本文選題：嗜熱鏈球菌 + 胞外多糖��； 參考：《哈爾濱工業(yè)大學(xué)》2017年碩士論文

【摘要】：嗜熱鏈球菌(Streptococcus thermophilus,S.thermophilus)是世界上公認(rèn)的最具經(jīng)濟(jì)價(jià)值的同型發(fā)酵乳酸菌之一,廣泛用于乳制品的生產(chǎn)。嗜熱鏈球菌胞外多糖(Exopolysaccharides,EPS)由于其具有改善乳制品的品質(zhì),并已被證實(shí)具有抗氧化、抑菌等生物活性,而具有廣泛的應(yīng)用前景。本研究首先采用苯酚硫酸法和3,5-二硝基水楊酸法對(duì)從本實(shí)驗(yàn)室前期分離獲得的22株S.thermophilus CS1~CS22產(chǎn)EPS能力進(jìn)行評(píng)價(jià),比較不同菌株所產(chǎn)EPS含量的差異,從中篩選確定高產(chǎn)EPS的優(yōu)良菌株CS5、CS6、CS9和CS18。本研究以產(chǎn)EPS含量較高的S.thermophilus CS6為研究對(duì)象,對(duì)該菌EPS合成條件進(jìn)行優(yōu)化。采用單因素實(shí)驗(yàn)確定傳代次數(shù)、接種量、發(fā)酵溫度、后熟時(shí)間和碳源(乳糖、半乳糖、果糖、葡萄糖和蔗糖)等影響因素的范圍。利用Plackett-Burman設(shè)計(jì)來(lái)篩選單因素中關(guān)鍵因素;利用響應(yīng)面的中心復(fù)合實(shí)驗(yàn)CCD法確定最佳培養(yǎng)條件如下:接種量4.34%,發(fā)酵溫度44°C,脫脂乳含量10%,后熟時(shí)間24 h,后熟溫度4°C,傳代次數(shù)3代,乳糖含量為1.7%,并進(jìn)行驗(yàn)證,得到EPS粗提取物的含量為343.75 mg/L,與預(yù)測(cè)值接近,且比優(yōu)化前提高了11.64%。本研究通過(guò)對(duì)S.thermophilus CS6進(jìn)行補(bǔ)充碳源(麥芽糖、甘露糖、甘露醇、木糖和核糖)和氮源(蛋白胨、大豆蛋白胨、胰蛋白胨、牛肉膏和乳清濃縮蛋白)優(yōu)化實(shí)驗(yàn),同時(shí)對(duì)最大影響EPS產(chǎn)量的碳源(麥芽糖)和氮源(大豆蛋白胨)進(jìn)行梯度實(shí)驗(yàn)設(shè)計(jì),得到它們的最佳濃度范圍,為碳源和氮源的進(jìn)一步優(yōu)化做準(zhǔn)備,最終為后續(xù)即將完成eps基因簇序列分析的全部22株S.thermophilus產(chǎn)EPS的培養(yǎng)條件優(yōu)化奠定基礎(chǔ)。本研究對(duì)22株S.thermophilus eps基因簇的測(cè)定:首先對(duì)已公布的eps基因簇進(jìn)行分析、歸類,并根據(jù)其5’端deoD和3’端orf14.9之間基因的保守序列設(shè)計(jì)引物(共計(jì)8對(duì));分別以22株S.thermophilus的基因組DNA為模板,擴(kuò)增eps基因簇,對(duì)PCR產(chǎn)物鑒定、純化及測(cè)序,序列進(jìn)行拼接,獲得完整的eps基因簇序列。本研究最終得到3株S.thermophilus(CS2、CS6和CS10)完整的eps基因簇序列信息,以及其它菌株大部分eps基因簇片段的序列信息。序列分析發(fā)現(xiàn),菌株CS2、CS6和CS10均與S.thermophilus ASCC 1275菌株(ID:CP006819.1)的eps基因簇具有高度同源性,含有額外的與EPS長(zhǎng)度相關(guān)的eps2C和eps2D基因,推測(cè)有利于EPS的合成;同時(shí)含有豐富的糖基轉(zhuǎn)移酶基因,負(fù)責(zé)葡萄糖、半乳糖、鼠李糖、UDP-N-乙酰葡萄糖胺和UDP-呋喃半乳糖的轉(zhuǎn)運(yùn),有利于形成獨(dú)特單體組成的EPS。以上研究,為進(jìn)一步研究eps基因簇在菌株產(chǎn)EPS差異中的作用奠定基礎(chǔ)。
[Abstract]:Streptococcus thermophilus S. thermophilus (S. thermophilus) is recognized as one of the most economically valuable homozygous lactic acid bacteria in the world and is widely used in dairy production. Streptococcus thermophilus Exopolysaccharide EPSs (EPSs) have a wide application prospect due to its improved quality of dairy products, and has been proved to have antioxidant, bacteriostatic and other biological activities. In this study, the EPS production ability of 22 S.thermophilus CS1 CS22 strains isolated from our laboratory was evaluated by phenol sulfuric acid method and 3zhudinitrosalicylic acid method, and the EPS content of different strains was compared. The high EPS producing strains CS5, CS6, CS9 and CS18 were screened. In this study, S. thermophilus CS6 with high EPS content was used as the research object to optimize the EPS synthesis conditions of S. thermophilus CS6. Single factor experiments were used to determine the range of factors affecting the passage, inoculation, fermentation temperature, post-ripening time and carbon sources (lactose, galactose, fructose, glucose and sucrose). Plackett-Burman design was used to screen the key factors in the single factor, and the optimum culture conditions were determined by the CCD method of central composite experiment of response surface as follows: inoculation amount 4.34, fermentation temperature 44 擄C, degreasing milk content 10, ripening time 24 h, post-ripening temperature 4 擄C, passage times 3 generations. The content of lactose was 1.7mg / L, and the content of crude extract of EPS was 343.75 mg / L, which was close to the predicted value and increased 11.64% than that before optimization. In this study, S. thermophilus CS6 was optimized for carbon sources (maltose, mannitol, xylose and ribose) and nitrogen sources (peptone, soybean peptone, tryptone, beef extract and whey concentrate protein). At the same time, the gradient experimental design of carbon source (maltose) and nitrogen source (soybean peptone), which has the greatest influence on EPS yield, was carried out, and the optimum concentration range was obtained, so as to prepare for the further optimization of carbon source and nitrogen source. The results laid a foundation for the optimization of the culture conditions of all 22 strains of S. thermophilus which were about to complete the sequence analysis of eps gene cluster. In this study, 22 strains of S. thermophilus eps gene cluster were determined. Firstly, the published eps gene cluster was analyzed and classified. According to the conserved sequence of 5 'end deoD and 3' terminal orf14.9, primers were designed (8 pairs in total), 22 S. thermophilus genomic DNA was used as template to amplify the eps gene cluster, and the PCR products were identified, purified, sequenced and sequenced. The complete eps gene cluster sequence was obtained. In this study, the complete sequence information of eps gene cluster and the sequence information of most eps gene cluster fragments of other strains were obtained. Sequence analysis showed that the eps gene cluster of S.thermophilus ASCC 1275 had high homology and contained additional EPS length related eps2C and eps2D genes, which suggested that CS2 + CS6 and CS10 were beneficial to the synthesis of eps, and there were abundant glycosyltransferase genes. The transport of glucose, galactose, rhamnose UDP-N-acetylglucosamine and UDP-furan galactose is beneficial to the formation of unique monomer EPSs. The above studies laid a foundation for further study on the role of eps gene cluster in the differences of EPS production.
【學(xué)位授予單位】：哈爾濱工業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：TS252.1

【參考文獻(xiàn)】

相關(guān)期刊論文 前8條

1 樊曉輝;趙春玲;張成軍;李陽(yáng);萬(wàn)端極;;3,5-二硝基水楊酸法測(cè)定枸杞提取液中多糖的含量[J];食品研究與開(kāi)發(fā);2014年21期

2 胡盼盼;宋微;單毓娟;杜明;張?zhí)m威;;影響乳酸菌胞外多糖產(chǎn)量的因素[J];食品科技;2014年09期

3 任曉輝;姜淑娟;錢方;牟光慶;;嗜熱鏈球菌胞外多糖的分離純化及理化性質(zhì)研究[J];食品與機(jī)械;2012年01期

4 王興華;鄭學(xué)瑞;邢會(huì)霞;任麗萍;;嗜熱鏈球菌產(chǎn)胞外多糖的研究[J];山西大學(xué)學(xué)報(bào)(自然科學(xué)版);2009年03期

5 劉宇;孟祥晨;;乳酸菌胞外多糖及其抗腫瘤活性[J];中國(guó)乳品工業(yè);2008年01期

6 劉宇峰,王金英,曲曉軍,于沖,孫建華,夏海華;西藏靈菇菌的菌相菌學(xué)的研究[J];中國(guó)乳品工業(yè);2005年09期

7 王蕾,鄭璞;DNS法定量測(cè)定海藻糖的研究[J];食品科技;2004年02期

8 趙玉臣,王迎俊,王忠鳳;乳酸菌胞外多糖[J];中國(guó)乳業(yè);2003年04期

相關(guān)碩士學(xué)位論文 前2條

1 張麗;嗜熱鏈球菌胞外多糖的多樣性與發(fā)酵乳黏度相互關(guān)系研究[D];哈爾濱工業(yè)大學(xué);2012年

2 齊沙沙;產(chǎn)粘嗜熱鏈球菌的篩選及其胞外多糖的純化與流變性質(zhì)研究[D];江南大學(xué);2011年

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