【作者】 葛向阳；

【导师】 张伟国；

【作者基本信息】 江南大学 ， 发酵工程， 2009， 博士

【摘要】 近年来,国内外对L-乳酸的需求日趋增加,而我国L-乳酸的生产技术比较落后。菊芋作为一种生长能力强、抗盐碱、耐沙漠的作物,其在我国中西部地区的种植面积正以基地的形式逐年加大。菊芋深加工的研究引起了国内外学者的广泛关注,然而开展菊芋发酵生产L-乳酸的研究尚未见报道。由于乳酸菌自身不能利用菊粉,本研究采用一株菊粉酶活力较强的黑曲霉为产酶菌株,以乳酸杆菌G-01为L-乳酸生产出发菌株,对以菊芋为原料的L-乳酸的发酵法生产进行了研究。主要内容及结果如下:⑴通过对黑曲霉产菊粉酶培养基组成的优化,得到最佳培养基组成:葡萄糖40 g/L,蛋白胨30 g/L和蔗糖脂4 g/L。并在该优化条件下于30℃,140 r/min下进行摇瓶发酵,96 h后菊粉酶酶活力达到230 U/mL。进一步研究表明菊粉酶是一种诱导酶,从诱导效果上看,蔗糖酯相对于菊粉是一种更有效的合成诱导剂,该物质可以激活用于生产菊粉酶的mRNA的合成。同时由于蔗糖酯在溶液中相对于菊粉,更难水解,所以有着更强的和持续的诱导作用。研究还发现在使用葡萄糖作为碳源,蔗糖酯作为合成促进剂时,更有利于菌丝体的生长和酶的合成。通过对该株菌的基因和形态学分析,确定其为黑曲霉。⑵菊粉酶的提取,先采用30%饱和度的硫酸铵盐析,沉淀除杂,然后采用70%饱和度盐析,经透析除盐,聚乙二醇20000浓缩,得到总酶活25253U,单位酶活1262 U/mL,比酶活64.37 U/mg,回收率83%,纯化倍数2.36。进一步纯化表明黑曲霉产菊粉酶是由两种分子量相差较小的内切酶和外切酶组成,前者分子量在165～210 kDa,而后者的分子量在160～170 kDa。比较转化酶和菊粉酶在30℃的催化动力学发现:转化酶的Km=34.9 mmol/L,Vm=909μmol/min,菊粉酶的Km=68.3 mmol/L,Vm=476.2μmol/min,这表明转化酶的最大反应速率接近菊粉酶的2倍,然而达到最大反应速率所需底物的浓度却接近菊粉酶的一半。⑶通过DES和NTG对乳酸杆菌G-01进行诱变,并进行耐渗透驯化,使菌株以葡萄糖为底物产酸达到92.8 g/L,提高了近1倍。乳酸菌G-02菌株经过多次传代接种,表现出较好的传代稳定性。通过对该菌株的基因、形态学及代谢产物分析,确定其为干酪乳杆菌(Lactobacillus casei),乳酸同型发酵,而且产物中L-乳酸的光学纯度达到95%以上,为一株较为理想的L-乳酸生产菌株。⑷为了消除菊粉酶合成体系中的产物抑制现象,降低发酵液中黑曲霉菌丝体密度,同时避免在同步糖化与发酵菊芋产乳酸工艺中的染菌问题,使乳酸杆菌在菊芋糖化酶液中提前形成优势菌群,本研究采用在黑曲霉产酶培养基中适时接入乳酸菌的混合培养工艺,并对混合培养工艺条件进行了优化。结果表明,在产酶培养开始12 h接入乳酸杆菌,可以显著提高黑曲霉后期产菊粉酶的活性,在优化的培养基中,经过60 h的培养,菊粉酶活力将达到250 U/mL,较参照水平(50 U/mL)提高了近4倍。进一步研究发现,乳酸菌的接入显著地降低了发酵液中可发酵性糖的浓度,发酵液中黑曲霉菌丝体浓度降低到参照的一半,而且黑曲霉的菌丝体形态发生了明显的改变,从而更有利于生存在竞争的环境中。⑸对采用同步糖化与发酵工艺利用菊芋生产L-乳酸的工艺进行了探索,研究发现最适发酵工艺为菊粉酶活量50 U/mL,接种量为35%,菊芋粉抑制浓度为230 g/L,碳酸钙浓度50 g/L。为了进一步提高乳酸发酵活力,将柠檬酸代谢引入乳酸发酵工艺,结果表明:柠檬酸代谢可以显著提高乳酸杆菌的抗酸胁迫能力和乳酸发酵活性,最适柠檬酸添加浓度为10 g/L。在最佳工艺条件下,通过采用50℃初始水解1h的补料发酵工艺,经40℃,30 h发酵,L-乳酸浓度达到141.5 g/L,得率为52.4g乳酸/100g菊芋粉。更多还原

【Abstract】 Recently, the sharply enlarged manufacture of the bio-degradable polylactide polymer has increased the global interest in the production of L-lactic acid by using fermentative route. Jerusalem artichoke can grow well in poor land and shows a high tolerance to frost and various plant diseases. However, there have been few studies on the L-lactic acid production from Jerusalem artichoke, which was not fermentable for Lactobacillus sp. In this dissertation, Aspergillus niger SL-09, an active inulinase producer was used to form the inulinase, and Lactobacillus sp. G-01 as a parent L-lactic acid production strain were used to investigate the production of L-lactic acid from Jerusalem artichoke. Its contents and results are as follows:⑴The optimization of medium contents and fermentation conditions for Aspergillus niger SL-09 to form inulinase was conducted. The optimum medium contains glucose 40g/L, peptone 30g/L, sucrose ester 4g/L. Under the optimum fermentation conditions, inulinase activity of 230U/mL was obtained at 30℃on reciprocating shaker with 140 rpm in 96 h. Further studies found that sucrose ester was a more effective inducer than inulin for inulinase production, which can promote the synthesis of mRNA for the formation of inulinase. Meanwhile, as sugar ester was more resistant to hydrolysis than inulin, so that sucrose ester acts as a persistent signal to initiate a response in the cell to provoke the transcription of mRNA available for fructanohydrolase synthesis. Moreover, it was found that the medium with glucose and sucrose ester as carbon source and inducer respectively, were more favorable for the growth and enzymes synthesis of this strain. The strain SL-09 was identified as Aspergillus niger by genetic and morphological analysis.⑵In the process of inulinase purification, salt out with 30% and 70% of ammonium sulfate were used respectively. Through the salting out by dialysis and concentrate, the enzymes were obtained. Further studies found that the enzyme form by Aspergillus niger was composed by inulinase and invertase, and the difference of molecular weight between the both enzyme was not significant. The molecular weight of the inulinase was around 165～210 kDa,and the invertase was 160～170 kDa. The Michaelis-Menten constant of the inulinase and invertase at 30℃were Km=34.9 mmol/L, and vm=909μmol/min for invertase, and Km=68.3 mmol/L, vm=476.2μmol/min for inulinase, respectively. Those results signified that the maximum reaction rate of invertase was near 2-fold higher than that of the inulinase, however, the substrate concentration required for maximum rate was only half of the inulinase.⑶The parent strain of Lactobacillus sp. G-01 was treated by diethyl sulfate (DES), N-methyl-N’-nitro-N- nitrosoguanidine (NTG), and domesticated with high concentration of fructose and lactic acid. A strain G-02 which could accumulate 92.8 g/L L-lactic acid using glucose as carbon source was obtained. The strains of G-02 also presented favorable genetics stability after several times of transfers. Strains G-02 was identified as Lactobacillus casei, homofermentation, and can produce L(+)-lactic acid with an optical purity of over 95%. Therefore, this strain was a favorable strain for L-lactic acid production.⑷To avoid the product repress in the inulinase production system and decrease the biomass of Aspergillus niger in the enzyme production medium, and to remove the contamination in the simultaneous saccharification and fermentation process for production of lactic acid from Jerusalem artichoke, the Lactobacillus sp. G-02 was inoculated to the enzyme production medium to form superiority bacterium before lactic acid fermentation and the compose of the medium was optimized. The results signified that when the Lactobacillus sp. strain was inoculated at 12 h of the culture, the enzyme activity was enhanced significantly. After 60 h of co-culture, inulinase activity reached 250 U/mL, which was 4-fold higher than that of the control (50 U/mL) with single strain. Further studies found that the inoculation of Lactobacillus sp. decreased the fermentable sugar in the medium dramatically, and the biomass of Aspergillus niger was decreased to half of the value in the control. The mycelial configuration of the Aspergillus niger changed obviously, which was more favorable to survive in the competitive environment.⑸The simultaneous saccharification and fermentation process for L-lactic acid production from Jerusalem artichoke was investigated. It was found that the optimized condition was inulinase activity 50 U/mL, inoculation volume 35%, Jerusalem artichoke concentration 230 g/L. It was also found that the citrate metabolism endowed cells with extra ability to counteract the acid toxicity, and then enhance the lactic acid productivity. The optimized citrate concentration was 10 g/L. Under the optimized condition in the fed-batch culture, the L-lactic acid with the concentration of 141.5 g/L was obtained at 40℃in 30 h, with a yield of the 52.4g lactic acid/100g Jerusalem artichoke flour.更多还原