畢業(yè)論文真菌發(fā)酵液對納塔爾鏈霉菌納他霉素產(chǎn)量的影響10647

1、安 徽 農(nóng) 業(yè) 大 學(xué) 畢 業(yè) 論 文（設(shè)計(jì)）論文題目 真菌發(fā)酵液對納塔爾鏈霉菌納他霉素產(chǎn)量的影響 姓 名 學(xué) 號(hào) 院 系 _生命科學(xué)學(xué)院_ 專 業(yè) 生物技術(shù)_指導(dǎo)教師 職 稱 講師_ _中國合肥二o 1 3 年 5 月目 錄摘要 1前言 11實(shí)驗(yàn)材料及方法 21.1實(shí)驗(yàn)材料 2 1.1.1實(shí)驗(yàn)菌種 2 1.1.2培養(yǎng)基 21.2實(shí)驗(yàn)方法 2 1.2.1納塔爾鏈霉菌的培養(yǎng) 2 1.2.2真菌發(fā)酵液的制備 2 1.2.3納他霉素產(chǎn)量的測定 2 1.2.4真菌發(fā)酵液添加時(shí)間對納他霉素產(chǎn)量的影響 2 1.2.5真菌發(fā)酵時(shí)間對納他霉素產(chǎn)量的影響 2 1.2.6真菌發(fā)酵液添加量對納他霉素產(chǎn)量的影響 32

2、結(jié)果與分析 32.1納他霉素標(biāo)準(zhǔn)曲線的制作 32.2發(fā)酵液加入時(shí)間對n5菌株納他霉素產(chǎn)量的影響 3 2.2.1產(chǎn)黃青霉發(fā)酵液加入時(shí)間對n5菌株納他霉素產(chǎn)量的影響 3 2.2.2黑曲霉發(fā)酵液加入時(shí)間對n5菌株納他霉素產(chǎn)量的影響 4 2.2.3釀酒酵母發(fā)酵液加入時(shí)間對n5菌株納他霉素產(chǎn)量的影響 52.3真菌培養(yǎng)時(shí)間對n5菌株納他霉素產(chǎn)量的影響 5 2.3.1 產(chǎn)黃青霉培養(yǎng)時(shí)間對n5菌株納他霉素產(chǎn)量的影響 5 2.3.2黑曲霉培養(yǎng)時(shí)間對n5菌株納他霉素產(chǎn)量的影響 6 2.3.3釀酒酵母培養(yǎng)時(shí)間對n5菌株納他霉素產(chǎn)量的影響 62.4真菌發(fā)酵液添加量對n5菌株納他霉素產(chǎn)量的影響 7 2.4.1產(chǎn)黃青霉發(fā)

3、酵液添加量對n5菌株納他霉素產(chǎn)量的影響 7 2.4.2黑曲霉發(fā)酵液添加量對n5菌株納他霉素產(chǎn)量的影響 8 2.4.3釀酒酵母發(fā)酵液添加量對n5菌株納他霉素產(chǎn)量的影響 83討論與結(jié)論 9參考文獻(xiàn) 9致謝 10英文摘要 10真菌發(fā)酵液對納塔爾鏈霉菌納他霉素產(chǎn)量的影響作者：潘 指導(dǎo)教師：曹 （安徽農(nóng)業(yè)大學(xué)生命科學(xué)學(xué)院2009級(jí)生物技術(shù)1班，學(xué)號(hào)09121012）摘要：納他霉素是由納塔爾鏈霉菌（streptomyces natalensis）、褐黃孢鏈霉菌（streptomyces gilvosporeus）、恰塔努加鏈霉菌（streptomyces chattanoogensis）等菌株發(fā)酵而產(chǎn)生的

4、一種廣譜抗真菌劑。因其低毒和極小改變食物本身味道可作為生物防腐劑。本實(shí)驗(yàn)通過在納塔爾鏈霉菌n5菌株中添加真菌發(fā)酵液的方法提高菌株納他霉素的產(chǎn)量。實(shí)驗(yàn)結(jié)果表明產(chǎn)黃青霉、黑曲霉、釀酒酵母三種菌的發(fā)酵液均對納塔爾鏈霉菌n5菌株的納他霉素產(chǎn)量有明顯的促進(jìn)作用。產(chǎn)黃青霉和黑曲霉培養(yǎng)1d時(shí)，其發(fā)酵液對n5菌株納他霉素的合成促進(jìn)效果最佳，而釀酒酵母在3d時(shí)促進(jìn)效果最好，此時(shí)菌株納他霉素產(chǎn)量較未加真菌發(fā)酵液處理樣品高出約1倍。關(guān)鍵詞：納塔爾鏈霉菌n5菌株 納他霉素 產(chǎn)黃青霉 黑曲霉 釀酒酵母 前言1955年struyk等人首次從南非natal州土壤中分離得到納塔爾鏈霉菌（streptomyces natale

5、nsis），并發(fā)現(xiàn)該菌種可以產(chǎn)生一種內(nèi)酯類抗生素，strnyk將其命名為匹馬菌素（pimaricin）1，后世界衛(wèi)生組織將其統(tǒng)一命名為納他霉素。該抗生素是一種二十六元多烯大環(huán)內(nèi)酯類抗生素，主要由納塔爾鏈霉菌（streptomyces natalensis）、褐黃孢鏈霉菌（streptomyces gilvosporeus）、恰塔努加鏈霉菌（streptomyces chattanoogensis）等菌發(fā)酵產(chǎn)生2。納他霉素是一種高效、廣譜的抗真菌劑，能有效抑制霉菌及酵母的生長，并能有效抑制絲狀真菌中黃曲霉素的形成，可廣泛應(yīng)用于食品保鮮及治療真菌引起的疾病3。由于其分子上的疏水部分可以范德華力與細(xì)

6、胞質(zhì)膜上的甾醇分子結(jié)合，形成抗生素甾醇復(fù)合物，破壞細(xì)胞質(zhì)膜滲透性，引起菌體細(xì)胞內(nèi)電解質(zhì)滲出，因而可導(dǎo)致菌體死亡，起到抑菌作用4。納他霉素在室溫下溶解度僅為30-100mg/l，微溶于甲醇，易溶于稀酸、冰醋酸和二甲基甲酰胺，不溶于非極性溶劑5。一般認(rèn)為，由于納他霉素難溶于水和油脂，所以很難被消化和吸收，因此人體攝入的納他霉素大部分會(huì)隨糞便排出6。因?yàn)榧{他霉素具有毒性低和不會(huì)對食品天然風(fēng)味有較大改變的性質(zhì)，美國食品及藥物管理局(fda)和我國衛(wèi)生部已經(jīng)先后于1982年與1997正式批準(zhǔn)納他霉素作為食品添加劑。迄今為止納他霉素已經(jīng)在食品工業(yè)中得到廣泛應(yīng)用7。由于各種因素的制約，國內(nèi)納他霉素的主要來源

7、至今仍然是從國外進(jìn)口。限制我國自主生產(chǎn)納他霉素的主要問題是菌株產(chǎn)量偏低，導(dǎo)致納他霉素的生產(chǎn)成本過高。這已成為急需解決的問題。提高納他霉素產(chǎn)量可以通過添加誘導(dǎo)子的方法。已有報(bào)道稱一些微生物的發(fā)酵代謝物可產(chǎn)生某種誘導(dǎo)子調(diào)節(jié)其它微生物的次級(jí)代謝，促進(jìn)其次級(jí)代謝產(chǎn)物的生成8,9,10。本實(shí)驗(yàn)將產(chǎn)黃青霉、黑曲霉、釀酒酵母的發(fā)酵液分別添加入納塔爾鏈霉菌（streptomyces natalensis）發(fā)酵液中，研究不同真菌發(fā)酵液對該菌株納他霉素產(chǎn)量的影響，以期提高菌株納他霉素產(chǎn)量。1實(shí)驗(yàn)材料與方法1.1實(shí)驗(yàn)材料1.1.1 菌種納塔爾鏈霉菌n5菌株由中國科學(xué)院合肥物質(zhì)科學(xué)研究院等離子所提供。黑曲霉、產(chǎn)黃青霉

8、、釀酒酵母由安徽農(nóng)業(yè)大學(xué)生命科學(xué)學(xué)院微生物學(xué)實(shí)驗(yàn)室保藏。1.1.2 培養(yǎng)基種子培養(yǎng)基 (g/l)：葡萄糖20、酵母浸膏5、蛋白胨6、氯化鈉10，ph 7.2發(fā)酵培養(yǎng)基 (g/l)：葡萄糖10、酵母浸膏5、大豆蛋白胨20、淀粉40，ph7.21.2實(shí)驗(yàn)方法1.2.1 納塔爾鏈霉菌的培養(yǎng) 用接種環(huán)刮取斜面菌種一環(huán)于裝有25ml種子培養(yǎng)基的250ml三角瓶中，28、 200r/min搖床培養(yǎng)，40h后以9%接種量接種于裝有30ml發(fā)酵培養(yǎng)基的250ml三角瓶中，28、200r/min搖床培養(yǎng) 4d。1.2.2 真菌發(fā)酵液的制備將產(chǎn)黃青霉、黑曲霉、釀酒酵母分別接種于裝有100ml發(fā)酵培養(yǎng)基的250ml

9、三角瓶中，28、150r/min搖床培養(yǎng)適當(dāng)時(shí)間，用八層紗布過濾，再在10000r/min下離心15min，取上清液進(jìn)行微孔濾膜過濾即可得到無菌的真菌發(fā)酵液。1.2.3 納他霉素產(chǎn)量的測定稱取納他霉素標(biāo)準(zhǔn)樣品，溶于5%冰乙酸的甲醇溶液中，使納他霉素濃度分別為1mg/l、2mg/l、3mg/l、4mg/l、5mg/l、6mg/l、7mg/l、8mg/l，用紫外分光光度計(jì)測其在303nm處的od值，以納他霉素濃度為橫坐標(biāo)，od303值為縱坐標(biāo)，繪制標(biāo)準(zhǔn)曲線。取n5菌株發(fā)酵液0.8ml于7.2ml 5%冰乙酸的甲醇溶液中。在震蕩器上震蕩5min后10000r/min離心15min。取上清液用紫外分光

10、光度計(jì)測量303nm波長下的吸光度，根據(jù)標(biāo)準(zhǔn)曲線計(jì)算納他霉素產(chǎn)量。1.2.4 真菌發(fā)酵液添加時(shí)間對納他霉素產(chǎn)量的影響將產(chǎn)黃青霉、黑曲霉、釀酒酵母培養(yǎng)3d后分別以5%接種量接種于納塔爾鏈霉菌n5菌株發(fā)酵液中，接種時(shí)間分別為0h、12h、24h、36h、48h、60h、72h，每個(gè)處理重復(fù)3次，并以不添加真菌發(fā)酵液的處理為對照。培養(yǎng)4d后測定菌株納他霉素產(chǎn)量。確定真菌發(fā)酵液最適添加時(shí)間。 1.2.5 真菌發(fā)酵時(shí)間對納他霉素產(chǎn)量的影響分別在產(chǎn)黃青霉、黑曲霉、釀酒酵母培養(yǎng)1d、2d、3d時(shí)取樣，制備無菌發(fā)酵液。將培養(yǎng)不同時(shí)間的真菌發(fā)酵液以5%接種量加入培養(yǎng)12h的納塔爾鏈霉菌n5菌株發(fā)酵培養(yǎng)液中，每個(gè)

11、處理重復(fù)3次，并以不添加真菌發(fā)酵液的處理為對照。繼續(xù)培養(yǎng)84h后測定菌株納他霉素產(chǎn)量。確定3種真菌的最佳發(fā)酵時(shí)間。1.2.6 真菌發(fā)酵液添加量對納他霉素產(chǎn)量的影響將產(chǎn)黃青霉、黑曲霉培養(yǎng)1d，釀酒酵母培養(yǎng)3d，制備無菌發(fā)酵液，并分別以1% 、2% 、5% 、8% 、10% 、15% 、20%的添加量加入到培養(yǎng)12h的納塔爾鏈霉菌n5菌株發(fā)酵培養(yǎng)液中，每個(gè)處理重復(fù)3次并以不添加真菌發(fā)酵液的處理為對照。培繼續(xù)培養(yǎng)84h后測定菌株納他霉素產(chǎn)量。確定3種真菌的最佳發(fā)酵時(shí)間。2結(jié)果及分析2.1 納他霉素標(biāo)準(zhǔn)曲線的制作以納他霉素濃度為橫坐標(biāo)，od303值為縱坐標(biāo)繪制納他霉素標(biāo)準(zhǔn)曲線，見圖1。由圖1可看出納他

12、霉素濃度與od303值呈線性相關(guān)，且r2=0.9988線性較好。因此可通過此標(biāo)準(zhǔn)曲線方程x=（y0.0065)/0.1489計(jì)算納他霉素產(chǎn)量。圖1 納他霉素標(biāo)準(zhǔn)溶液曲線fig1 the standard curve of natamyctn2.2 發(fā)酵液加入時(shí)間對n5菌株納他霉素產(chǎn)量的影響2.2.1產(chǎn)黃青霉發(fā)酵液加入時(shí)間對n5菌株納他霉素產(chǎn)量的影響將產(chǎn)黃青霉發(fā)酵培養(yǎng)基進(jìn)行適當(dāng)處理后接種于培養(yǎng)不同時(shí)間的納塔爾鏈霉菌n5菌株發(fā)酵液中，繼續(xù)培養(yǎng)，當(dāng)菌株培養(yǎng)4d時(shí)用紫外分光光度法測定納他霉素產(chǎn)量，結(jié)果如圖2所示。由圖2可看出產(chǎn)黃青霉發(fā)酵液對n5菌株納他霉素產(chǎn)量有明顯的促進(jìn)作用，當(dāng)在發(fā)酵培養(yǎng)12h時(shí)添加

13、效果最佳，此時(shí)菌株納他霉素產(chǎn)量達(dá)1.42g/l，較對照提高65.96。 圖2 產(chǎn)黃青霉發(fā)酵液添加時(shí)間對納他霉素產(chǎn)量的影響fig 2 effects of addition time of fermentation broth of penicillium chrysogenum on the natamycin production of n5 strain2.2.2黑曲霉發(fā)酵液加入時(shí)間對n5菌株納他霉素產(chǎn)量的影響將培養(yǎng)3d的黑曲霉發(fā)酵培養(yǎng)液適當(dāng)處理后于不同時(shí)間添加入n5菌株發(fā)酵培養(yǎng)液中，繼續(xù)培養(yǎng)，當(dāng)菌株培養(yǎng)4d時(shí)測定納他霉素產(chǎn)量，結(jié)果見圖3。從圖3中可以看出黑曲霉發(fā)酵液對n5菌株納他霉素產(chǎn)量

14、的提高有顯著的促進(jìn)作用。當(dāng)在發(fā)酵培養(yǎng)12h時(shí)添加效果最佳，此時(shí)菌株納他霉素產(chǎn)量達(dá)1.44g/l，較對照提高67.44。圖3黑曲霉發(fā)酵液添加時(shí)間對納他霉素產(chǎn)量的影響fig 3 effects of addition time of fermentation broth of aspergillus nigeron the natamycin production of n5 strain2.2.3釀酒酵母發(fā)酵液加入時(shí)間對n5菌株納他霉素產(chǎn)量的影響釀酒酵母發(fā)酵液加入時(shí)間對n5菌株納他霉素產(chǎn)量的影響見圖4。由圖可見，釀酒酵母發(fā)酵液也可明顯促進(jìn)n5菌株納他霉素的合成，最適接入時(shí)間也是菌株培養(yǎng)的12h。

15、 圖4 釀酒酵母發(fā)酵液添加時(shí)間對納他霉素產(chǎn)量的影響fig 4 effects of addition time of fermentation broth of saccharomyces cerevisiae on the natamycin production of n5 strain2.3 真菌培養(yǎng)時(shí)間對n5菌株納他霉素產(chǎn)量的影響2.3.1 產(chǎn)黃青霉培養(yǎng)時(shí)間對n5菌株納他霉素產(chǎn)量的影響將分別培養(yǎng)1d、2d、3d的產(chǎn)黃青霉發(fā)酵液進(jìn)行適當(dāng)處理，添加至培養(yǎng)12h的n5菌株發(fā)酵培養(yǎng)液中，繼續(xù)培養(yǎng)84h后測定菌株納他霉素產(chǎn)量，結(jié)果如圖5所示。由圖5可以看出培養(yǎng)1d的產(chǎn)黃青霉發(fā)酵液對n5菌株納他霉

16、素產(chǎn)量的促進(jìn)作用最顯著。隨著產(chǎn)黃青霉培養(yǎng)時(shí)間的延長，發(fā)酵液對n5納他霉素產(chǎn)量的促進(jìn)作用呈下降趨勢。因此，產(chǎn)黃青霉的最佳培養(yǎng)時(shí)間為1d。圖5產(chǎn)黃青霉培養(yǎng)時(shí)間對納他霉素產(chǎn)量的影響fig 5 effects of culture time of penicillium chrysogenum on the natamycin production of n5 strain2.3.2黑曲霉培養(yǎng)時(shí)間對n5菌株納他霉素產(chǎn)量的影響 將培養(yǎng)不同時(shí)間的黑曲霉發(fā)酵液添加入n5菌株發(fā)酵液中，培養(yǎng)后測定納他霉素產(chǎn)量，結(jié)果見圖6。由圖6可以看出當(dāng)在n5菌株發(fā)酵液中添加培養(yǎng)1d的黑曲霉發(fā)酵液時(shí)，納他霉素產(chǎn)量最高，因此，黑

17、曲霉的最佳培養(yǎng)時(shí)間確定為1d。圖6黑曲霉培養(yǎng)時(shí)間對納他霉素產(chǎn)量的影響fig 6 effects of culture time of aspergillus niger on the natamycin production of n5 strain2.3.3釀酒酵母培養(yǎng)時(shí)間對n5菌株納他霉素產(chǎn)量的影響釀酒酵母培養(yǎng)時(shí)間對n5菌株納他霉素產(chǎn)量的影響如圖7所示。由圖7可見，當(dāng)在n5菌株發(fā)酵液中添加培養(yǎng)3d的釀酒酵母發(fā)酵液時(shí)，納他霉素產(chǎn)量最高，因此，釀酒酵母的最佳培養(yǎng)時(shí)間確定為3d。圖7釀酒酵母培養(yǎng)時(shí)間對納他霉素產(chǎn)量的影響fig 7 effects of culture time of saccha

18、romyces cerevisiae on the natamycin production of n5 strain2.4真菌發(fā)酵液添加量對n5菌株納他霉素產(chǎn)量的影響2.4.1產(chǎn)黃青霉發(fā)酵液添加量對n5菌株納他霉素產(chǎn)量的影響將培養(yǎng)1d的產(chǎn)黃青霉發(fā)酵液處理后以不同添加量添加于培養(yǎng)12h的納塔爾鏈霉菌n5菌株發(fā)酵培養(yǎng)液中，于28、200r/min條件下繼續(xù)培養(yǎng)84h，測定納他霉素產(chǎn)量。以產(chǎn)黃青霉發(fā)酵液添加量為橫坐標(biāo)，納他霉素產(chǎn)量為縱坐標(biāo)做圖，見圖8。從圖8中可以添加5的產(chǎn)黃青霉發(fā)酵液對n5菌株納他霉素的積累效果最顯著，此時(shí)納他霉素產(chǎn)量達(dá)1.69g/l，較對照提高94.25%。圖8產(chǎn)黃青霉發(fā)酵液添

19、加量對納他霉素產(chǎn)量的影響的影響fig 8 effects of addition amount of penicillium chrysogenum fermentation broth on the natamycin production of n5 strain2.4.2黑曲霉發(fā)酵液添加量對n5菌株納他霉素產(chǎn)量的影響將培養(yǎng)1d的黑曲霉發(fā)酵液處理后以不同添加量添加于培養(yǎng)12h的納塔爾鏈霉菌n5菌株發(fā)酵培養(yǎng)液中，于28、200r/min條件下繼續(xù)培養(yǎng)84h，測定納他霉素產(chǎn)量結(jié)果如圖8所示。由圖可看出5的黑曲霉發(fā)酵液對n5菌株納他霉素的積累效果最佳，因此，黑曲霉發(fā)酵液的最適添加量確定為5。 圖

20、9黑曲霉發(fā)酵液添加量對納他霉素產(chǎn)量的影響的影響fig 9 effects of addition amount of aspergillus niger fermentation broth onthe natamycin production of n5 strain2.4.3釀酒酵母發(fā)酵液添加量對n5菌株納他霉素產(chǎn)量的影響釀酒酵母發(fā)酵液添加量對n5菌株納他霉素產(chǎn)量的影響見圖10。從圖10中可以看出5的釀酒酵母發(fā)酵液對n5菌株納他霉素的積累效果最佳，因此，黑曲霉發(fā)酵液的最適添加量確定為5。圖10釀酒酵母發(fā)酵液添加量對納他霉素產(chǎn)量的影響的影響fig 10 effects of addition

21、 amount of saccharomyces cerevisiae fermentation broth onthe natamycin production of n5 strain3討論與結(jié)論 隨著人們對健康、安全問題的日益關(guān)注，食品安全問題越來越受到人們的重視，化學(xué)防腐劑的缺點(diǎn)逐漸暴露，納他霉素受到人們的青睞。納他霉素是和乳酸鏈球菌素并列的僅有的兩種生物防腐劑，其具有毒性低且不會(huì)造成食品風(fēng)味巨大改變的特性，是一種極具有使用、生產(chǎn)價(jià)值的抗生素。但納他霉素產(chǎn)生菌株產(chǎn)量不高一直是我國納他霉素自主生產(chǎn)的瓶頸，過低的單株產(chǎn)量會(huì)使生產(chǎn)成本過高而不利于工廠生產(chǎn)。本實(shí)驗(yàn)將產(chǎn)黃青霉、黑曲霉、釀酒酵母發(fā)

22、酵液添加入納塔爾鏈霉菌n5菌株發(fā)酵液中，研究這3種真菌培養(yǎng)時(shí)間、發(fā)酵液添加時(shí)間及添加量對n5菌株納他霉素產(chǎn)量的影響，以促進(jìn)菌株納他霉素的合成。實(shí)驗(yàn)結(jié)果表明這三種真菌的發(fā)酵液均對納塔爾鏈霉菌n5菌株的納他霉素產(chǎn)量有明顯促進(jìn)作用。產(chǎn)黃青霉和黑曲霉培養(yǎng)1d時(shí)，其發(fā)酵液對n5菌株納他霉素的合成促進(jìn)效果最佳，而釀酒酵母在3d時(shí)促進(jìn)效果最好。三種真菌最佳發(fā)酵液添加時(shí)間及添加量均為12h和5。具體這些真菌發(fā)酵液中何種因素對n5菌株納他霉素生產(chǎn)起到促進(jìn)作用還不清楚，有待于進(jìn)一步實(shí)驗(yàn)研究。參考文獻(xiàn)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 陳曉麗、呂振岳、黃東東等。新型天然食品防腐劑納他霉素的研究進(jìn)展j食品研究與開發(fā).2002.23（4）：23；4 盧國營、張朝輝、洪偉杰，新型防腐劑納他霉素的研究進(jìn)展j中國食品添加劑 2005（3）：3032；5 魏寶東、孟寬軍、天然生物性食品防腐劑納他霉素的特性及應(yīng)用j，遼寧農(nóng)業(yè)科學(xué) 2004 （2）：2426；6 賀家亮、張敏、趙勝娟，納他霉素的研究現(xiàn)狀j中國食品添加劑2005（3）：3032；7 martin j f demain al control of antibiotic biosynthesis 1980（02）；8 葉輝、王兆慧、陳佩林等 微生物誘導(dǎo)子對卡

25、諾氏菌屬n05205 菌株的影響j，生物技術(shù)，2004 14（6）：5758；9 邊猛、田黎、張久明等，誘導(dǎo)子對海洋真菌penicillium crustosum 204215菌株生物堿代謝的調(diào)節(jié)作用j海洋學(xué)報(bào)，2008.30（2）：117123；10 邊猛 海洋真菌產(chǎn)生生物堿代謝調(diào)節(jié)的研究d：碩士學(xué)位論文青島科技大學(xué) 生物化工系 。11 史強(qiáng) 真菌代謝產(chǎn)物中促進(jìn)納他霉素合成的誘導(dǎo)子的研究d：碩士學(xué)位論文江南大學(xué)。致謝感謝曹老師對于本試驗(yàn)的悉心指導(dǎo)，她對生物學(xué)科領(lǐng)域中的基本問題和發(fā)展前景的敏銳洞察力，對學(xué)術(shù)思想的重視，對選題和研究內(nèi)容的嚴(yán)格要求，以及對論文工作中遇到的關(guān)鍵問題和疑難問題的指導(dǎo)使

26、我受益匪淺。她對試驗(yàn)的研究態(tài)度和嚴(yán)謹(jǐn)?shù)墓ぷ髯黠L(fēng)給予我今后的學(xué)習(xí)和工作很深的啟示。同時(shí)我還要感謝一起做實(shí)驗(yàn)的同學(xué)，他們在我的實(shí)驗(yàn)過程中給了我很大的幫助。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 cerevisiaeemloyment tribunals sort out disagreements between employers and employees. you may need to make a claim to an employment tribunal if: you dont agr

31、ee 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 formal than some other courts, but

32、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 from one of the organisations list

33、ed 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 may reduce any compensation award

34、ed 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, the tribunal will not usually

35、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 agreement. in england and wales

36、, 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, including those that give advice by e-

37、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 150th anniversary celebrations and

38、 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 hasopened disciplinary proceedings

39、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 complaint. in a statement the russian

40、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 belief disillusionment, these days,

41、 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.一字開頭的年齡已經(jīng)到了尾聲?；蛟S是愧疚于自己似乎把轉(zhuǎn)瞬即逝的很多個(gè)不同的日子過成了同一天的樣子；或許是追溯過去，對自己那些近乎偏執(zhí)的怪異信念的醒悟，這些天以來，思緒一直很凌亂，在腦海中不斷糾纏?？傆X得自

42、己似乎應(yīng)該去做點(diǎn)什么，或者寫點(diǎn)什么。二十年的人生軌跡深深淺淺，突然就感覺到有些事情，非做不可了。the end of our life, and can meet many things really do?而窮盡我們的一生，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 found that those things are option

43、al; 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 vent to out your inner voice ca

44、n 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 remember his appearance.童年時(shí)，覺得壓歲錢和新

45、衣服是過年必備，但是隨著年齡的推進(jìn)，會(huì)越來越發(fā)現(xiàn)，那些東西根本就可有可無；初中時(shí)，以為要有一場暗戀才意味著真正的成長，但三年過去后，自己心平氣和的寫同學(xué)錄的時(shí)候，突然就發(fā)現(xiàn)是不是真正的成長了，好像并沒有那么重要了；然后到了高中，覺得非要吐露出自己的心聲才能為高中生涯里的懵懂情愫劃上一個(gè)句點(diǎn)，但畢業(yè)晚會(huì)的時(shí)候最終還是被梗塞在了咽喉，后來再次站在他曾經(jīng)揮汗如雨的球場，看著他投過籃球的球框時(shí)，突然間發(fā)現(xiàn)自己已經(jīng)想不起他的容顏。originally, this world, can produce a chemical reaction to an event, in addition to reso

46、lutely, have to do, and time.原來，這個(gè)世界上，對某個(gè)事件能產(chǎn)生化學(xué)反應(yīng)的，除了非做不可的堅(jiān)決，還有，時(shí)間。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 last. dislike his cowardice, fin

47、ally 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.一個(gè)人的時(shí)候，自己的想法總是特別地清晰。想要的，不想要的，界限明確，好像沒有什么可以撼動(dòng)自己。也曾經(jīng)好像已經(jīng)下定了決心去做某件事，但更多的時(shí)候是最后又打起了退堂鼓。嫌惡過自己的怯懦，最終卻

48、發(fā)現(xiàn)有很多緣分，有很多錯(cuò)過，好像冥冥之中真的已經(jīng)注定。那些曾經(jīng)所謂的非做不可，只是青蔥年華里自己給自己注射的一支強(qiáng)心劑，或者說，是自以為是的精神寄托罷了。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 beginning of deep friendship; have

49、 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，天空是陰暗的，空氣里有著剛下過雨之后的清新因子。突然想到那件藍(lán)格子襯衫；那些被折成各種各樣形狀的信紙；那段從街角深巷伊始的友誼；還有那場還沒有開始就宣告了終結(jié)的邂逅計(jì)劃那些年那些天的非做不可，終于和青春一樣，都將在我們的人生中謝幕。baumgartner the disappointing news: mission a

50、borted. 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 atmosphere (the troposphere) whe

51、re 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 layer (called the tropopause),e c

52、an 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 can try this all we want, in the

53、 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.生活發(fā)生不幸時(shí)，我們常常會(huì)關(guān)上心門；世界不僅沒能慰藉我們，反倒使我們更加消沉。我們假裝一切仿佛都不曾發(fā)生，以此試圖忘卻傷痛，可就算隱藏得再好，最終也還是騙不了自己。既然如此，何不嘗試打開心門，擁抱生活中的各種可能，讓世界感化我們呢？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.當(dāng)恐懼與焦慮來襲時(shí)，我們應(yīng)該退后一步，重新反思自己的言行。下面六個(gè)方法