【作者】 戴传云；

【导师】 王伯初；

【作者基本信息】 重庆大学 ， 生物医学工程， 2003， 硕士

【摘要】 超声波是一种物理能，在低强度和适当的频率条件下，其特有的空化作用形成振荡，涡流式超声波作用，致使液体中形成局部的暂时负压区，产生非稳定的空泡和气泡，当它们突然闭合时，会产生激波，局部压强很大，达上千个大气压，破坏了物质间的相互引力吸附。微弱的超声空化作用对细胞产生的破坏作用很小，可以在细胞表面瞬间造成微伤，使细胞壁局部破裂，从而改变细胞膜的通透性，使胞内物质释放或胞外物质进入细胞内，从而加强细胞的物质运输，加速细胞的新陈代谢和目标产物的生物合成。本文以阿氏假囊酵母 (Ecemothecium ashbyii )为实验材料，寻找不同的刺激方式，来研究超声刺激对菌株的生长和次生代谢的影响，为超声波在发酵工业生产上的应用提供一些参考。本文的主要研究内容和方法如下：1. 研究超声对E. ashbyii)生长的基本规律的影响。选用功率一定，频率分别为18、20、24、28、30 kHz的超声波加载于该菌的摇瓶发酵过程中，加载方式是每12h刺激一次，每次全程30 min，刺激30 s，间隔30 s。结果表明：在已知作用频率的刺激下，都对阿氏假囊酵母的生长有促进作用；同时核黄素的发酵得率也有提高。该结果在24 kHz的刺激作用下最为明显，在此频率超声作用下，发酵时间比对照组提前了36 h，核黄素的发酵得率提高了2倍左右。2. 研究一定频率（24 kHz）的超声波在阿氏假囊酵母生长的不同阶段刺激，对核黄素生物合成的影响。即分别在菌丝体生长和核黄素生物合成阶段施加超声的作用，与空白对照。结果表明，在菌丝体生长阶段，超声刺激是由于促进了微生物生长量的增加而使核黄素的发酵得率提高，效果显著；但在核黄素的生物合成阶段，超声刺激促进核黄素向胞外分泌，抑制代谢产物核黄素对次生代谢的反馈作用，从而导致核黄素的发酵得率的极其显著提高。在核黄素的生物合成阶段，研究不同刺激时间和周期对核黄素生物合成的影响，结果表明，在阿氏假囊酵母发酵的104 h开始刺激时，超声对核黄素向胞外分泌的效果才开始明显，可能的原因是在这以前代谢产物很低，不足以对核黄素的生物合成产生抑制，因此刺激效果不明显，随着发酵时间的推移，细胞内产生大量的核黄素，开始抑制核黄素的生物合成，加载超声刺激促进核黄素向胞外分泌，使核黄素对次生代谢的反馈作用受到抑制，从而导致核黄素的发酵得率的显著提高，在108 h左右，超声作用最为明显，此时核黄素的相对含量提高了40 %左右。116 h处理前后，核黄素的相对含量变化不大。因此可以得出，在发酵的104～112 h是超<WP=5>3. 声处理的最佳时间段。4. 用超声处理发酵液后，核黄素被释放到发酵液中，但随着发酵的继续进行，细胞内进一步合成核黄素，产物的浓度又逐渐升高，另一方面发酵液中的核黄素又有可能被胞壁吸附，再次形成产物对核黄素生物合成的抑制。研究结果表明，在104～112 h时间段，每隔1.5 h就需要再次超声处理。5. 用高效液相色谱（HPLC）法监视了超声刺激在阿氏假囊酵母发酵过程中丙酮酸含量的变化。结果表明，超声刺激会引起细胞内丙酮酸发生变化，同时发现，丙酮酸含量的变化与菌丝体干重和核黄素的含量的变化成一定的关系，说明超声刺激可能影响到三羧酸循环过程，从而改变整个代谢过程，最终导致核黄素的生物合成。更多还原

【Abstract】 Ultrasound is physics energy. Under the condition of low intensity and proper frequency, cavitation will be come into being, which give birth to surge and whirlpool. And part and temporarily negative suppress will be formed in liquid which produce non-stabilization vacuole and bladder. When those vacuole and bladder is closed, it will produce fierce wave and make pressure aggrandizement to thousands of atmospheric pressure around liquid, which will destroy gravitation adsorption among many substance or molecule. Power of slender cavitation destroying cell is very little and may produce tiny injure in the face of cell, cell wall breaking partly, which have an increase of cell membrane penetrability, and the cell membrane allows substance within to leave and that outside to enter the cell easily. So it strengthens matter transport and accelerates cell metabolism and target outcome biosynthesize. In this thesis, low frequency effecting growth and second metabolize of Ecemothecium ashbyii are researched, aim to cast about for different stimulation manner effect fermentation of E. ashbyii and provide some reference for ultrasound application in fermentation industry.The main research contents and results are as follows:Firstly, ultrasound effecting growth basic rule of E. ashbyii was investigated. Ultrasonic power known and frequency being 18、20、24、28、30 kHz respectively were load during the fermentation of E. ashbyii . All of the media were stimulated once 12 h, and treatment time was 30 min including 30 s working and 30 s resting time in turns. Several key biochemical parameters related to cell growth were measured. The result of the present show the all of the given different frequency of ultrasonic treatment could promote mycelium growth, at the same time also improve output of riboflavin, and the optimum frequency was about 24 kHz. After 24 kHz ultrasonic incontinuous treatments, the fermentation time was shortened by 36 hours and productivity rate of riboflavin was increased by about 2 times compared with the control groups. Secondly, during the different growth phase, ultrasonic of 24 kHz effecting riboflavin biosynthesize was investigated. It was found that at the phase of mycelium growth, because ultrasonic treatments promote mycelium quantity accumulate; the outcome of riboflavin was increased. It was significant difference; at the phase of riboflavin biosynthesize, owing to ultrasonic stimulation promote riboflavin secrete to outside and keep riboflavin from feedback second metabolize, it was great significant<WP=7>difference.Thirdly, at the phase of riboflavin biosynthesize, ultrasonic treatment at different time effecting riboflavin biosynthesize was investigated. Until the fermentation time was 14 h, ultrasonic stimulation promote evidently riboflavin secrete to outside. The likelihood is that before 104 h, an outcome of riboflavin was not enough to withdrawn riboflavin biosynthesize. Along with fermentation processing, a lot of riboflavin was produced within cell and begin to withdrawn riboflavin biosynthesize. Ultrasonic loaded could promote riboflavin secrete to outside and enhance riboflavin output.Fourthly, after, in the broth, ultrasonic was treated, riboflavin was released into the broth. But following the fermentation time was coming, within cell riboflavin was biosynthesize and content of offspring was increasingly increased, at the same time, offspring in the broth could be adsorbed by cell wall. So riboflavin biosynthesize was restrained again. From 104 h to 112 h, ultrasonic treatment should be loaded every 1.5 h.Lastly, a HPLC method was presented for simultaneous determination of pyruvate during ultrasonic stimulation. It was found that ultrasonic could give birth to pyruvate change, at the same time, concentration of pyruvate change relation to dry weight of mycelium and content of riboflavin. It showed that ultrasonic treatment might effect TCA cycle and influence the whole metabolize course, in the end, result in riboflavin biosynthesize.更多还原