毕业论文-真菌发酵液对纳塔尔链霉菌纳他霉素产量的影响

1、安 徽 农 业 大 学 毕 业 论 文（设计）论文题目 真菌发酵液对纳塔尔链霉菌纳他霉素产量的影响 姓 名 学 号 院 系 _生命科学学院_ 专 业 生物技术_指导教师 职 称 讲师_ _中国合肥二o 1 3 年 5 月目 录摘要 1前言 11实验材料及方法 21.1实验材料 2 1.1.1实验菌种 2 1.1.2培养基 21.2实验方法 2 1.2.1纳塔尔链霉菌的培养 2 1.2.2真菌发酵液的制备 2 1.2.3纳他霉素产量的测定 2 1.2.4真菌发酵液添加时间对纳他霉素产量的影响 2 1.2.5真菌发酵时间对纳他霉素产量的影响 2 1.2.6真菌发酵液添加量对纳他霉素产量的影响 32

2、结果与分析 32.1纳他霉素标准曲线的制作 32.2发酵液加入时间对N5菌株纳他霉素产量的影响 3 2.2.1产黄青霉发酵液加入时间对N5菌株纳他霉素产量的影响 3 2.2.2黑曲霉发酵液加入时间对N5菌株纳他霉素产量的影响 4 2.2.3酿酒酵母发酵液加入时间对N5菌株纳他霉素产量的影响 52.3真菌培养时间对N5菌株纳他霉素产量的影响 5 2.3.1 产黄青霉培养时间对N5菌株纳他霉素产量的影响 5 2.3.2黑曲霉培养时间对N5菌株纳他霉素产量的影响 6 2.3.3酿酒酵母培养时间对N5菌株纳他霉素产量的影响 62.4真菌发酵液添加量对N5菌株纳他霉素产量的影响 7 2.4.1产黄青霉发

3、酵液添加量对N5菌株纳他霉素产量的影响 7 2.4.2黑曲霉发酵液添加量对N5菌株纳他霉素产量的影响 8 2.4.3酿酒酵母发酵液添加量对N5菌株纳他霉素产量的影响 83讨论与结论 9参考文献 9致谢 10英文摘要 10真菌发酵液对纳塔尔链霉菌纳他霉素产量的影响作者：潘 指导教师：曹 （安徽农业大学生命科学学院2009级生物技术1班，学号09121012）摘要：纳他霉素是由纳塔尔链霉菌（streptomyces natalensis）、褐黄孢链霉菌（streptomyces gilvosporeus）、恰塔努加链霉菌（streptomyces chattanoogensis）等菌株发酵而产生的

4、一种广谱抗真菌剂。因其低毒和极小改变食物本身味道可作为生物防腐剂。本实验通过在纳塔尔链霉菌N5菌株中添加真菌发酵液的方法提高菌株纳他霉素的产量。实验结果表明产黄青霉、黑曲霉、酿酒酵母三种菌的发酵液均对纳塔尔链霉菌N5菌株的纳他霉素产量有明显的促进作用。产黄青霉和黑曲霉培养1d时，其发酵液对N5菌株纳他霉素的合成促进效果最佳，而酿酒酵母在3d时促进效果最好，此时菌株纳他霉素产量较未加真菌发酵液处理样品高出约1倍。关键词：纳塔尔链霉菌N5菌株 纳他霉素 产黄青霉 黑曲霉 酿酒酵母 前言1955年struyk等人首次从南非natal州土壤中分离得到纳塔尔链霉菌（streptomyces natale

5、nsis），并发现该菌种可以产生一种内酯类抗生素，strnyk将其命名为匹马菌素（pimaricin）1，后世界卫生组织将其统一命名为纳他霉素。该抗生素是一种二十六元多烯大环内酯类抗生素，主要由纳塔尔链霉菌（streptomyces natalensis）、褐黄孢链霉菌（streptomyces gilvosporeus）、恰塔努加链霉菌（streptomyces chattanoogensis）等菌发酵产生2。纳他霉素是一种高效、广谱的抗真菌剂，能有效抑制霉菌及酵母的生长，并能有效抑制丝状真菌中黄曲霉素的形成，可广泛应用于食品保鲜及治疗真菌引起的疾病3。由于其分子上的疏水部分可以范德华力与细

6、胞质膜上的甾醇分子结合，形成抗生素甾醇复合物，破坏细胞质膜渗透性，引起菌体细胞内电解质渗出，因而可导致菌体死亡，起到抑菌作用4。纳他霉素在室温下溶解度仅为30-100mg/L，微溶于甲醇，易溶于稀酸、冰醋酸和二甲基甲酰胺，不溶于非极性溶剂5。一般认为，由于纳他霉素难溶于水和油脂，所以很难被消化和吸收，因此人体摄入的纳他霉素大部分会随粪便排出6。因为纳他霉素具有毒性低和不会对食品天然风味有较大改变的性质，美国食品及药物管理局(FDA)和我国卫生部已经先后于1982年与1997正式批准纳他霉素作为食品添加剂。迄今为止纳他霉素已经在食品工业中得到广泛应用7。由于各种因素的制约，国内纳他霉素的主要来源

7、至今仍然是从国外进口。限制我国自主生产纳他霉素的主要问题是菌株产量偏低，导致纳他霉素的生产成本过高。这已成为急需解决的问题。提高纳他霉素产量可以通过添加诱导子的方法。已有报道称一些微生物的发酵代谢物可产生某种诱导子调节其它微生物的次级代谢，促进其次级代谢产物的生成8,9,10。本实验将产黄青霉、黑曲霉、酿酒酵母的发酵液分别添加入纳塔尔链霉菌（streptomyces natalensis）发酵液中，研究不同真菌发酵液对该菌株纳他霉素产量的影响，以期提高菌株纳他霉素产量。1实验材料与方法1.1实验材料1.1.1 菌种纳塔尔链霉菌N5菌株由中国科学院合肥物质科学研究院等离子所提供。黑曲霉、产黄青霉

8、、酿酒酵母由安徽农业大学生命科学学院微生物学实验室保藏。1.1.2 培养基种子培养基 (g/L)：葡萄糖20、酵母浸膏5、蛋白胨6、氯化钠10，pH 7.2发酵培养基 (g/L)：葡萄糖10、酵母浸膏5、大豆蛋白胨20、淀粉40，pH7.21.2实验方法1.2.1 纳塔尔链霉菌的培养 用接种环刮取斜面菌种一环于装有25mL种子培养基的250mL三角瓶中，28、 200r/min摇床培养，40h后以9%接种量接种于装有30mL发酵培养基的250mL三角瓶中，28、200r/min摇床培养 4d。1.2.2 真菌发酵液的制备将产黄青霉、黑曲霉、酿酒酵母分别接种于装有100mL发酵培养基的250mL

9、三角瓶中，28、150r/min摇床培养适当时间，用八层纱布过滤，再在10000r/min下离心15min，取上清液进行微孔滤膜过滤即可得到无菌的真菌发酵液。1.2.3 纳他霉素产量的测定称取纳他霉素标准样品，溶于5%冰乙酸的甲醇溶液中，使纳他霉素浓度分别为1mg/L、2mg/L、3mg/L、4mg/L、5mg/L、6mg/L、7mg/L、8mg/L，用紫外分光光度计测其在303nm处的OD值，以纳他霉素浓度为横坐标，OD303值为纵坐标，绘制标准曲线。取N5菌株发酵液0.8mL于7.2mL 5%冰乙酸的甲醇溶液中。在震荡器上震荡5min后10000r/min离心15min。取上清液用紫外分光

10、光度计测量303nm波长下的吸光度，根据标准曲线计算纳他霉素产量。1.2.4 真菌发酵液添加时间对纳他霉素产量的影响将产黄青霉、黑曲霉、酿酒酵母培养3d后分别以5%接种量接种于纳塔尔链霉菌N5菌株发酵液中，接种时间分别为0h、12h、24h、36h、48h、60h、72h，每个处理重复3次，并以不添加真菌发酵液的处理为对照。培养4d后测定菌株纳他霉素产量。确定真菌发酵液最适添加时间。 1.2.5 真菌发酵时间对纳他霉素产量的影响分别在产黄青霉、黑曲霉、酿酒酵母培养1d、2d、3d时取样，制备无菌发酵液。将培养不同时间的真菌发酵液以5%接种量加入培养12h的纳塔尔链霉菌N5菌株发酵培养液中，每个

11、处理重复3次，并以不添加真菌发酵液的处理为对照。继续培养84h后测定菌株纳他霉素产量。确定3种真菌的最佳发酵时间。1.2.6 真菌发酵液添加量对纳他霉素产量的影响将产黄青霉、黑曲霉培养1d，酿酒酵母培养3d，制备无菌发酵液，并分别以1% 、2% 、5% 、8% 、10% 、15% 、20%的添加量加入到培养12h的纳塔尔链霉菌N5菌株发酵培养液中，每个处理重复3次并以不添加真菌发酵液的处理为对照。培继续培养84h后测定菌株纳他霉素产量。确定3种真菌的最佳发酵时间。2结果及分析2.1 纳他霉素标准曲线的制作以纳他霉素浓度为横坐标，OD303值为纵坐标绘制纳他霉素标准曲线，见图1。由图1可看出纳他

12、霉素浓度与OD303值呈线性相关，且R2=0.9988线性较好。因此可通过此标准曲线方程x=（y0.0065)/0.1489计算纳他霉素产量。图1 纳他霉素标准溶液曲线Fig1 The standard curve of natamyctn2.2 发酵液加入时间对N5菌株纳他霉素产量的影响2.2.1产黄青霉发酵液加入时间对N5菌株纳他霉素产量的影响将产黄青霉发酵培养基进行适当处理后接种于培养不同时间的纳塔尔链霉菌N5菌株发酵液中，继续培养，当菌株培养4d时用紫外分光光度法测定纳他霉素产量，结果如图2所示。由图2可看出产黄青霉发酵液对N5菌株纳他霉素产量有明显的促进作用，当在发酵培养12h时添加

13、效果最佳，此时菌株纳他霉素产量达1.42g/L，较对照提高65.96。 图2 产黄青霉发酵液添加时间对纳他霉素产量的影响Fig 2 Effects of addition time of fermentation broth of Penicillium chrysogenum on the natamycin production of N5 strain2.2.2黑曲霉发酵液加入时间对N5菌株纳他霉素产量的影响将培养3d的黑曲霉发酵培养液适当处理后于不同时间添加入N5菌株发酵培养液中，继续培养，当菌株培养4d时测定纳他霉素产量，结果见图3。从图3中可以看出黑曲霉发酵液对N5菌株纳他霉素产量

14、的提高有显著的促进作用。当在发酵培养12h时添加效果最佳，此时菌株纳他霉素产量达1.44g/L，较对照提高67.44。图3黑曲霉发酵液添加时间对纳他霉素产量的影响Fig 3 Effects of addition time of fermentation broth of Aspergillus Nigeron the natamycin production of N5 strain2.2.3酿酒酵母发酵液加入时间对N5菌株纳他霉素产量的影响酿酒酵母发酵液加入时间对N5菌株纳他霉素产量的影响见图4。由图可见，酿酒酵母发酵液也可明显促进N5菌株纳他霉素的合成，最适接入时间也是菌株培养的12h。

15、 图4 酿酒酵母发酵液添加时间对纳他霉素产量的影响Fig 4 Effects of addition time of fermentation broth of Saccharomyces cerevisiae on the natamycin production of N5 strain2.3 真菌培养时间对N5菌株纳他霉素产量的影响2.3.1 产黄青霉培养时间对N5菌株纳他霉素产量的影响将分别培养1d、2d、3d的产黄青霉发酵液进行适当处理，添加至培养12h的N5菌株发酵培养液中，继续培养84h后测定菌株纳他霉素产量，结果如图5所示。由图5可以看出培养1d的产黄青霉发酵液对N5菌株纳他霉

16、素产量的促进作用最显著。随着产黄青霉培养时间的延长，发酵液对N5纳他霉素产量的促进作用呈下降趋势。因此，产黄青霉的最佳培养时间为1d。图5产黄青霉培养时间对纳他霉素产量的影响Fig 5 Effects of culture time of Penicillium chrysogenum on the natamycin production of N5 strain2.3.2黑曲霉培养时间对N5菌株纳他霉素产量的影响 将培养不同时间的黑曲霉发酵液添加入N5菌株发酵液中，培养后测定纳他霉素产量，结果见图6。由图6可以看出当在N5菌株发酵液中添加培养1d的黑曲霉发酵液时，纳他霉素产量最高，因此，黑

17、曲霉的最佳培养时间确定为1d。图6黑曲霉培养时间对纳他霉素产量的影响Fig 6 Effects of culture time of Aspergillus Niger on the natamycin production of N5 strain2.3.3酿酒酵母培养时间对N5菌株纳他霉素产量的影响酿酒酵母培养时间对N5菌株纳他霉素产量的影响如图7所示。由图7可见，当在N5菌株发酵液中添加培养3d的酿酒酵母发酵液时，纳他霉素产量最高，因此，酿酒酵母的最佳培养时间确定为3d。图7酿酒酵母培养时间对纳他霉素产量的影响Fig 7 Effects of culture time of Saccha

18、romyces cerevisiae on the natamycin production of N5 strain2.4真菌发酵液添加量对N5菌株纳他霉素产量的影响2.4.1产黄青霉发酵液添加量对N5菌株纳他霉素产量的影响将培养1d的产黄青霉发酵液处理后以不同添加量添加于培养12h的纳塔尔链霉菌N5菌株发酵培养液中，于28、200r/min条件下继续培养84h，测定纳他霉素产量。以产黄青霉发酵液添加量为横坐标，纳他霉素产量为纵坐标做图，见图8。从图8中可以添加5的产黄青霉发酵液对N5菌株纳他霉素的积累效果最显著，此时纳他霉素产量达1.69g/L，较对照提高94.25%。图8产黄青霉发酵液添

19、加量对纳他霉素产量的影响的影响Fig 8 Effects of addition amount of Penicillium chrysogenum fermentation broth on the natamycin production of N5 strain2.4.2黑曲霉发酵液添加量对N5菌株纳他霉素产量的影响将培养1d的黑曲霉发酵液处理后以不同添加量添加于培养12h的纳塔尔链霉菌N5菌株发酵培养液中，于28、200r/min条件下继续培养84h，测定纳他霉素产量结果如图8所示。由图可看出5的黑曲霉发酵液对N5菌株纳他霉素的积累效果最佳，因此，黑曲霉发酵液的最适添加量确定为5。 图

20、9黑曲霉发酵液添加量对纳他霉素产量的影响的影响Fig 9 Effects of addition amount of Aspergillus Niger fermentation broth onthe natamycin production of N5 strain2.4.3酿酒酵母发酵液添加量对N5菌株纳他霉素产量的影响酿酒酵母发酵液添加量对N5菌株纳他霉素产量的影响见图10。从图10中可以看出5的酿酒酵母发酵液对N5菌株纳他霉素的积累效果最佳，因此，黑曲霉发酵液的最适添加量确定为5。图10酿酒酵母发酵液添加量对纳他霉素产量的影响的影响Fig 10 Effects of addition

21、 amount of Saccharomyces cerevisiae fermentation broth onthe natamycin production of N5 strain3讨论与结论 随着人们对健康、安全问题的日益关注，食品安全问题越来越受到人们的重视，化学防腐剂的缺点逐渐暴露，纳他霉素受到人们的青睐。纳他霉素是和乳酸链球菌素并列的仅有的两种生物防腐剂，其具有毒性低且不会造成食品风味巨大改变的特性，是一种极具有使用、生产价值的抗生素。但纳他霉素产生菌株产量不高一直是我国纳他霉素自主生产的瓶颈，过低的单株产量会使生产成本过高而不利于工厂生产。本实验将产黄青霉、黑曲霉、酿酒酵母发

22、酵液添加入纳塔尔链霉菌N5菌株发酵液中，研究这3种真菌培养时间、发酵液添加时间及添加量对N5菌株纳他霉素产量的影响，以促进菌株纳他霉素的合成。实验结果表明这三种真菌的发酵液均对纳塔尔链霉菌N5菌株的纳他霉素产量有明显促进作用。产黄青霉和黑曲霉培养1d时，其发酵液对N5菌株纳他霉素的合成促进效果最佳，而酿酒酵母在3d时促进效果最好。三种真菌最佳发酵液添加时间及添加量均为12h和5。具体这些真菌发酵液中何种因素对N5菌株纳他霉素生产起到促进作用还不清楚，有待于进一步实验研究。参考文献1 Marta V Mendes ，sedef Tunca ，Nuria Anton et al The two-c

23、omponent phoR-phoP system of streptomyces natalensis ;inactivation or deletion of phoP reduces the negative phosphate regulation of pimaricin biosynthesis metabolic engineering 2007 ,9 (2)：217227；2 PEDERSEN J C Natamycin as a fungicide imager media JApplied and environment 。microbiobogy 1992 ，58（3）.

24、10641066；3 陈晓丽、吕振岳、黄东东等。新型天然食品防腐剂纳他霉素的研究进展J食品研究与开发.2002.23（4）：23；4 卢国营、张朝辉、洪伟杰，新型防腐剂纳他霉素的研究进展J中国食品添加剂 2005（3）：3032；5 魏宝东、孟宽军、天然生物性食品防腐剂纳他霉素的特性及应用J，辽宁农业科学 2004 （2）：2426；6 贺家亮、张敏、赵胜娟，纳他霉素的研究现状J中国食品添加剂2005（3）：3032；7 Martin J F DEMAIN AL control of antibiotic biosynthesis 1980（02）；8 叶辉、王兆慧、陈佩林等 微生物诱导子对卡

25、诺氏菌属N05205 菌株的影响J，生物技术，2004 14（6）：5758；9 边猛、田黎、张久明等，诱导子对海洋真菌penicillium crustosum 204215菌株生物碱代谢的调节作用J海洋学报，2008.30（2）：117123；10 边猛 海洋真菌产生生物碱代谢调节的研究D：硕士学位论文青岛科技大学 生物化工系 。11 史强 真菌代谢产物中促进纳他霉素合成的诱导子的研究D：硕士学位论文江南大学。致谢感谢曹老师对于本试验的悉心指导，她对生物学科领域中的基本问题和发展前景的敏锐洞察力，对学术思想的重视，对选题和研究内容的严格要求，以及对论文工作中遇到的关键问题和疑难问题的指导使

26、我受益匪浅。她对试验的研究态度和严谨的工作作风给予我今后的学习和工作很深的启示。同时我还要感谢一起做实验的同学，他们在我的实验过程中给了我很大的帮助。The effect of fungus fermented liquid on the output of Streptomyces natalensis natamycinAuthor: instructor: (2009 College of life science of Anhui agricultural University-level biotechnology class 1, 09121012,)Abstract: Natam

27、ycin is a broad-spectrum antifungal agent generated by the fermentation of the streptomyces natalensis,streptomyces gilvosporeus,streptomyces chattanoogensis and other strains.It can be used as biological preservatives because it has low toxicity and change food taste lightly.This experiment adopts

28、the methods of adding Fungal fermentation broth to streptomyces natalensis N5 strains to improve natamycin production of the strains.The results reveal that Fermentation broth of Penicillium chrysogenum, Aspergillus niger, Saccharomyces cerevisiae can all improve Production of natamycin well.It has

29、the best effect on the improvement of the synthesis of natamycin when Penicillium chrysogenum and Aspergillus niger are cultured for one day.However,Saccharomyces cerevisiae has the best effect of improvement when culture for three days,and when the production of natamycin is one time higher than sa

30、mples without treatment of fungal fermentation broth.Key words: natamycin productivity Penicillium chrysogenum Aspergillus niger Saccharomyces cerevisiaeag an employment tribunal clai Emloyment tribunals sort out disagreements between employers and employees. You may need to make a claim to an emplo

31、yment tribunal if: you dont agree with the disciplinary action your employer has taken against you your employer dismisses you and you think that you have been dismissed unfairly. For more informu, take advice from one of the organisations listed underFurther help. Employment tribunals are less form

32、al than some other courts, but it is still a legal process and you will need to give evidence under an oath or affirmation. Most people find making a claim to an employment tribunal challenging. If you are thinking about making a claim to an employment tribunal, you should get help straight away fro

33、m one of the organisations listed underFurther help. ation about dismissal and unfair dismissal, seeDismissal. You can make a claim to an employment tribunal, even if you haventappealedagainst the disciplinary action your employer has taken against you. However, if you win your case, the tribunal ma

34、y reduce any compensation awarded to you as a result of your failure to appeal. Remember that in most cases you must make an application to an employment tribunal within three months of the date when the event you are complaining about happened. If your application is received after this time limit,

35、 the tribunal will not usually accept i. If you are worried about how the time limits apply to you If you are being represented by a solicitor at the tribunal, they may ask you to sign an agreement where you pay their fee out of your compensation if you win the case. This is known as adamages-based

36、agreement. In England and Wales, your solicitor cant charge you more than 35% of your compensation if you win the case.youre clear about the terms of the agreement. It might be best to get advice from an experienced adviser, for example, at a Citizens Advice Bureau. To find your nearest CAB, includi

37、ng those that give advice by e-mail, click onnearest CAB.For more information about making a claim to an employment tribunal, seeEmployment tribunals.The (lack of) air up there Watch m Cay man Islands-based Webb, the head of Fifas anti-racism taskforce, is in London for the Football Associations 150

38、th anniversary celebrations and will attend Citys Premier League match at Chelsea on Sunday.I am going to be at the match tomorrow and I have asked to meet Ya ya Toure, he told BBC Sport.For me its about how he felt and I would like to speak to him first to find out what his experience was.Uefa haso

39、pened disciplinary proceedings against CSKAfor the racist behaviour of their fans duringCitys 2-1 win.Michel Platini, president of European footballs governing body, has also ordered an immediate investigation into the referees actions.CSKA said they were surprised and disappointed by Toures complai

40、nt. In a statement the Russian side added: We found no racist insults from fans of CSKA.Age has reached the end of the beginning of a word. May be guilty in his seems to passing a lot of different life became the appearance of the same day; May be back in the past, to oneself the paranoid weird beli

41、ef disillusionment, these days, my mind has been very messy, in my mind constantly. Always feel oneself should go to do something, or write something. Twenty years of life trajectory deeply shallow, suddenly feel something, do it.一字开头的年龄已经到了尾声。或许是愧疚于自己似乎把转瞬即逝的很多个不同的日子过成了同一天的样子；或许是追溯过去，对自己那些近乎偏执的怪异信念

42、的醒悟，这些天以来，思绪一直很凌乱，在脑海中不断纠缠。总觉得自己似乎应该去做点什么，或者写点什么。二十年的人生轨迹深深浅浅，突然就感觉到有些事情，非做不可了。The end of our life, and can meet many things really do?而穷尽我们的一生，又能遇到多少事情是真正地非做不可？During my childhood, think lucky money and new clothes are necessary for New Year, but as the advance of the age, will be more and more fou

43、nd that those things are optional; Junior high school, thought to have a crush on just means that the real growth, but over the past three years later, his writing of alumni in peace, suddenly found that isnt really grow up, it seems is not so important; Then in high school, think dont want to give

44、vent to out your inner voice can be in the high school children of the feelings in a period, but was eventually infarction when graduation party in the throat, later again stood on the pitch he has sweat profusely, looked at his thrown a basketball hoops, suddenly found himself has already cant reme

45、mber his appearance.童年时，觉得压岁钱和新衣服是过年必备，但是随着年龄的推进，会越来越发现，那些东西根本就可有可无；初中时，以为要有一场暗恋才意味着真正的成长，但三年过去后，自己心平气和的写同学录的时候，突然就发现是不是真正的成长了，好像并没有那么重要了；然后到了高中，觉得非要吐露出自己的心声才能为高中生涯里的懵懂情愫划上一个句点，但毕业晚会的时候最终还是被梗塞在了咽喉，后来再次站在他曾经挥汗如雨的球场，看着他投过篮球的球框时，突然间发现自己已经想不起他的容颜。Originally, this world, can produce a chemical reaction t

46、o an event, in addition to resolutely, have to do, and time.原来，这个世界上，对某个事件能产生化学反应的，除了非做不可的坚决，还有，时间。A persons time, your ideas are always special to clear. Want, want, line is clear, as if nothing could shake his. Also once seemed to be determined to do something, but more often is he backed out at l

47、ast. Dislike his cowardice, finally found that there are a lot of love, there are a lot of miss, like shadow really have been doomed. Those who do, just green years oneself give oneself an arm injection, or is a self-righteous spiritual.一个人的时候，自己的想法总是特别地清晰。想要的，不想要的，界限明确，好像没有什么可以撼动自己。也曾经好像已经下定了决心去做某件

48、事，但更多的时候是最后又打起了退堂鼓。嫌恶过自己的怯懦，最终却发现有很多缘分，有很多错过，好像冥冥之中真的已经注定。那些曾经所谓的非做不可，只是青葱年华里自己给自己注射的一支强心剂，或者说，是自以为是的精神寄托罢了。At the moment, the sky is dark, the air is fresh factor after just rained. Suddenly thought of blue plaid shirt; Those were broken into various shapes of stationery; From the corner at the beg

49、inning of deep friendship; Have declared the end of the encounter that havent start planning. Those years, those days of do, finally, like youth, will end in our life.此刻，天空是阴暗的，空气里有着刚下过雨之后的清新因子。突然想到那件蓝格子衬衫；那些被折成各种各样形状的信纸；那段从街角深巷伊始的友谊；还有那场还没有开始就宣告了终结的邂逅计划那些年那些天的非做不可，终于和青春一样，都将在我们的人生中谢幕。Baumgartner th

50、e disappointing news: Mission aborted. r plays an important role in this mission. Starting at the ground, conditions have to be very calm - winds less than 2 mph, with no precipitation or humidity and limited cloud cover. The balloon, with capsule attached, will move through the lower level of the a

51、tmosphere (the troposphere) where our day-to-day weather lives. It will climb higher than the tip of Mount Everest (5.5 miles/8.85 kilometers), drifting even higher than the cruising altitude of commercial airliners (5.6 miles/9.17 kilometers) and into the stratosphere. As he crosses the boundary la

52、yer (called the tropopause),e can expect a lot of turbulence. We often close ourselves off when traumatic events happen in our lives; instead of letting the world soften us, we let it drive us deeper into ourselves. We try to deflect the hurt and pain by pretending it doesnt exist, but although we c

53、an try this all we want, in the end, we cant hide from ourselves. We need to learn to open our hearts to the potentials of life and let the world soften us.生活发生不幸时，我们常常会关上心门；世界不仅没能慰藉我们，反倒使我们更加消沉。我们假装一切仿佛都不曾发生，以此试图忘却伤痛，可就算隐藏得再好，最终也还是骗不了自己。既然如此，何不尝试打开心门，拥抱生活中的各种可能，让世界感化我们呢？Whenever we start to let our fears and seriousness get the best of us, we should take a step back and re-evaluate our behavior. The items listed below are six ways you can open your heart more fully and completely.当