米曲霉不同培养方式的发酵探究

利用10L发酵罐在摇瓶实验基础上进行了米曲霉(Aspergillusoryzae)放大实验。根据不同菌体形态对代谢产物的影响,在米曲霉菌丝生长到絮状前,终止发酵。指数补料实验结果表明,分批发酵在第14小时菌体干质量达到最大值15.06g/L,比摇瓶发酵提高了33%;指数补料在第16小时菌体干质量达到20.43g/L,比分批发酵提高26%。     

分批补料高密度培养米根霉As3.819产L-乳酸的研究

采用分批补料方式对米根霉As3.819高密度培养产L-乳酸的效果进行研究。摇瓶实验确定的最佳培养条件为：接种量5%、接种时间24h、装液量40%、补料液中葡萄糖与硫酸铵的质量比为20:1。罐发酵的最佳补料方式为葡萄糖浓度反馈流加。通过流加培养获得了较高的菌体密度,菌体干重达18.45g/L;重复发酵5批次,产L-乳酸效率达2.25g/L·h;菌体干重和产酸效率较分批培养分别提高了92.9%和82.9%。     

L-亮氨酸摇瓶补料分批发酵实验

进行了补葡萄糖、补醋酸铵和硫酸铵和全组分补料的摇瓶补料分批发酵实验。实验结果显示,以初始葡萄糖浓度为50g/L,初始醋酸铵和硫酸铵浓度分别为10g/L,于发酵过程第24h起每隔12h分4次补加全组分发酵培养基,摇瓶发酵72h产L-亮氨酸21.32g/L。     

纳豆激酶分批补料发酵的研究

在摇瓶和发酵罐上研究了分批补料发酵对枯草芽孢杆菌LSSE-22发酵生产纳豆激酶的影响。通过摇瓶分批发酵,确定最优碳源和氮源分别为葡萄糖和大豆蛋白胨。在优化初始葡萄糖和大豆蛋白胨浓度的基础上,进一步研究了补料底物、补料方式和补料时间对产酶的影响。结果表明,采用分批补糖发酵工艺,纳豆激酶产量可达到1 437.34 IU/m L,比分批培养提高了21.38%。在7.5 L发酵罐上进行分批补料发酵放大实验,纳豆激酶产量可达2 046.47 IU/m L,明显优于分批培养。     

发酵法生产桑黄胞外多糖条件的研究

以单因子摇瓶实验和响应面分析对药用真菌桑黄培养基进行了优化,确定了优化培养基(g/L):蔗糖50、玉米浆3.0、KH2PO410.0、MgSO41.0、CaCl23.0、VB1200μg/L。在优化条件下,研究了0~168h,桑黄发酵pH、总糖、还原糖、胞外多糖、生物量的变化情况。摇瓶发酵结果表明,整个过程中pH变化不明显,总糖含量先降后升,生物量先增后趋于稳定,于120h达最大值11.012 g/L,胞外多糖在144h达最大2.594 g/L,生产强度为0.018 g/(L.h)。经7 L发酵罐放大实验表明,桑黄代谢与摇瓶存在一定差异,胞外多糖的合成速度更快,于120 h达最大值3.283 g/L,生产强度为0.027 g/(L.h),与摇瓶相比分别提高了26.5%和50%。     

产朊假丝酵母高产GSH发酵条件优化研究

对产朊假丝酵母发酵产GSH进行研究,考察了胞内GSH的抽提方法、培养基组成及补加葡萄糖对GSH产量的影响。结果显示:最佳的抽提方法为乙醇法;酵母粉与硫酸铵对胞内谷胱甘肽含量影响最大,以硫酸铵为氮源时GSH含量可达到25.48mg/g;补加葡萄糖的时间为接种后10h、补加量为16mL时。最佳条件下得到菌体干重为8.14g/L,通过摇瓶发酵GSH产量为113.45mg/L,GSH产量提高了2.89倍。     

富硒酵母发酵工艺的优化

以富硒酿酒酵母（Saccharomyces cerevisiae）SY2为发酵菌种,利用5 L发酵罐培养富硒酵母。以富硒酵母生长及硒转化率为评价指标,优化其发酵工艺条件,比较溶氧反馈补料与拟指数补料两种方式对富硒酵母生长、硒转化率等的影响。结果表明,富硒酵母最适发酵条件为：初始pH 5.0,发酵温度30 ℃,接种量8%,硒添加量30 mg/L,发酵时间24 h。在此优化条件下,富硒酵母生物量为9.8 g/L,硒转化率为77.5%。在拟指数补料方式下,培养周期为34 h,富硒酵母得率为0.252 g/g,硒含量为1 920.5 μg/g,硒的转化率为82.7%;在溶氧反馈补料方式下,培养周期为46 h,富硒酵母得率为0.317 g/g,硒含量为1 759.5 μg/g,硒转化率为90.1%。结果显示,拟指数补料培养周期短,生产强度、酵母硒含量较高,是适宜补料方式。     

红曲霉JR液体发酵产红曲色素的工艺研究

对红曲霉JR液体发酵产红曲色素的发酵丁艺进行了研究.首先,考察了培养基装量、接种量以及培养基初始pH值等因素对红曲霉JR液体发酵产红曲色素色阶的影响,结果表明30mL/250mL三角瓶的培养基装量5%～20%的接种量、培养基初始pH7.0最有利于红曲色素的合成;此外,对红曲霉爪摇瓶分批发酵与摇瓶分批补料发酵的培养模式进行了比较,结果表明:摇瓶分批补料培养所得的最大菌体干重和红曲色素色阶分别为44.57g/L和350.7U/mL,均显著高于摇瓶分批培养下的25.59g/L和160.83U/mL.     

产辅酶Q_(10)白地霉菌的罐发酵条件优化

以白地霉菌(Geotrichum candidum)为出发菌株,根据摇瓶发酵结果控制pH值进行罐发酵。采用间歇补料分批发酵的方法,考察溶氧量、搅拌转速、空气流量及补糖因素对菌体生物量及辅酶Q10产量的影响。确定发酵罐发酵的最适条件为控制溶氧在35%～40%,补糖控制在发酵液糖质量浓度为2g/L,该发酵条件下辅酶Q10产量从分批发酵的78.75mg/L提高到106.26mg/L,较摇瓶发酵提高了69.8%。     

灵芝菌体液态深层发酵条件的优化

研究摇瓶灵芝菌体液态深层发酵温度和初始pH 值,在此基础上进行5L 发酵罐批次培养,研究发酵过程pH 值控制、溶氧控制对灵芝菌体生长和灵芝胞外多糖的影响。结果表明：发酵温度30℃,初始pH 值为6.0;过程pH 值控制策略：菌体生长前期(0～40h)控制pH 值为5.5,40～48h 控制pH 5.0,48h 后至发酵结束控制pH4.5;溶氧控制策略为：搅拌转速160r/min,通风量0.75vvm。优化后的验证实验结果：灵芝菌体生物量最高达到19.7g/L,胞外多糖最高达到3.23g/L,较优化前灵芝菌体生物量12.8g/L 和灵芝胞外多糖2.39g/L 分别提高了53.9% 和35.1%。     

凝集型酵母酒精发酵的研究

通过Saccharomyces 1～5号菌株间歇发酵试验发现,间歇通气发酵与摇床发酵方式的发酵速率远高于静置发酵,摇床发酵的糖醇转化率48h超过80%。说明适量通风可使酵母菌在反应器中保持高密度和高活性,进而加快反应的进行,并提高糖醇转化率,尤其是在发酵前期可以使酵母菌快速增殖建立优势环境。在二级反应器中的连续发酵试验中,当稀释速率为0.05/h时,流出末级反应器的发酵液酒精浓度达到77.4g/L,糖醇转化率达到89.93%,反应器发酵速率为3.87g/(L.h),残余还原糖浓度为8.2 g/L。     

粉被虫草液体培养条件的优化

通过摇瓶液体培养正交试验获得了粉被虫草的液体培养条件,即蔗糖2.5%,马铃薯淀粉2%,黄豆0.5%,牛肉膏0.5%,酵母膏0.1%,K2HPO4 0.1%,KCl 0.02%,MgSO4•7H2O 0.05%,pH5～7,装液量(100ml/250ml摇瓶),加15颗玻璃珠作分散剂,5%接种量,旋转式摇床150r/min,28℃培养7d,达到最大菌丝干重31.86g/L,于9d获得最大胞内产物3.13g/L。同时,对摇瓶液体培养和生物反应器液体深层分批培养的动力学做了较系统的研究,无论生物反应器还是摇瓶,其培养过程中相对应的pH、残糖、氨基氮、菌丝干重、胞内产物等参数变化趋势基本一致,但生物反应器培养时间大大缩短,48h即可达到最大菌丝干重29.97g/L,54h达到4.95g/L的最高胞内产物产率。生物反应器设定培养温度28℃、装液量65%、接种量10%、0.2%的消泡剂、搅拌转速350r/min、通气量(12±3)L/min、罐压1.1MPa、初始pH6.5,培养基配方用食用蔗糖3%、蔗糖糖蜜2%、花生0.5%分别替代蔗糖、马铃薯淀粉、牛肉膏以降低成本。实验表明,粉被虫草的液体培养条件基本能达到工业发酵水平的要求。     

茅台酒酒曲中5株真菌的分离、纯化、鉴定初报

实验对贵州茅台酒酒曲中5株真菌（菌种编号为R1、R2、R3、R4,R5）进行了分离、纯化培养,旨在为贵州茅台酒酒曲的微生物组成进行深入研究提供基础实验依据。通过观察菌落形态与个体形态,参照KlichMA、RaperKB和张纪忠的方法对5株真菌进行了鉴定。鉴定结果表明：霉菌菌株R1为米曲霉（Aspergillus Oryzae）,R2为红色红曲菌（Monascus）,R3黑曲霉（Aspergillus Niger）,R4为炭黑曲霉（Aspergillus carbonarius）,R5为米根霉（Rhizopus Oryzae）。     

Production of Citric Acid from Beet Molasses by Immobilized Cells of Aspergillus niger

Citric acid was produced from beet molasses by immobilized cells in shake flasks and glass bioreactor. Maximum concentration of citric acid (35 g/L) was observed from immobilized A. niger cells in shake flasks after 28 days fermentation. In repeated batch fermentations, the A. niger cells entrapped in Ca-alginate gel beads retained ability to produce citric acid for up to 84 days.     

烟曲霉酸的发酵培养基优化和反应器放大研究

烟曲霉酸是一种四环三萜类结构的化合物,具有良好的抗菌活性和抗癌效果。该研究在单因素试验的基础上,对来源于海洋的烟曲霉菌（Aspergillus fumigatus）CY018发酵生产烟曲霉酸的培养基进行了优化及放大培养。结果表明,优化后发酵培养基为甘露醇39.88 g/L,硝酸钠2.25 g/L,牛肉膏3.54 g/L,丁二酸钠8.4 g/L,硫酸镁0.3 g/L,硫酸亚铁0.01 g/L,磷酸二氢钾0.67 g/L。初始pH值调至4.2,摇瓶装液量50 mL/250 mL,转速160 r/min,28 ℃培养9 d。在此优化培养基及发酵条件下,烟曲霉酸的产量达到68.49 mg/L,为优化之前的4.1倍。并在此基础上进行了5 L反应器的放大研究,在发酵第7天得到了烟曲霉酸的最大产量为54.81 mg/L。该实验为HA生产的放大制备与应用推广打下了基础。     

Comparison of Galactooligosaccharide Production in Free-Enzyme Ultrafiltration and in Immobilized-Enzyme Systems

ABSTRACT:  Galactooligosaccharides (GOSs) are prebiotics that have been shown to reduce colon cancer risk and enhance immunity. The GOSs can be formed enzymatically from whey lactose using β-galactosidase, but commercial application has been limited. Free- and immobilized-enzyme recycle batch processes were investigated and compared in this study. Optimum initial conditions were estimated using batch solutions. Using these optimum conditions, an ultrafiltration (UF) free-enzyme system was developed, and UF fluid pressure (100 to 400 p.s.i.) effect on enzyme performance was studied and compared with a 0 p.s.i. batch control. The optimum conditions were also used to develop an immobilized-enzyme process using polyethyleneimine (PEI), glutaraldehyde (GA), and cotton cloth. The PEI and GA immobilized agents were studied for their effect on enzyme inactivation. Finally, compatible free- and immobilized-enzyme recycle batch systems were compared for GOS production. Optimum initial enzyme conditions were estimated as 270 g/L initial lactose, 4.5 g/L initial enzyme ( Aspergillus oryzae, 9400 U/g), and 30-min incubation. Fluid pressure within the free-enzyme UF membrane system had no significant effect on enzyme performance. The highly agitated UF enzyme system was found to be superior to the less agitated 0 p.s.i batch control. In the immobilization process, approximately 50% to approximately 90% enzyme inactivation was found with the combination of PEI and GA. Equivalent free- and immobilized-enzyme systems showed very similar maximum GOS production of approximately 22% and approximately 20% (w/v) at approximately 15 to 17 min, or 50% conversion for free- and immobilized-systems, respectively.     

Development of a fed-batch cultivation strategy for the enhanced production and secretion of cutinase by a recombinant Saccharomyces cerevisiae SU50 strain

Saccharomyces cerevisiae SU50 strain was cultivated with different concentrations of glucose and galactose with the aim of increasing cutinase activity, cutinase yield on the carbon source, and bioreactor productivity. Cultivations in shake flasks with galactose as the sole carbon source, with sugar concentrations between 10 and 40 g/l, exhibited growth-associated cutinase production and a constant specificity of cutinase secretion. Furthermore, as the galactose concentration increased to values higher than 15 g/l, a progressively higher maximum specific galactose consumption rate and a consequent higher alcoholic fermentation occurred, resulting in progressively lower biomass yields on the carbon source and cutinase yields on biomass. Using high glucose and galactose concentrations in a well-aerated bioreactor resulted in a high biomass productivity (0.5 g dcw/l/h), a high cutinase yield on biomass (21.5 U/mg dew), a final high cutinase secretion efficiency (97%), and plasmid stability (99%). Based on these studies, a two phase fed-batch cultivation strategy was developed. A batch phase with high glucose and galactose concentrations, followed by a fed-batch with a constant feed rate with galactose as the sole carbon source in order to minimize the repression of the GAL 7 promoter, were established. The feed rate was estimated to maintain a pre-determined concentration of galactose (20 g/l) on the culture medium in order to maximize the efficiency of cutinase secretion and minimize the galactose alcoholic fermentation. By this cultivation strategy, enhancements of 3.6-fold in cutinase activity, 1.2-fold in cutinase yield on the carbon source, and 8.7-fold culture productivity were obtained in relation to a batch cultivation performed in shake flasks with 20 g/l of galactose.     

分批与流加发酵法生产纤维素酶的研究

采用里氏木霉ＲｕｔＣ－３０,对２．５Ｌ罐分批和流加发酵产酶条件及优化进行了系统研究。通过研究不同浓度的ＳｏｌｋａＦｌｏｃ（纤维素粉）对分批发酵产酶的影响,发现菌体浓度与产酶量随底物浓度增加而增加,当采用５０ｇ／ＬＳｏｌｋａＦｌｏｃ复合１０ｇ／Ｌ麸皮为碳源时,菌体浓度和产酶量最大,最大ＤＣＷ１３．８２ｇ／Ｌ,ＣＭＣａｓｅ２３４．２Ｕ／ｍｌ,ＦＰＡ２１．２５Ｕ／ｍｌ,但ＳｏｌｋａＦｌｏｃ增加至６０ｇ／Ｌ,高浓度底物对菌体初始生长产生强烈抑制,产酶下降。通过研究不同初始Ｓｏｌ－ｋａＦｌｏｃ浓度对流加发酵产酶的影响,发现当初始底物浓度为５０ｇ／ＬＳｏｌｋａＦｌｏｃ复合１０ｇ／Ｌ麸皮时,菌体量和产酶均达到最大值,分别为ＤＣＷ１５．４１ｇ／Ｌ,ＣＭＣａｓｅ３５９．７Ｕ／ｍｌ,ＦＰＡ３０．６Ｕ／ｍｌ,高菌体量是获得纤维素酶高产的关键因素之一。此外用硫铵盐析法对纤维素酶进行了提取。     

雅致放射毛霉YYS-15菌株产菊粉酶发酵条件的研究

在摇瓶条件下对雅致放射毛霉YYS-15产菊粉酶的条件(不同的碳源、氮源、无机盐、初始pH值以及发酵量)进行了研究.经正交试验对其发酵条件优化后,确立了最适宜的发酵条件为菊粉20g/L、蛋白胨25g/L、KNO36g/L、KCl 5g/L、FeSO4·7H2O 0.1g/L、pH值为8.0,在28℃、振荡培养96h后酶活性达到27.36U/mL.