以甘油为底物发酵生产黄原胶及其特性和应用研究

发布时间：2018-05-12 16:32

本文选题：野油菜黄单胞菌 + 驯化　；参考：《江南大学》2017年博士论文

【摘要】：黄原胶的安全性、稳定性、悬浮性、乳化性、假塑性和增稠性使其作为一种“工业味精”被应用于食品、医药、纺织、农业和石油开采等众多领域。黄原胶生产以玉米淀粉为主要原料,随着全球人口不断增加和世界范围内粮食短缺,国内外许多学者都在研究采用工农业产品副产物来代替玉米淀粉实现黄原胶生产。甘油是生物柴油酯交换生产过程中不可避免的一种副产物,随着生物柴油产业的发展而产量巨大。微生物转化法条件温和、简单、易操作等特点,使甘油在发酵领域的应用受到广泛关注。如果甘油可以被用于黄原胶生产,将为缓解全球粮食危机做出巨大贡献。本文以Xanthomonas campestris NRRL B-1459为出发菌株,经驯化得到了一株可以利用甘油发酵生产黄原胶的驯化株X.campestris CCTCC M2015714,且首次从基因水平对野油菜黄单胞菌中与黄原胶合成相关的甘油代谢基因进行了研究。经继续驯化,驯化株对甘油耐受能力提高到了100 g·L-1,并采用多阶段控制流加甘油发酵策略,使黄原胶产量(33.9 g·L-1)和发酵周期(60 h)与当前以淀粉为原料黄原胶工业生产水平相当。同时,对驯化株以甘油为底物发酵得到黄原胶的分子特性、结构特征、流变学特性和潜在应用进行了研究。主要研究结果如下:(1)以X.campestris NRRL B-1459为出发菌,经驯化得到了一株可以利用甘油发酵生产黄原胶的优良菌株X.campestris CCTCC M2015714。采用RT-PCR对驯化株和原始菌中甘油代谢相关基因研究发现:原始菌中甘油代谢相关基因(glp F、glp K、glp D和fbp)相对转录水平均为1.0,而驯化株中相关基因相对转录水平均高于1.0,依次为:glp D(4.76)glp F(3.36)glp K(3.05)fbp(2.53),说明甘油代谢相关基因的增强表达是驯化株能够利用甘油生长并合成黄原胶的可能原因。通过在培养基中添加5 g·L-1蔗糖或葡萄糖做启动物质,X.campestris CCTCC M2015714菌体生长时间从36 h缩短至24 h,黄原胶产量从11.0 g·L-1增加到12.5 g·L-1。此外,实验所用粗甘油中钠盐、甲醇、灰分等杂质对X.campestris CCTCC M2015714生产性能基本无影响。(2)经继续驯化,X.campestris CCTCC M2015714对甘油耐受能力提高到了100g·L-1,且其体内甘油代谢相关基因的表达进一步增强,依次为glp D(8.56)glp F(7.73)glp K(6.48)fbp(5.31)。采用多阶段控制流加甘油发酵策略:低的初始甘油浓度(40g·L-1)、变搅拌转速和变通气量(0~24 h,0.5 vvm和200 rpm;24~60 h,1.0 vvm和400 rpm)以及变速流加甘油(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),不仅解除了底物浓度对驯化株生长抑制,还维持了黄原胶合成过程中高C/N,使黄原胶产量达到33.9 g·L-1,发酵周期缩短为60 h,这一生产能力与当前以淀粉为原料黄原胶工业生产水平相当,且33.9 g·L-1是目前报道以甘油为底物发酵生产黄原胶的最高产量。(3)X.campestris CCTCC M2015714以甘油为底物发酵得到的胞外多糖中只含有葡萄糖、甘露糖和葡萄糖醛酸,且三种单糖摩尔比为2.0:1.65:1.0,这一比例与商品级黄原胶(2.0:1.85:1.0)十分接近。此外,甘油产胞外多糖的红外光谱和核磁共振图谱与商品级黄原胶相吻合。上述结果说明驯化株以甘油为底物发酵得到的胞外多糖是黄原胶。新型黄原胶分子量(3.0×106 Da)是商品级黄原胶(6.4×106 Da)一半左右,1.0%(w/v)新型黄原胶溶液稠度系数(1.7958)不足商品级黄原胶(21.0842)十分之一,但其流态特性指数(0.235)小于1.0,说明其仍是假塑性流体。原子力显微镜结果显示:新型黄原胶在水中形成不连续、间断结构,而商品级黄原胶在水中形成蜂窝形网状结构。同时,扫描电子显微镜和差示量热扫描仪结果显示:新型黄原胶空间结构纤细且松散,而商品级黄原胶空间结构致密、杆状。(4)新型黄原胶的低粘度可以提高发酵液中色素、菌体细胞和不溶性杂质等去除率而提高透明性,新型黄原胶透光率达到95%,商品级黄原胶透光率为80%左右;新型黄原胶的低粘度和低分子量可以加速水分子与其结合速度,使其水化速率快于商品级黄原胶;新型黄原胶与商品级黄原胶溶液粘度随胶浓度增加而增大,且两者均对p H、温度和盐稳定;当盐浓度低于0.5 g·L-1时,新型黄原胶的低粘度和低分子量使盐离子可以通过中和黄原胶侧链上羧基所带负电荷,减小黄原胶分子间静电排斥作用而增大溶液粘度,且二价盐离子还可在黄原胶分子间形成“盐桥”而使溶液粘度增大效果强于一价盐离子;新型黄原胶的丙酮酸含量(5.2%)高于商品级黄原胶(4.1%),这使新型黄原胶在-20℃反复冻融处理过程中分子间交联作用增强而增大溶液粘度,且冻融处理3次之后溶液粘度趋于稳定。(5)新型黄原胶的低粘度可以增加在食品中添加量,使其成为一种具有潜在作为膳食纤维功能可能性的微生物多糖。对新型黄原胶潜在应用研究发现:新型黄原胶对不饱和脂肪和饱和脂肪吸附量为2.15±0.26 g·g-1和2.08±0.21 g·g-1;阳离子交换能力1.15±0.08 mmol·g-1;在1 h时间内对Cu、Cd和Pb三种重金属离子吸附去除率均超过50%,4 h时吸附去除效果达到75%;对胆固醇最大吸附量为12.36 mg·g-1(p H 2.0)和11.72 mg·g-1(p H 7.0);对胆酸钠吸附量与胆酸钠浓度之间存在一种动态平衡;对亚硝酸根离子吸附去除率分别为80%(p H 2.0)和60%(p H 7.0);同时,可以有效延缓葡萄糖在水中运行速率和淀粉水解速率,并维持淀粉溶液粘度;最后,新型黄原胶与可溶性低聚果糖和水不溶性大豆拉丝蛋白均可良好复配。
[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.

【学位授予单位】：江南大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TQ920.6

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