本文選題：葡萄糖耐量因子 + YSI-3.7酵母菌株　； 參考：《天津科技大學(xué)》2017年碩士論文

【摘要】：鉻是人體必需的微量元素,對(duì)人體糖脂代謝具有重要的作用。糖尿病患者及老年人通過(guò)適量補(bǔ)鉻(Cr3+),可顯著恢復(fù)糖耐量損傷狀況,維持體內(nèi)糖代謝平衡。從酵母菌中提取純化出的天然葡萄糖耐量因子(Glucose tolerance factor,GTF),其活性中心也為Cr3+。因此,本文通過(guò)提取純化GTF,并將其應(yīng)用于胰島素抵抗型Hep-G2細(xì)胞模型中,研究GTF結(jié)構(gòu)及其對(duì)細(xì)胞糖脂代謝的作用。全文旨在建立優(yōu)良、高效的GTF純化方法,得到高產(chǎn)、高純度的GTF物質(zhì),同時(shí)探究GTF在降糖降脂功能上的作用。首先,本文通過(guò)鉻平板篩選出最高可在鉻離子濃度為1000 μg/mL平板上生長(zhǎng)的耐鉻啤酒酵母菌YSI-3.7,將其接種于含鉻發(fā)酵液中,并逐步增加培養(yǎng)基中鉻離子濃度。結(jié)果表明,當(dāng)發(fā)酵液中鉻添加量達(dá)到500 μg/mL時(shí),測(cè)得其有機(jī)鉻積累最多,達(dá)到1818μg/g,且有機(jī)鉻百分含量達(dá)到了 90.34%。同時(shí),將富鉻馴化得到的高產(chǎn)GTF酵母菌株分別進(jìn)行不同規(guī)模的發(fā)酵實(shí)驗(yàn),包括搖瓶發(fā)酵、15 L發(fā)酵、50 L發(fā)酵以及500 L發(fā)酵,通過(guò)測(cè)定其有機(jī)鉻的含量發(fā)現(xiàn),有機(jī)鉻含量顯著下降,由搖瓶發(fā)酵的2160μg/mL下降至500L中試發(fā)酵的765 μg/mL;有機(jī)鉻占總鉻含量的百分比也由87.17%下降至61.55%;菌體干生物量由0.89g/100mL到1.01g/100mL,反而有所增加�？傮w來(lái)看,隨著發(fā)酵規(guī)模的擴(kuò)大,酵母菌中有機(jī)鉻和總鉻含量呈下降趨勢(shì)。其次,將制備得到的高產(chǎn)GTF的酵母菌粉經(jīng)氨水提取,30%乙醇、0℃冰浴沉淀20 min,離心取上清液,測(cè)得其鉻含量與蛋白含量的比值為5.02,相比單獨(dú)使用氨水提取得到的0.818, GTF的提取率提高了六倍以上。將乙醇沉淀后的粗提物依次經(jīng)過(guò)SuperdexTM75和Sephadex G25凝膠層析分離,得到GTF純化物質(zhì),之后對(duì)其進(jìn)行高效液相色譜(High performance liquid chromatography,HPLC)分析。結(jié)果表明,經(jīng)過(guò)氨水提取,乙醇沉淀及兩級(jí)柱層析純化分離后,可得到純度較高的GTF物質(zhì)。同時(shí),本文通過(guò)HPLC及核磁共振(Nuclear magnetic resonance,NMR)實(shí)驗(yàn)對(duì)GTF結(jié)構(gòu)進(jìn)行分析,得出谷氨酸和天門(mén)冬氨酸為GTF的主要氨基酸成分。采用HPLC和電感耦合等離子體質(zhì)譜(Inductively coupled plasma - Mass spectrometry,ICP-MS )技術(shù)聯(lián)用對(duì)GTF中鉻價(jià)態(tài)進(jìn)行分析,得出GTF中只存在Cr3+。最后,本文還采用濃度為10-6mol/L胰島素作用Hep-G2細(xì)胞48 h,建立胰島素抵抗細(xì)胞模型,此細(xì)胞模型可在48h內(nèi)穩(wěn)定維持胰島素抵抗特性。建模成功后,將GTF及其他鉻制劑應(yīng)用于模型細(xì)胞中,通過(guò)測(cè)定細(xì)胞葡萄糖消耗量、甘油三酯含量(Triglyceride,TG)及游離脂肪酸含量(Non-esterified fatty acid,NEFA),對(duì)細(xì)胞的糖脂代謝作用進(jìn)行評(píng)價(jià),得出GTF鉻含量在1.0 μg/mL,作用24 h時(shí),對(duì)細(xì)胞葡萄糖代謝的促進(jìn)作用最明顯,葡萄糖消耗量達(dá)到18.07mmol/L,和對(duì)照組相比增加了 5%;同時(shí),還將鉻含量為1.0 μg/mL的GTF及其他鉻制劑共同作用于胰島素抵抗模型細(xì)胞中,結(jié)果顯示,鉻制劑對(duì)細(xì)胞糖脂代謝均有促進(jìn)作用,其中GTF作用最顯著(P 0.01 )。綜上所述,本文通過(guò)富鉻馴化,篩選得到高耐鉻啤酒酵母菌株YSI-3.7,經(jīng)過(guò)氨水提取、30%乙醇沉淀、兩級(jí)層析柱分離,提取純化GTF,通過(guò)氨基酸組成分析及NMR試驗(yàn)得出天門(mén)冬氨酸和谷氨酸為GTF主要成分;HPLC-ICP-MS技術(shù)聯(lián)用得出GTF中只存在Cr3+一種鉻價(jià)態(tài);采用高胰島素作用Hep-G2細(xì)胞建立胰島素抵抗模型,將GTF及其它鉻制劑分別作用于模型細(xì)胞中,研究分析其對(duì)細(xì)胞糖脂代謝的作用。
[Abstract]:Chromium is an essential trace element in human body, which plays an important role in the metabolism of glycolipid in the human body. By using a proper amount of chromium supplementation (Cr3+), the diabetic patients and the elderly can significantly restore the impaired glucose tolerance and maintain the balance of glucose metabolism in the body. The natural glucose tolerance factor (Glucose tolerance factor, GTF) extracted from the yeast and its activity The heart also is Cr3+., therefore, the purpose of this paper is to extract and purify GTF and apply it to the insulin resistance Hep-G2 cell model to study the structure of GTF and its effect on the metabolism of glycemic cells. The full text aims to establish an excellent and efficient GTF purification method, to obtain high yield and high purity GTF substances, and to explore the role of GTF in reducing the function of glucose lowering and lipid lowering. A chromium resistant Saccharomyces cerevisiae YSI-3.7, which can grow on the 1000 g/mL plate of chromium ion concentration, was screened by chromium plate, and inoculated into the chromium containing fermented liquid and gradually increased the concentration of chromium ion in the medium. The results showed that the accumulation of chromium in the fermentation broth was up to 500 g /mL, and the accumulation of organic chromium was up to 1818 mu. G/g, and the content of organic chromium was 90.34%., and the high yield GTF yeast strains were fermented at different sizes, including shake flask fermentation, 15 L fermentation, 50 L fermentation and 500 L fermentation. By measuring the content of organic chromium, the content of organic chromium decreased significantly, and 2160 g/mL decreased from shake flask fermentation. The 765 micron g/mL was fermented in 500L, and the percentage of organic chromium in the total chromium content decreased from 87.17% to 61.55%; the dry biomass of the bacteria increased from 0.89g/100mL to 1.01g/100mL. As a whole, the content of chromium and total chromium in the yeast decreased with the expansion of the fermentation scale. Secondly, the high yield GTF yeast was prepared. The bacteria powder was extracted by ammonia water, 30% ethanol and 20 min in 0 centigrade ice bath. The ratio of chromium content to protein content was 5.02, and the extraction rate of GTF was six times higher than that obtained by the extraction of ammonia water alone. The crude extract after ethanol precipitation was separated by SuperdexTM75 and Sephadex G25 gel chromatography in turn. After purifying the substance to GTF, the high performance liquid chromatography (High performance liquid chromatography, HPLC) was analyzed. The results showed that the purity of GTF substance could be obtained after the extraction of ammonia, the ethanol precipitation and the purification and separation of the two column chromatography. At the same time, the experiment was carried out by HPLC and nuclear magnetic resonance (Nuclear magnetic resonance, NMR). The TF structure is analyzed and the main amino acid components of GTF are glutamic acid and aspartic acid. HPLC and inductively coupled plasma mass spectrometry (Inductively coupled plasma Mass spectrometry, ICP-MS) are used to analyze the valence state of chromium in GTF. It is concluded that GTF is in the last of Cr3+., and the concentration is also used as the islet. Hep-G2 cells were treated with 48 h to establish an insulin resistance cell model. This cell model could maintain insulin resistance stability in 48h. After the modeling, GTF and other chromium preparations were applied to the model cells, and the glucose consumption, triglyceride content (Triglyceride, TG) and free fatty acid content (Non-esterified f) were measured. Atty acid, NEFA), to evaluate the glycolipid metabolism of cells, it is concluded that the content of GTF chromium is 1 u g/mL, when the action of 24 h, the promotion of glucose metabolism is the most obvious, the consumption of glucose reaches 18.07mmol/L, which is increased by 5% compared with the control group; meanwhile, the chromium content of 1 u g/mL and other chromium preparations are combined in the pancreas. In the island element resistance model cells, the results showed that the chromium preparation had the promotion effect on the cell glycolipid metabolites, and the GTF effect was most significant (P 0.01). In summary, the high chromium resistant Saccharomyces cerevisiae strain YSI-3.7 was screened through the acclimation of chromium rich, through the ammonia extraction, the 30% ethanol precipitation, the two level chromatography column separation, the extraction and purification of GTF, through the amino acid extraction. The composition analysis and NMR test showed that aspartic acid and glutamic acid were the main components of GTF; HPLC-ICP-MS was used to conclude that only a chromium valence state in Cr3+ was found in GTF; the insulin resistance model was established by high insulin action Hep-G2 cells, and GTF and other chromium preparations were used in the model cells respectively, and the metabolism of cell glycolipid was studied and analyzed. Effect.
【學(xué)位授予單位】：天津科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R587.1

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