本文選題：野油菜黃單胞菌 + 馴化�。� 參考：《江南大學(xué)》2017年博士論文

【摘要】：黃原膠的安全性、穩(wěn)定性、懸浮性、乳化性、假塑性和增稠性使其作為一種“工業(yè)味精”被應(yīng)用于食品、醫(yī)藥、紡織、農(nóng)業(yè)和石油開采等眾多領(lǐng)域。黃原膠生產(chǎn)以玉米淀粉為主要原料,隨著全球人口不斷增加和世界范圍內(nèi)糧食短缺,國內(nèi)外許多學(xué)者都在研究采用工農(nóng)業(yè)產(chǎn)品副產(chǎn)物來代替玉米淀粉實現(xiàn)黃原膠生產(chǎn)。甘油是生物柴油酯交換生產(chǎn)過程中不可避免的一種副產(chǎn)物,隨著生物柴油產(chǎn)業(yè)的發(fā)展而產(chǎn)量巨大。微生物轉(zhuǎn)化法條件溫和、簡單、易操作等特點,使甘油在發(fā)酵領(lǐng)域的應(yīng)用受到廣泛關(guān)注。如果甘油可以被用于黃原膠生產(chǎn),將為緩解全球糧食危機做出巨大貢獻。本文以Xanthomonas campestris NRRL B-1459為出發(fā)菌株,經(jīng)馴化得到了一株可以利用甘油發(fā)酵生產(chǎn)黃原膠的馴化株X.campestris CCTCC M2015714,且首次從基因水平對野油菜黃單胞菌中與黃原膠合成相關(guān)的甘油代謝基因進行了研究。經(jīng)繼續(xù)馴化,馴化株對甘油耐受能力提高到了100 g·L-1,并采用多階段控制流加甘油發(fā)酵策略,使黃原膠產(chǎn)量(33.9 g·L-1)和發(fā)酵周期(60 h)與當(dāng)前以淀粉為原料黃原膠工業(yè)生產(chǎn)水平相當(dāng)。同時,對馴化株以甘油為底物發(fā)酵得到黃原膠的分子特性、結(jié)構(gòu)特征、流變學(xué)特性和潛在應(yīng)用進行了研究。主要研究結(jié)果如下:(1)以X.campestris NRRL B-1459為出發(fā)菌,經(jīng)馴化得到了一株可以利用甘油發(fā)酵生產(chǎn)黃原膠的優(yōu)良菌株X.campestris CCTCC M2015714。采用RT-PCR對馴化株和原始菌中甘油代謝相關(guān)基因研究發(fā)現(xiàn):原始菌中甘油代謝相關(guān)基因(glp F、glp K、glp D和fbp)相對轉(zhuǎn)錄水平均為1.0,而馴化株中相關(guān)基因相對轉(zhuǎn)錄水平均高于1.0,依次為:glp D(4.76)glp F(3.36)glp K(3.05)fbp(2.53),說明甘油代謝相關(guān)基因的增強表達是馴化株能夠利用甘油生長并合成黃原膠的可能原因。通過在培養(yǎng)基中添加5 g·L-1蔗糖或葡萄糖做啟動物質(zhì),X.campestris CCTCC M2015714菌體生長時間從36 h縮短至24 h,黃原膠產(chǎn)量從11.0 g·L-1增加到12.5 g·L-1。此外,實驗所用粗甘油中鈉鹽、甲醇、灰分等雜質(zhì)對X.campestris CCTCC M2015714生產(chǎn)性能基本無影響。(2)經(jīng)繼續(xù)馴化,X.campestris CCTCC M2015714對甘油耐受能力提高到了100g·L-1,且其體內(nèi)甘油代謝相關(guān)基因的表達進一步增強,依次為glp D(8.56)glp F(7.73)glp K(6.48)fbp(5.31)。采用多階段控制流加甘油發(fā)酵策略:低的初始甘油濃度(40g·L-1)、變攪拌轉(zhuǎn)速和變通氣量(0~24 h,0.5 vvm和200 rpm;24~60 h,1.0 vvm和400 rpm)以及變速流加甘油(24~34 h,3 g·L-1·h-1;34~44 h,2 g·L-1·h-1;44~54 h,1g·L-1·h-1),不僅解除了底物濃度對馴化株生長抑制,還維持了黃原膠合成過程中高C/N,使黃原膠產(chǎn)量達到33.9 g·L-1,發(fā)酵周期縮短為60 h,這一生產(chǎn)能力與當(dāng)前以淀粉為原料黃原膠工業(yè)生產(chǎn)水平相當(dāng),且33.9 g·L-1是目前報道以甘油為底物發(fā)酵生產(chǎn)黃原膠的最高產(chǎn)量。(3)X.campestris CCTCC M2015714以甘油為底物發(fā)酵得到的胞外多糖中只含有葡萄糖、甘露糖和葡萄糖醛酸,且三種單糖摩爾比為2.0:1.65:1.0,這一比例與商品級黃原膠(2.0:1.85:1.0)十分接近。此外,甘油產(chǎn)胞外多糖的紅外光譜和核磁共振圖譜與商品級黃原膠相吻合。上述結(jié)果說明馴化株以甘油為底物發(fā)酵得到的胞外多糖是黃原膠。新型黃原膠分子量(3.0×106 Da)是商品級黃原膠(6.4×106 Da)一半左右,1.0%(w/v)新型黃原膠溶液稠度系數(shù)(1.7958)不足商品級黃原膠(21.0842)十分之一,但其流態(tài)特性指數(shù)(0.235)小于1.0,說明其仍是假塑性流體。原子力顯微鏡結(jié)果顯示:新型黃原膠在水中形成不連續(xù)、間斷結(jié)構(gòu),而商品級黃原膠在水中形成蜂窩形網(wǎng)狀結(jié)構(gòu)。同時,掃描電子顯微鏡和差示量熱掃描儀結(jié)果顯示:新型黃原膠空間結(jié)構(gòu)纖細且松散,而商品級黃原膠空間結(jié)構(gòu)致密、桿狀。(4)新型黃原膠的低粘度可以提高發(fā)酵液中色素、菌體細胞和不溶性雜質(zhì)等去除率而提高透明性,新型黃原膠透光率達到95%,商品級黃原膠透光率為80%左右;新型黃原膠的低粘度和低分子量可以加速水分子與其結(jié)合速度,使其水化速率快于商品級黃原膠;新型黃原膠與商品級黃原膠溶液粘度隨膠濃度增加而增大,且兩者均對p H、溫度和鹽穩(wěn)定;當(dāng)鹽濃度低于0.5 g·L-1時,新型黃原膠的低粘度和低分子量使鹽離子可以通過中和黃原膠側(cè)鏈上羧基所帶負電荷,減小黃原膠分子間靜電排斥作用而增大溶液粘度,且二價鹽離子還可在黃原膠分子間形成“鹽橋”而使溶液粘度增大效果強于一價鹽離子;新型黃原膠的丙酮酸含量(5.2%)高于商品級黃原膠(4.1%),這使新型黃原膠在-20℃反復(fù)凍融處理過程中分子間交聯(lián)作用增強而增大溶液粘度,且凍融處理3次之后溶液粘度趨于穩(wěn)定。(5)新型黃原膠的低粘度可以增加在食品中添加量,使其成為一種具有潛在作為膳食纖維功能可能性的微生物多糖。對新型黃原膠潛在應(yīng)用研究發(fā)現(xiàn):新型黃原膠對不飽和脂肪和飽和脂肪吸附量為2.15±0.26 g·g-1和2.08±0.21 g·g-1;陽離子交換能力1.15±0.08 mmol·g-1;在1 h時間內(nèi)對Cu、Cd和Pb三種重金屬離子吸附去除率均超過50%,4 h時吸附去除效果達到75%;對膽固醇最大吸附量為12.36 mg·g-1(p H 2.0)和11.72 mg·g-1(p H 7.0);對膽酸鈉吸附量與膽酸鈉濃度之間存在一種動態(tài)平衡;對亞硝酸根離子吸附去除率分別為80%(p H 2.0)和60%(p H 7.0);同時,可以有效延緩葡萄糖在水中運行速率和淀粉水解速率,并維持淀粉溶液粘度;最后,新型黃原膠與可溶性低聚果糖和水不溶性大豆拉絲蛋白均可良好復(fù)配。
[Abstract]:The safety, stability, suspension, emulsification, pseudoplasticity and thickening of xanthan gum make it used as a kind of "industrial monosodium" in many fields, such as food, medicine, textile, agriculture and petroleum exploitation. The main raw material of xanthan gum is corn starch, with the increasing global population and the food shortage worldwide, it is permitted at home and abroad. Many scholars are studying the production of xanthan gum by using the by-products of industrial and agricultural products instead of corn starch. Glycerol is an inevitable by-product in the process of biodiesel oil transesterification. With the development of biodiesel industry, the production is huge. The conditions of microbial transformation are mild, simple and easy to operate, so that glycerol is used in the fermentation collar. The application of domain is widely concerned. If glycerol can be used in xanthan gum production, it will make a great contribution to mitigate the global food crisis. This paper, taking Xanthomonas campestris NRRL B-1459 as the starting strain, has been domesticated to produce a domesticated strain of X.campestris CCTCC M2015714, which can be fermented by glycerol to produce xanthan, and for the first time The gene level was studied in the glycerol metabolism gene related to xanthan gum in Xanthomonas campestris. After continuing domestication, the tolerance of glycerol was increased to 100 g. L-1, and the multi stage control flow and glycerol fermentation strategy were adopted to make the xanthan gum yield (33.9 G. L-1) and fermentation period (60 h) with the starch as raw material. The industrial production level of xanthan gum is equal. At the same time, the molecular properties, structural characteristics, rheological properties and potential applications of the tamed plants with glycerol as substrates are studied. The main results are as follows: (1) a strain of xanthan gum can be produced by taming X.campestris NRRL B-1459 by acclimation. The good strain X.campestris CCTCC M2015714. used RT-PCR to study the glycerol metabolism related genes in the acclimated and primitive bacteria: the relative transcriptional level of glycerol metabolism related genes (GLP F, GLP K, GLP D and FBP) in the primitive bacteria were 1, while the relative transcriptional level of the related genes in the domesticated strain was higher than that of 1, and the sequence of the relative genes in the domesticated strain was: GLP (4.76) (3.36) LP K (3.05) FBP (2.53) indicates that the enhanced expression of glycerol metabolism related genes is a possible reason for the growth and synthesis of xanthan gum by the acclimated strain of glycerol. By adding 5 g. L-1 sucrose or glucose as the starting substance in the medium, the growth time of X.campestris CCTCC M2015714 bacteria is shortened from 36 h to 24 h, and the xanthan gum yield is from 11 g L-1. In addition to 12.5 g. L-1., the sodium salt, methanol, ash and other impurities in the crude glycerol had no effect on the production performance of X.campestris CCTCC M2015714. (2) after continued acclimatization, the tolerance of X.campestris CCTCC M2015714 to glycerol tolerance was increased to 100g L-1, and the expression of glycerol metabolism related genes in the body was further enhanced, followed by G. LP D (8.56) GLP F (7.73) GLP K (6.48) FBP (5.31). Using a multi stage control flow plus glycerol fermentation strategy: low initial glycerol concentration (40g. L-1), variable stirring speed and variable gas volume (0~24 h, 0.5 VVM and 200), and variable speed flow plus glycerin. The growth inhibition of the acclimated strain was only lifted, and the high C/N was maintained during the synthesis of xanthan gum. The production of Huang Yuan gum was 33.9 G. L-1 and the fermentation period was shortened to 60 h. The production capacity was equivalent to the industrial level of xanthan gum with starch as raw material, and 33.9 G. L-1 was reported to produce xanthan with glycerol as substrate at present. The highest yield of glue. (3) X.campestris CCTCC M2015714 contains only glucose, mannose and glucuronic acid, and three monosaccharide mole ratios are 2.0:1.65:1.0, and the proportion of the polysaccharide is very close to the commodity grade xanthan gum (2.0:1.85:1.0). The results show that the extracellular polysaccharide produced by glycerol as substrate is xanthan gum. The molecular weight of the new xanthan gum (3 x 106 Da) is about half of commercial xanthan gum (6.4 x 106 Da), and 1% (w/v) new xanthan gum solution consistency coefficient (1.7958) is less than commodity grade xanthan gum (21.0842). One of them, but its flow characteristic index (0.235) is less than 1, indicating that it is still a pseudoplastic fluid. The results of atomic force microscopy show that the new xanthan gum is discontinuous and discontinuous in water, and the product grade xanthan gum forms a cellular network in water. At the same time, the results of the scanning electric microscope and differential thermal scanner show that the new xanthan is a new xanthan. The space structure of the adhesive is thin and loose, but the spatial structure of the commercial xanthan gum is compact and rod like. (4) the low viscosity of the new xanthan gum can improve the transparency of the pigment, the cell and insoluble impurities in the fermentation broth, the transmittance of the new xanthan gum is 95%, the light transmittance of the product grade xanthan gum is about 80%, and the low viscosity of the new xanthan gum is low. The degree and low molecular weight can accelerate the water molecules and their bonding speed, making the hydration rate faster than the commercial xanthan gum. The viscosity of the new xanthan gum and the commercial xanthan gum is increased with the adhesive concentration, both of which are stable to P H, temperature and salt. When the salt concentration is lower than 0.5 g. L-1, the low viscosity and low molecular weight of the new xanthan gum make the salt away from the salt. By neutralizing the negative charge of the carboxyl group on the side chain of the xanthan gum, it can reduce the electrostatic repulsion between the xanthan molecules and increase the viscosity of the solution, and the two valence salt ions can also form a "salt bridge" between the xanthan molecules and make the solution viscosity more effective than the monovalent ion; the content of pyruvic acid (5.2%) of the new xanthan gum is higher than that of the commercial yellow. The original glue (4.1%) makes the intermolecular crosslinking effect of the new xanthan gum in the process of repeated freezing and thawing at -20 C and increase the viscosity of the solution. And after 3 times freeze-thaw treatment, the viscosity of the solution tends to be stable. (5) the low viscosity of the new xanthan gum can be added to the food, making it a potential functional possibility of dietary fiber. The potential application of microbial polysaccharides. The potential application of the new xanthan gum found that the adsorption of unsaturated fats and unsaturated fats was 2.15 + 0.26 G. G-1 and 2.08 + 0.21 G. G-1, and the cation exchange capacity was 1.15 + 0.08 mmol. G-1. The adsorption removal efficiency of Cu, Cd and Pb three heavy metals were more than 50% and 4 h adsorption removal efficiency in 1 h time. The maximum amount of fruit reached to 75%; the maximum adsorption amount of cholesterol was 12.36 mg. G-1 (P H 2) and 11.72 mg. G-1 (P H 7). There was a dynamic balance between the adsorption capacity of sodium cholate and the concentration of sodium cholate; the removal rate of nitrite ion adsorption was 80% (P H 2) and 60% (P H 7), respectively. At the same time, it could effectively delay the operation rate and starch of glucose in water. Finally, the new xanthan gum and soluble fructo oligosaccharide and water insoluble soybean protein can be well mixed.

【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】：TQ920.6

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