 0.6%, which indicates that the BMPS molecular chain is more easily entangled than the general semi rigid chain polymer. Through the dynamic rheological study, it is found that the BMPS solution is viscous mainly in the low frequency region, while the frequency is oscillating in the high frequency region. A transient network structure is formed by increasing the concentration, and with the increase of concentration, the stronger the ability to form a transient network structure. (5) by examining the effect of the concentration of polyvinyl alcohol (PVA) and the spinning voltage on the PVA nanofibers, the PVA solution of 8% is selected as a spinning aid, the spinning voltage is 14 kV, and the mass ratio of BMPS to PVA is 3/10 to 3/10. With the increase of the proportion of 0,5/100,10/100,20/100, the electrical conductivity and viscosity of the mixed solution increase, the diameter of the nanofibers gradually becomes smaller and the spinning process is gradually difficult. The weak hydrogen bonds between BMPS and PVA can be found from the infrared atlas. The BMPS/PVA blend membrane of different proportions can be found. Through crosslinking 10min with glutaraldehyde, it can change its water solubility and maintain fiber morphology. Through the cell compatibility test, it is found that both the PVA fiber membrane and the BMPS/PVA blend membrane can promote the proliferation of NIH3T3 cells, and the PVA fiber membrane and the BMPS/PVA blend membrane are more suitable for the cell growth and adhesion. Therefore, the nano fiber produced by the blending of BMPS and PVA is made. The membrane is expected to be a new type of wound repair material.

【學(xué)位授予單位】：江南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：Q936

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