重离子诱变酵母培养基的优化与研究

利用12C6+离子诱变技术对面包酵母菌种进行诱变,得到一株粗蛋白含量达到55% 以上的菌株,借助Minitab16.0,采用Plackett-Burman 实验设计法及响应面分析法,对诱变后面包酵母菌发酵培养基的成分进行了优化,得到3 个最为显著的主要影响因子:葡萄糖、酵母抽提物和硫酸镁。利用最陡爬坡实验逼近最大响应区域后,利用Box-Behnken 实验设计及响应面分析法进行回归分析。通过求解回归方程,得到优化发酵的条件为,葡萄糖:11.03 g/L、酵母抽提物:6.53 g/L、硫酸镁:5.59 g/L。面包酵母生物量为4.84 g/L,相比未进行优化时的生物量提高了15%。A mutant bread yeast strain with high protein content of 55% was gained by use of 12C6+ ions. The MINITAB 16.0 software, Plackett-Burman experimental design and response surface methodology were applied to optimize the culture medium for the irradiated yeast. The most important three factors which influenced the culture results were identified as glucose, magnesium sulphate and yeast extract. The path of the steepest ascent was undertaken to approach the optimal region of the three significant factors. Box-Behnken design and response surface methodology were used for the regression analysis. Finally, the optimal fermentation conditions were identified as glucose 11.03 g/L, yeast extract 6.53 g/L and magnesium sulphate 5.59 g/L by the regression analysis. It was found that the biomass of the bread yeasts reached 4.84 g/L and increased by 15% compared to original conditions.     

阿佛曼链霉菌的~(12)C离子辐照效应及高产菌株选育

选用12C6+离子辐照诱变阿维菌素B1a产生菌ZJAV-A1,研究其诱变效应。实验结果表明,12C6+离子辐照剂量50Gy时致死率97%,正突变率最高可达到34.2%。通过12C6+离子诱变处理,结合平板培养基及斜面培养基的正突变菌株筛选,最终获得一株稳定性良好,阿维菌素B1a组分产量稳定在4460—4588μg/ml之间,较出发菌株提高11.1%—14.7%的突变株ZJAV-Y1-203。     

重离子辐照结合α-纤维素适应性进化提高黑曲霉真菌纤维素酶活性研究

为获得可有效降解植物细胞壁中纤维素的高产纤维素酶菌株,将具有安全可靠、不产生毒素、生长速度快且发酵周期短等优势的黑曲霉作为出发菌株。利用重离子12C6+束流按照0 , 40 , 70 , 100 , 130 , 160, 190 Gy的辐照剂量对黑曲霉原始菌株进行辐照,经平板初筛后得到两株优良菌株;然后添加α-纤维素进行了8周自适应性进化,通过连续5代发酵该菌株生长性能稳定;将得到的每代菌株进行胞内蛋白分子量的对比,得到一株最优菌株CJH-JWSFZh-W122,将其摇瓶发酵测定纤维素酶活性,其产纤维素酶的滤纸酶活(FPA)到了223.5 U/mL,内切葡聚糖酶活(CMC)达到了440.8 U/mL,分别比原始菌株提高了6.07%和8.01%。突变株在连续5代发酵后仍保持了良好的发酵性能。对突变菌株CJH-JWSFZh-W122和原始菌株进行了RAPD分析和SDS-PAGE电泳分析,证实了该菌株存在基因突变。     

激光诱变选育面包酵母菌的研究

采用He-Ne激光对面包酵母菌进行诱变处理,筛选到3株低糖面团起发活力大幅度提高的菌株XD198、XD193和XD174,其低糖面团起发活力比出发菌株分别提高31.3%、32.7%和35.3%.海藻糖含量分别较出发菌株提高-1.37%、10.83%、5.10%.而且筛选到的高活力菌株遗传性能稳定.证明激光诱变效果明显,是进行面包酵母菌种选育的理想方法之一.     

He-Ne激光和紫外线对PHB产生菌的复合诱变效应

采用紫外线、He-Ne激光及紫外线与He-Ne激光复合作用的方法, 对本实验室的一株产PHB的细菌G-Q7进行诱变, 以提高其产量. 结果表明, 紫外线和He-Ne激光单独作用, 诱变效果不佳, 而二者的复合作用却能产生很好的效果. 用He-Ne激光辐照20 min再经紫外线照射150 s, 正变菌株的细胞量为11.5g/L, PHB含量为65%, 并具有良好的遗传稳定性. 对诱变前后菌株积累的PHB产物的外观、热性质和13C-NMR图谱进行分析, 结果表明该诱变使代谢途径中的某种酶发生了改变.     

C离子束诱变产生甜高粱汁酒精酵母高产菌株的研究

利用100 MeV/u C离子束对高产酒精酵母菌株进行了辐照诱变的研究。 采用红四氮唑作为筛选指示剂, 得到了5株产酒能力有所提高的突变酵母菌。 利用甜高粱汁的发酵结果表明, T4突变菌株的产酒精能力比原始出发菌株提高了18.6%, 且发酵液中的残糖含量也有所降低。 随后对T4菌株在甜高粱汁中的最适宜发酵条件做了初步探索, 结果表明: 最适发酵温度和pH值分别为30 ℃和4.5。 通过10 l发酵罐的验证试验表明: 在同样发酵条件下, T4菌株的发酵率和产酒精能力都比原始出发菌株提高了12%。 Five mutants with high ability of producing alcohol were selected out by using TTC as an indicator after irradiation of the alcohol yeast with 100 MeV/u carbon ions. The fermentation experiment in sweet sorghum juice showed that the alcohol production ability of mutant T4 strain increased 18.6% compared to the control strain. The residual sugar content in the juice was decreased too. After that, the optimum fermentation conditions of the T4 strain in sweet sorghum juice were investigated. The results showed that the optimum temperature and pH value for fermentation were 30 ℃ and 4.5, respectively. The verification experiment was fermented in a 10 l bio reactor and the obtained data indicated that the fermentative rate and the ability of producing alcohol in T4 strain was higher than that in the control strain under the same fermentation condition.     

缓苗期遮荫对雌雄银杏幼苗抗氧化酶系统及生化物质含量的影响

本实验采用双因素完全随机设计,对银杏(Gingko biloba L.)移栽苗处于缓苗期的衰老叶片进行57%遮荫处理。利用紫外分光光度法测得结果:(1)遮荫处理降低了银杏植株的SOD活性,雌株降低了32.69%,雄株降低了27.51%;(2)遮荫处理降低了POD活性:雌株降低了76.14%,雄株降低了53.88%;(3)雌雄株的可溶性蛋白含量也分别降低12.02%和2.74%;(4)遮荫后雌雄株MDA的含量分别提高了79.06%和51.34%。雌株对遮荫的反应更为敏感,各项指标变化均大于雄株,因此雄株对遮荫胁迫的抗性较雌株强。本结论对移栽银杏幼苗起到一定的指导作用,可优先选用雄株。     

火焰原子吸收光谱法测定饲料中的铬

5g/L氯化铵抑制共存元素的干扰,火焰原子吸收光谱法测定饲料中铬的含量。方法简便、快捷,加标回收率在92.8%—102.3%之间,相对标准偏差小于3%,检出限为6.26μg/L。     

提高拮抗菌WB3对番茄灰霉病菌拮抗能力的He-Ne激光与氯化锂复合诱变技术

利用He-Ne激光(波长632.8 nm,功率12 mW)与氯化锂复合诱变一株番茄灰霉病菌拮抗茵WB3,获得14株拮抗效果突出的菌株,其最大抑菌带宽为15.7 mm.综合单一诱变组里菌株致死率以及拮抗能力的大小,其中辐照时间为30 min,氯化锂浓度为1.2%时为复合诱变最佳条件.经过复合诱变,L8拮抗茵最高抑菌带宽可...     

提高拮抗菌WB3对番茄灰霉病菌拮抗能力的He-Ne激光与氯化锂复合诱变技术

利用He-Ne激光(波长632.8 nm,功率12 mW)与氯化锂复合诱变一株番茄灰霉病菌拮抗菌WB3,获得14株拮抗效果突出的菌株,其最大抑菌带宽为15.7 mm.综合单一诱变组里菌株致死率以及拮抗能力的大小,其中辐照时间为30 min,氯化锂浓度为1.2%时为复合诱变最佳条件.经过复合诱变,L8拮抗菌最高抑菌带宽可提高14%以上,而单一He-Ne激光诱变后抑菌带宽提高8.8%,单一氯化锂诱变后抑菌带宽提高9%.L8菌株生长迅速,经传代培养证明其抑菌性能具有稳定遗传性.该实验研究He-Ne激光与氯化锂复合诱变枯草芽孢杆菌WB3的方法可应用于番茄灰霉病的生物防治领域.     

细菌纤维素菌株超高压诱变选育及其发酵培养基的优化

 为了获得高产细菌纤维素菌株,对初选的细菌纤维素菌株J₂进行超高压诱变,运用Plackett-Burman设计对影响高压诱变菌株生产细菌纤维素的因素效应进行评价,采用Box-Behnken试验优化发酵培养基组成。试验结果表明,超高压诱变压力、时间对细菌纤维素菌株有显著或极显著影响。细菌纤维素菌株高压诱变条件为压力250 MPa、时间15 min、温度25 ℃。经超高压诱变,获得产纤维素能力高、遗传稳定性好的诱变菌株M₄₃₈。影响诱变菌株M₄₃₈发酵生产细菌纤维素的关键因子是酵母浸出汁、MgSO₄和无水乙醇。优化的发酵培养基为碳源5%（葡萄糖∶蔗糖为4∶1）、酵母浸出汁1.25%、CaCl₂0 15%、ZnSO₄ 0.20%、K₂HPO₄ 0.20%、MgSO₄ 0.93%、富马酸0.30%、无水乙醇0.50%。利用此培养基培养诱变细菌纤维素菌株M₄₃₈,其纤维素产量是优化前的1.84倍,是超高压诱变之前的2.69倍。超高压技术用于细菌纤维素菌株的诱变育种是可行的。发酵培养基的优化可显著提高菌株M₄₃₈发酵生产细菌纤维素的能力。     

重离子束12C6+累进辐照诱变柠檬酸菌株选育研究

应用大剂量重离子束12C6+ 对菌株H3001 进行二次辐照选育,对初选获得的高产柠檬酸菌株进行摇瓶发酵试验及10~ 100 L 中试发酵罐试验,采用酸碱中和法测定发酵液中柠檬酸的含量。结果表明:当二次重离子12C6+剂量为857.8 Gy 时,致死率和正突变率达到最大值,分别为94.5% 和8%。通过摇瓶发酵试验,最终获得一株高产柠檬酸菌株hw317,控制该菌株发酵周期为60 h,柠檬酸酸度能达到19.2±0.2%。Heavy 12C6+ ion beams in various high doses were employed to irradiate H3001 strain for screening Aspergillus niger strain for hyper citric acid production. Three high-yield strains were obtained after shaker fermentation test. Among the three strains, the strain hw317 was implemented shaker fermentation for stability test and 10~100 L pilot fermentation tank for citric acid productive maximization. Acid-base neutralization method was applied to determinate the content of citric acid in fermented liquid. The results showed that: when the secondary heavy ion 12C6+ dose was 857.8 Gy, both of the fatality rate (94.5%) and the positive mutation rate (8%) were highest. Through the shaker fermentation tests and 10 ~ 100 L pilot fermentation test, one strain hw317 was screened and obtained for hyper citric acid production. Consequently, the final citric acid acidity can reach up to 19.2±0.2% with controlling fermentation cycle for 60 h.     

酵母同步细胞的Percoll连续密度梯度改良方法分离及其单细胞拉曼光谱鉴别

利用改良后的Percoll连续密度梯度分离方法,分离不同酵母菌株稳定期的同步细胞,并在单细胞水平上,应用激光光镊拉曼光谱对分离的同步细胞进行鉴别。采用高速离心机角式转头和2 mL聚丙烯离心管,20 ℃,19 320 g离心15 min,将大约1.75 mL Percoll溶液形成1.00～1.31 g·mL-1连续密度梯度;将样品均匀铺加在离心管连续密度梯度液顶层,20 ℃,400 g离心60 min,酵母分成明显的2层细胞带;微分干涉差显微镜（DIC）、相差显微镜以及同步生长曲线分析显示,下层酵母细胞以单个细胞为主,大小均匀、致密,折光性强,生长呈现阶梯式增长,具有同步细胞生长的特性,确定其为同步的G₀细胞。利用单细胞光镊拉曼光谱系统对所分离的G₀同步细胞与非G₀细胞进行分析,结果显示G₀同步细胞和非G₀细胞的特征峰位置基本一致,但G₀细胞对应的蛋白质、糖类、核酸等生物大分子特征峰强度大于非G₀细胞,说明G₀细胞大分子物质含量要比非G₀细胞的高;对G₀细胞和非G₀细胞进行主成分分析,结果显示,分离的同步化G₀细胞之间成分含量差异少,比较均一,而非同步细胞之间内含物质差异大,呈不均一性,说明单细胞光镊拉曼光谱系统可以鉴别同步和非同步细胞。改良后的Percoll连续密度梯度分离方法,能够分离多数酵母菌株同步化细胞,是一种易操作、成本低、效率高的细胞分离方法。     

Influence of ultrasonic amplitude,temperature, time and solvent concentration on bioactive compounds extraction from propolis

An ultrasound assisted method was investigated to extract bioactive compounds from propolis. This method was based on a simple ultrasound treatment using ethanol as an extraction medium to facilitate the disruption of the propolis cells. Four different variables were chosen for determining the influence on the extraction efficiency: ultrasonic amplitude, ethanol concentration, temperature and time; the variables were selected by Box-Behnken design experiments. These parameters were optimised in order to obtain the highest yield, and the results exhibited the optimum conditions for achieving the goal as 100% amplitude of ultrasonic treatment, 70% solvent concentration, 58 °C and 30 min. The extraction yield under modified optimum extraction conditions was, as follows: 459.92 mg GAE/g of TPC, 220.62 mg QE/g of TFC and 1.95% of balsam content. The results showed that the ultrasound assisted extraction was suitable for bioactive compounds extraction from propolis. The most abundant phenolic compound was kaempferol (228.8 mg/g propolis) followed by myricetin (115.5 mg/g propolis), luteolin (27.2 mg/g propolis) and quercetin (25.2 mg/g propolis).     

He-Ne激光对钝顶螺旋藻的诱变效应

利用He-Ne激光(波长632 nm，功率10 mW)诱变钝顶螺旋藻IS（Spirulina platensis IS），经过一定时间的辐照，选取生长较快的藻种测量其β-胡萝卜素、蛋白质及多糖含量，进一步筛选出生长快、高产胡萝卜素或高产蛋白质的藻种.实验结果表明：与出发藻种IS相比，经He-Ne激光15min和25 min照射后的藻种MS-1、MS-2和 MS-3，藻丝形态发生变化，藻丝变短、螺旋变紧密，生长速度明显加快，其中 MS-1的β-胡萝卜素含量增幅为18.1%，MS-3的蛋白质和多糖含量均有较大增加.此外通过对出发藻种和诱变藻种的叶绿素a和胡萝卜素的紫外吸收光谱进行比较，发现诱变藻种与出发藻种相比最大吸收峰值略有变化，从而说明He-Ne激光对于钝顶螺旋藻的诱变效应.     

Fermentation of Saccharomyces cerevisiae in a 7.5 L ultrasound-enhanced fermenter: Effect of sonication conditions on ethanol production,intracellular Ca2+ concentration and key regulating enzyme activity in glycolysis

In this study, the effect of sonication on the fermentation process of a single-celled fungus was examined. During the experiment, Saccharomyces cerevisiae (S. cerevisiae) was used as the starting strain for ethanol fermentation (batch fermentation) in a 7.5 L automated fermentation tank. The fermentation tank connected with a six-frequency ultrasonic equipment. Non-sonication treatment was set up as the control. Sonication treatment with power density of 280 W/L and 48 h of treatment time were set up as trial groups for investigating the influence of different ultrasound frequency including 20, 23, 25, 28, 33 and 40 kHz on the changes in dry cell-weight, glucose consumption rate, and ethanol yield. The results showed that the dry cell-weight, glucose consumption rate, and ethanol content reached the best results under the ultrasonic condition of 28 kHz ultrasound frequency in comparison with other ultrasound frequency. The dry cell-weight and ethanol content of the 28 kHz ultrasonic treatment group increased by 17.30% and 30.79%, respectively in comparison with the control group The residual sugar content dropped to a lower level within 24 h, which was consistent with the change in ethanol production. Besides, the results found that the glucose consumption rate increased compared to the control. It indicated that ultrasound accelerated glucose consumption contributed to increase the rate of ethanol output. In order to explore the mechanism of sonication enhanced the content of ethanol output by S. cerevisiae, the morphology, permeability of S. cerevisiae and key enzyme activities of ethanol synthesis were investigated before and after sonication treatment. The results showed that after sonication treatment, the extracellular nucleic acid protein content and intracellular Ca²⁺ concentration increased significantly. The morphology of S. cerevisiae was observed by SEM and found that the surface of the strain had wrinkles and depressions after ultrasonic treatment. furthermore after sonication treatment, the activities of three key enzymes which catalyze three irreversible reactions in glycolysis metabolism, namely, hexokinase, phosphofructokinase and pyruvate kinase increased by 59.02%, 109.05% and 87.27%, respectively. In a word, low-intensity ultrasound enhance the rate of ethanol output by S. cerevisiae might due to enhancing the growth and cell permeability of strains, and increasing the activities of three key enzymes of ethanol biosynthesis.