【作者】 李妍；

【导师】 张和平；

【作者基本信息】 内蒙古农业大学 ， 畜产品安全生产， 2008， 博士

【摘要】 Lactobacillus casei Zhang是从中国内蒙古地区传统酸马奶中分离的一株具有良好益生特性的新生益生菌。因为益生菌产品中高浓度的活性菌体细胞是其发挥功效的必要条件,所以本研究针对L. casei Zhang的高密度培养技术展开研究,同时采用实时定量PCR技术从转录水平上研究了发酵过程中不同培养条件和不同生长阶段对菌体的影响。研究了不同碳源、氮源、碳氮比例、微量元素及缓冲盐对L. casei Zhang增殖培养的效果,并采用响应面法对优选的碳源、氮源和缓冲盐类的组成含量进行优化,得到L. casei Zhang的增殖培养基为:葡萄糖20.9g/L、大豆蛋白胨10.4g/L、酵母粉10.4 g/L、K2HPO43.5 g/L、醋酸钠14.6 g/L、柠檬酸钠2.3 g/L、MgSO4·7H2O 1 g/L、MnSO4·5H2O 54mg/L、CuSO4·5H2O 10mg/L、吐温80 1g/L。通过对温度,初始pH、摇床转速等条件的研究得到菌体在此培养基中适宜的试管培养条件为:初始pH6.5,37℃,保温静止发酵。经18～20h培养后,L. casei Zhang活菌数可达到4.78×109 CFU/mL,比在MRS中(4.8×108 CFU/mL)提高近10倍。在优化增殖培养基的基础上,研究了不同中和剂、缓冲盐浓度、葡萄糖浓度、pH值控制、通气条件和接种量对菌体在恒pH条件下发酵的影响情况,根据不同条件下菌体的比生长速率、菌体密度和活菌数情况,确定L. casei Zhang较适宜的高密度培养条件为:培养基葡萄糖浓度为80 g/L,以氨水为中和剂使pH保持5.9,采用间歇通氮气的方法保持环境厌氧,接种量7%以分批培养方式下37℃保温发酵,10～12h后,L. casei Zhang细胞干重达到7.45 g/L,活菌数3.68×1010 CFU/mL,较优化前提高7倍以上。分别在不同pH和不同乳酸浓度条件下接种L. casei Zhang进行培养结果表明乳酸和低pH都抑制菌体生长并且两者有协同作用。不控制pH条件下,乳酸浓度20 g/L(pH4.0)就足以抑止菌体的生长,如果添加中和剂保持pH6则乳酸达到120 g/L才完全抑止菌体的生长根据发酵过程中菌体生长和代谢曲线变化规律,采用Origin7.5软件非线性拟合建立L. casei Zhang的生长、葡萄糖代谢和产乳酸的动力学模型,模型与试验值拟合良好,平均误差小于10%,能够真实地反应发酵过程。分别以100 mL/h和200 mL/L补料速度流加新鲜培养基研究了补料分批培养,结果表明100 mL/h补料分批培养与分批培养结果无差异,200 mL/h补料在发酵8h时出现稀释效应。在L. casei Zhang高密度培养小试基础上,进行50 L(有效工作容积30 L)到200 L(有效工作容积150 L)逐级放大中试生产工艺验证。200 L规模发酵罐发酵菌体密度可达2.9×1010 CFU/mL,与小试水平无差异。证明本研究所得到的L. casei zhang高密度培养技术切实可行,能够实现工业化生产。初步探讨发酵后菌体的离心和冷冻干燥过程对菌体的影响,虽然发酵液经离心收集菌体并冷冻干燥可得到平均活菌数2.65×1011 CFU/g的菌粉,能够满足益生菌制剂和发酵剂对高活菌数的要求,但冻干前后活菌得率仅49.97%。有必要针对L. casei Zhang的冻干保护剂和冻干工艺进一步优化,以提高菌体存活率得到更高菌体浓度的益生菌粉。研究了不同培养条件下,gapd、gyrB、ldh、16s rRNA和recA 5个常用管家基因的表达情况,并应用GeNorm软件分析了管家基因的稳定性,结果表明5个管家基因在本试验所研究条件下表现都比较稳定,尤以gapd和gyrB最稳定,可以作为实时定量PCR的内参。采用荧光实时定量PCR技术研究了pH、乳酸浓度、通气条件和生长阶段的变化对L. casei Zhang一些关键酶基因(涉及葡萄糖转运、糖酵解和分子伴侣蛋白等方面的13个基因)表达变化的影响,结果表明不同培养条件对不同基因表达的影响程度不一,从转录水平描述了各种不同的发酵条件对菌体生长代谢的影响,为后续深入研究该菌生长代谢的分子机制奠定基础。更多还原

【Abstract】 Lactobacillus casei Zhang is a new probiotic strain isolated from home-made koumiss in Inner Mongolia, China. High number of viable cells is a prerequisite for the effectiveness of probiotics. Therefore, we studied the high cell density culture (HCDC) of L. casei Zhang in this paper in order to use it for probiotic products. At the same time, we also studied the influences of cultural conditions and growth phases on this strain at genetic level.The effects of various carbon source, nitrogen source, microelement and buffer salts on the growth of L. casei Zhang were studied. Then the composition of enrichment medium for L. casei Zhang was optimized by using response surface analysis, as follows: glucose 20.9 g/L, soy peptone10.4 g/L, yeast extract 10.4g/L, K2HPO4 3.5 g/L, sodium acetate 14.6g/L, citrate sodium 2.35 g/L, MgSO4.7H2O 1.0 g/L, MnSO4.5H2O 54 mg/L, CuSO4.5H2O 10 mg/L, tween 80 1.0 g/L. By investigating the effects of start pH, temperature and shaking speed on the strain, the suitable test-tube cultural conditions were determined as: start pH 6.5, cultured at 37℃without shaking. After cultivated in these conditions for 18h-20h, the viable cells of L. casei Zhang can reach to 4.78×109 CFU/mL, which was about 10 times higher than in MRS (4.8×108 CFU/mL).The influences of neutralizing agents, concentration of buffer salts and glucose in the medium, pH, and aeration on the growth of L. casei Zhang during pH-stat batch culture were investigated to optimize the cultural conditions. Then, according to the values of maximum specific growth rate, biomass and viable cells count of the strain growing under above cultural conditions, the appropriate cultural conditions were determined as: temperature controlled at 37℃, glucose concentration of the medium set as 80 g/L, pH maintained at 5.9 by automatic addition of aqua ammonia and anaerobic condition kept by injection of nitrogen periodically. By cultured under these conditions for 10-12 h, the biomass and viable cells count of L. casei Zhang can reach to 7.45 g/L and 3.68×1010 CFU/mL respectively, which were seven times as high as the before optimization.To figure out the inhibition of lactate and low pH on the growth of L. casei Zhang, it was fermented under different lactate concentration and pH. The results showed that both low pH and lactate inhibited the growth of this strain and act synergistically. Cultured in non-regulated pH, lactate concentration of 20 g/L (pH4.0) in the medium is enough to repress the growth of cells absolutely. However, cultured in pH maintained at 6.0 with neutralizer, the growth of cells can not stop until the concentration of lactate arrived at 120 g/L.The kinetic models of growth, glucose consumption, and lactic acid production for L. casei Zhang were built up with nonlinear curve fitting method using Origin 7.5 software and fit well with the experiment data as relative error less than 10%. These models can forecast the actual fermentation process well.Fed-batch culture was carried out with feed rate of 100 mL/h and 200 mL/h respectively. When feed at 100 mL/h, there is no different between fed-batch and batch fermentation, but when feed at 200 mL/h, dilution effect was found after cultured for 8 h.Based on lab scale experimentation, the pilot plant scale test (50L and 200L scale) of the high cell density culture of L. casei Zhang was studied to get the optimal fermentation technology for industrial production. The viable cells count of fermented liquid from pilot plant test was 2.9×1010 CFU/mL and no significant different from that in the lab scale test. So it can be concluded that the HCDC technology obtained in this study was operable.Preliminary study was carried out on the influences of centrifugation and freeze-drying processes on the cells after fermentation. Although the viable cells count of the lyophilized powder was 2.65×1011 CFU/g, which can meet the requirements of the probiotics and starter cultures, only 49.97% of L. casei Zhang cells were survived from freeze drying. So it’s necessary to make further study on how to improve the survival of L. casei Zhang during freeze-drying.In order to evaluate the gene expression pattern in L. casei Zhang, a study was conducted to evaluate different cultural conditions and growth phases on the expression levels of five housekeeping genes. The expression stability of HKG candidates were analyzed by GeNorm software. The result showed that all these five tested HKG candidates (gapd, gyrB, ldh, 16s rRNA, and recA) were proved to be stable, and the gapd and gyrB were the most stable genes, which were suggested as reference genes for real-time PCR of L. casei Zhang.The influences of various cultural conditions and growth phases on expressions of thirteen key enzyme genes of L. casei Zhang involved in glucose transport, glycolysis and molecular chaperone were investigated by real-time quantitative PCR. The results showed that the influences of different cultural conditions on gene expression were depended on the kinds of genes. These results can provide reference for the further study on the molecular mechanism of growth and metabolism of L. casei Zhang.更多还原