【作者】 朱至；

【导师】 陈坚；

【作者基本信息】 江南大学 ， 发酵工程， 2008， 硕士

【摘要】 开菲尔多糖(Kefiran)是由一种特殊发酵乳—开菲尔(Kefir)的发酵剂—开菲尔粒(Kefir grain)产生的乳酸菌胞外多糖。Kefiran作为一种新型的活性多糖,不仅可以作为增稠剂、稳定剂、乳化剂和凝胶剂,而且具有抗真菌、抗细菌、降压、降血糖、降胆固醇和抗肿瘤的特性,是一种很好的消炎降压及免疫激活物质,在临床医药、食品工业及有关生物研究领域都有着广泛的用途。本论文在实验室规模上以一株能在胞外高积累Kefiran的开菲尔基质乳杆菌(Lactobacillus kefiranofaciens JCM6985T)为生产菌株,以细胞和Kefiran的高产量,高得率和高生产强度为目标,对Kefiran发酵过程进行了优化。主要研究结果如下:(1)优化得到了摇瓶条件下Kefrian生产的最优碳氮源种类及浓度:碳源为100 g/L蔗糖,氮源为100 g/L酵母粉。(2)不同pH控制方式对Kefiran分批发酵的影响有较大差异。通过对pH 4.0~5.5范围内Kefiran分批发酵的各过程参数进行比较,确定Kefiran分批发酵最适宜pH为5.0。(3)考察不同温度条件下Kefiran分批发酵过程,并在深入分析各动力学参数的基础上提出两阶段温度控制策略:发酵起始控制温度33oC,发酵10 h后切换至28oC并保持到发酵结束。该温度控制策略的实施可进一步提高Kefiran的合成能力,比温度控制在28oC和33oC时分别提高了12%和46%,其生产强度比28oC和33oC分别提高了36%和33%。具有很好的实用性。(4)考察不同初糖浓度下Kefiran分批发酵过程,发现仅通过分批培养难以实现细胞和Kefiran高产量,高得率和高生产强度的有机统一。分批补料、恒速流加和指数流加等几种培养方式都可以实现乳酸菌细胞和Kefiran的高产。综合比较来看,4 g/(L·h)的蔗糖恒速流加方式下L. kefiranofaciens细胞干重63.6 g/L,Kefiran产量4.95 g/L,比较适合Kefiran的发酵生产。(5)为降低乳酸菌发酵过程中乳酸对菌体生长和产物合成的抑制作用,考察了乳好氧共培养合成Kefiran,使Kefiran发酵强度达到94.19 mg/(L·h),比单菌厌氧培养提高了53.7%;L. kefiranofaciens与C. utilis好氧共培养合成Kefiran使Kefiran发酵强度达到86.44 mg/(L·h),比单菌厌氧培养提高了41.1%,并且产生了250 mg/L的GSH。采用这种共培养方式不仅可以降低乳酸菌发酵过程中乳酸对菌体生长和产物合成的抑制而且可以合成高附加值的共同产物,对其他终端产物抑制发酵类型有较好借鉴作用。更多还原

【Abstract】 Lactobacillus kefiranofaciens, which was isolated from kefir grains, produces an exopolysaccharides called Kefiran. Not only can Kefiran be used as thickener, stabilizer, emulsifier or gelling agent, but it also has an antitumor activity. Because of this, the procedure for the mass production of Kefiran from this bacterium is currently being extensively studied.In this paper, Lactobacillus kefiranofaciens JCM6985T, a strain could accumulate high concentration of exopolysaccharide(Kefiran) was selected for Kefiran production. Optimize the L. kefiranofaciens cultivation processes to achieve high product concentration, high yield and high productivity of Kefiran. Main results of this dissertation include following aspects:(1)The required carbon and nitrogen sources for Kefiran production by L. kefiranofaciens in shaking flasks were investigated. The optimal concentrations of the carbon and nitrogen sources were gained as following: sucrose 100 g/L, yeast extract 100 g/L.(2)The mode of pH control is an important factor for batch Kefiran fermentation. It was shown that pH 5.0 was the optimum condition to improve the Kefiran production by comparing the parameters of five batch processes controlled at the pH ranging from 4.0 to 5.5 and without pH control.(3)The effect of temperature, varied from 25oC to 33oC, on the batch fermentation of Kefiran was investigated. Owing to the difference occurred in the optimal temperature for cell growth and Kefiran production by thoroughly analyzing the kinetics of batch Kefiran production under different temperatures, a two-stage temperature control strategy, in which the temperature was kept at 33oC during the first 10 hours of cultivation, followed by a shift from 33oC to 28oC, then maintained at 28oC until the end of the cultivation, was developed in order to enhance the production of Kefiran. The result indicated that, the Kefiran production was 4.65 g/L,which was respectively increased by 12% and 46% compared with that at 28oC and 33oC, and Kefiran productivity was increased by 36% and 33% compared with that at above two temperature respectively.(4)Five initial sucrose concentrations were studied to determine their effects on microbial growth and substrate consumption. It was concluded that high concentration, high yield and high productivity of both cell and Kefiran could not be consolidated in a batch process. The effects of different cultivation modes such as batch feeding, constant-rate feeding, exponential rate feeding on Kefiran production were also studied. High production of cell and Kefiran were all realized in these cultivation modes of sucrose feeding. As a whole, fed-batch culture with constant feeding rate at 4 g/(L·h) was the most suitable approach to attain not only high concentrations but also high yields and high productivities of cell and Kefiran in the cultivation of L. kefiranofaciens under which the maximum dry cell weight and Kefiran reached 63.6 g/L and 4.95 g/L respectively.(5)The high concentration of lactic acid produced during the course of L. kefiranofaciens fermentation inhibits kefiran production. Yeast was cocultured with L. kefiranofaciens to improve kefiran production by consuming lactic acid to decrease the inhibition effect.The productivity of the coculture of L. kefiranofaciens and S. cerevisiae under aerobic condition reached 94.19 mg/(L·h), 53.7% higher than which of the pure culture. And the productivity of the coculture of L. kefiranofaciens and C. utilis under aerobic condition reached 86.44 mg/(L·h), 41.1% higher than which of the pure culture, another product of glutathione also reached 250 mg/L.更多还原