锁掷酵母发酵产物中类胡萝卜素的分离、鉴定及体外活性研究
发布时间：2019-05-10 14:26
【摘要】：类胡萝卜素因其广泛的生理功能和难以替代的食用、药用价值,被认为是最具发展潜力的天然色素类食品添加剂之一。本实验室前期研究发现锁掷酵母(Sporidiobolus pararoseus)菌株的发酵产物可能含有β-胡萝卜素、γ-胡萝卜素、圆酵母素(torulene)、红酵母红素(torularhodin)等多种类胡萝卜素。其中圆酵母素和红酵母红素与番茄红素化学结构相似,可能具有抗氧化、抗癌等多种生物活性,但目前尚未见到国内外相关报道。 因此,本研究首先采用有机溶剂提取得到色素混合溶液,后利用硅胶柱分离纯化。纯化的色素通过薄层层析、高效液相色谱及质谱分析(LC-ESI-MS)对其种类进行鉴定。而后,采用DDPH(1,1-二苯基-2-三硝基苯肼)自由基清除体系、亚油酸抗氧化体系及过氧化氢诱导离体人肝癌细胞SK-HEP-1氧化损伤体系进行纯化色素抗氧化性的评价;并通过体外培养细胞(人肝癌细胞SK-HEP-1)形态学观察、细胞存活力实验分析纯化色素对体外培养细胞的作用,乳酸脱氢酶(LDH)检测、细胞周期分析其作用机理。通过以上研究得到以下结果: (1)锁掷酵母发酵产物中的色素通过硅胶柱层析可以得到有效分离,根据其色谱结果判定所得色素的性质符合类胡萝卜素的特性,圆酵母素和红酵母红素是锁掷酵母发酵产物。 (2) DDPH自由基清除体系和亚油酸抗氧化体系结果表明,圆酵母素和红酵母红素均具有较强的体外抗氧化活性,且圆酵母素的抗氧化活性尤为显著。过氧化氢诱导离体人肝癌细胞SK-HEP-1氧化损伤体系的结果表明实验浓度范围内,圆酵母素和红酵母红素并不单一表现出抗氧化修复的作用,高浓度条件下可能会出现协同损伤的现象。 (3)经圆酵母素和红酵母红素处理的细胞可导致SK-HEP-1细胞发生皱缩、细胞颗粒增多等形态学改变。细胞存活力实验显示圆酵母素和红酵母红素可以显著抑制SK-HEP-1细胞存活力,作用强度呈时间-剂量效应,并且红酵母红素的48 h的半抑制浓度为2.4μmol/L,显著强于番茄红素。 (4)圆酵母素和红酵母红素处理SK-HEP-1细胞后,均未观测到LDH释放增加的现象,说明该两种类胡萝卜素对细胞存活力的抑制和细胞毒性无关。流式分析结果显示圆酵母素和红酵母红素都是通过抑制细胞周期,使SK-HEP-1细胞阻滞在G0/G1期达到抑制增殖的结果,细胞增殖指数明显降低。 综上所述,圆酵母素和红酵母红素是锁掷酵母的发酵产物,具有较强的体外抗氧化活性,可通过阻滞细胞周期来抑制体外培养人肝癌细胞SK-HEP-1的增殖,且红酵母红素的抑制作用显著强于番茄红素。
[Abstract]:Carotenoids are considered to be one of the most potential natural pigment food additives because of their extensive physiological functions and irreplaceable edible and medicinal value. Previous studies in our laboratory found that the fermentation products of (Sporidiobolus pararoseus) strain may contain many kinds of carotene, such as 尾-carotene, gamma-carotene, circular yeast (torulene), red yeast lycopene (torularhodin) and so on. Among them, the chemical structure of circular yeast and red yeast lycopene is similar to that of lycopene, and may have many biological activities, such as antioxidation, anticancer and so on, but there are no reports at home and abroad. Therefore, in this study, the pigment mixed solution was extracted by organic solvent, and then separated and purified by silica gel column. The purified pigment was identified by thin layer chromatography, high performance liquid chromatography and mass spectrometry (LC-ESI-MS). Then, DDPH (1,1-diphenyl-2-trinitrophenylhydrazine) free radical scavenging system, linoleic acid antioxidant system and hydrogen peroxide-induced SK-HEP-1 oxidative damage system were used to evaluate the antioxidant activity of purified pigments. The morphological observation of human hepatoma cell line (SK-HEP-1) was carried out in vitro. The effect of purified pigment on the cultured cells was analyzed by cell viability test. Lactate dehydrogenase (LDH) (LDH) and cell cycle analysis were used to analyze the mechanism of action of the purified pigment on the cultured cells in vitro. The results are as follows: (1) the pigment of yeast fermentation product can be separated effectively by silica gel column chromatography, and the properties of the pigment can accord with the character of carotenoid, according to the chromatographic results, the results show that the pigment in yeast fermentation product can be separated effectively by silica gel column chromatography. Saccharomyces cerevisiae and Saccharomyces cerevisiae are the fermentation products of lock-throw yeast. (2) the results of DDPH free radical scavenging system and linoleic acid antioxidant system showed that both lycopene and lycopene had strong antioxidant activity in vitro, and the antioxidant activity of lycopene was especially significant. The results of oxidative damage system of human hepatocellular carcinoma cell line SK-HEP-1 induced by hydrogen peroxide showed that Saccharomyces cerevisiae and Saccharomyces cerevisiae did not have the effect of antioxidant repair in the range of experimental concentration. The phenomenon of synergistic damage may occur under the condition of high concentration. (3) the cells treated with Saccharomyces cerevisiae and Saccharomyces cerevisiae could cause morphological changes such as shrinkage and increase of cell particles in SK-HEP-1 cells. Cell viability test showed that lycopene and lycopene could significantly inhibit the viability of SK-HEP-1 cells in a time-dose dependent manner, and the semi-inhibitory concentration of lycopene was 2.4 渭 mol / L for 48 h. Significantly stronger than lycopene. (4) the increase of LDH release was not observed in SK-HEP-1 cells treated with circular yeast and mycopene, which indicated that the inhibitory effect of these two carotenoids on cell viability was not related to cytotoxicity. The results of flow analysis showed that both circular yeast and red yeast lycopene could inhibit the proliferation of SK-HEP-1 cells in G0/G1 phase by inhibiting the cell cycle, and the cell proliferation index was significantly decreased. In conclusion, Saccharomyces cerevisiae and Saccharomyces cerevisiae are the fermentation products of Saccharomyces cerevisiae, which have strong antioxidant activity in vitro and can inhibit the proliferation of human hepatocellular carcinoma cell line SK-HEP-1 by blocking the cell cycle. The inhibitory effect of Saccharomyces cerevisiae was significantly stronger than that of lycopene.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R151
本文编号：2473718
[Abstract]:Carotenoids are considered to be one of the most potential natural pigment food additives because of their extensive physiological functions and irreplaceable edible and medicinal value. Previous studies in our laboratory found that the fermentation products of (Sporidiobolus pararoseus) strain may contain many kinds of carotene, such as 尾-carotene, gamma-carotene, circular yeast (torulene), red yeast lycopene (torularhodin) and so on. Among them, the chemical structure of circular yeast and red yeast lycopene is similar to that of lycopene, and may have many biological activities, such as antioxidation, anticancer and so on, but there are no reports at home and abroad. Therefore, in this study, the pigment mixed solution was extracted by organic solvent, and then separated and purified by silica gel column. The purified pigment was identified by thin layer chromatography, high performance liquid chromatography and mass spectrometry (LC-ESI-MS). Then, DDPH (1,1-diphenyl-2-trinitrophenylhydrazine) free radical scavenging system, linoleic acid antioxidant system and hydrogen peroxide-induced SK-HEP-1 oxidative damage system were used to evaluate the antioxidant activity of purified pigments. The morphological observation of human hepatoma cell line (SK-HEP-1) was carried out in vitro. The effect of purified pigment on the cultured cells was analyzed by cell viability test. Lactate dehydrogenase (LDH) (LDH) and cell cycle analysis were used to analyze the mechanism of action of the purified pigment on the cultured cells in vitro. The results are as follows: (1) the pigment of yeast fermentation product can be separated effectively by silica gel column chromatography, and the properties of the pigment can accord with the character of carotenoid, according to the chromatographic results, the results show that the pigment in yeast fermentation product can be separated effectively by silica gel column chromatography. Saccharomyces cerevisiae and Saccharomyces cerevisiae are the fermentation products of lock-throw yeast. (2) the results of DDPH free radical scavenging system and linoleic acid antioxidant system showed that both lycopene and lycopene had strong antioxidant activity in vitro, and the antioxidant activity of lycopene was especially significant. The results of oxidative damage system of human hepatocellular carcinoma cell line SK-HEP-1 induced by hydrogen peroxide showed that Saccharomyces cerevisiae and Saccharomyces cerevisiae did not have the effect of antioxidant repair in the range of experimental concentration. The phenomenon of synergistic damage may occur under the condition of high concentration. (3) the cells treated with Saccharomyces cerevisiae and Saccharomyces cerevisiae could cause morphological changes such as shrinkage and increase of cell particles in SK-HEP-1 cells. Cell viability test showed that lycopene and lycopene could significantly inhibit the viability of SK-HEP-1 cells in a time-dose dependent manner, and the semi-inhibitory concentration of lycopene was 2.4 渭 mol / L for 48 h. Significantly stronger than lycopene. (4) the increase of LDH release was not observed in SK-HEP-1 cells treated with circular yeast and mycopene, which indicated that the inhibitory effect of these two carotenoids on cell viability was not related to cytotoxicity. The results of flow analysis showed that both circular yeast and red yeast lycopene could inhibit the proliferation of SK-HEP-1 cells in G0/G1 phase by inhibiting the cell cycle, and the cell proliferation index was significantly decreased. In conclusion, Saccharomyces cerevisiae and Saccharomyces cerevisiae are the fermentation products of Saccharomyces cerevisiae, which have strong antioxidant activity in vitro and can inhibit the proliferation of human hepatocellular carcinoma cell line SK-HEP-1 by blocking the cell cycle. The inhibitory effect of Saccharomyces cerevisiae was significantly stronger than that of lycopene.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R151
【引证文献】
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2 杜超;锁掷酵母中圆酵母素和红酵母红素对前列腺疾病干预机制的研究[D];江南大学;2016年
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2 应丹瑜;S. pararoseus JD-2酵母油对血脂异常小鼠脂代谢影响的研究[D];江南大学;2017年
3 吴佳伶;红酵母红素对氧化受损PC12细胞的保护作用[D];江南大学;2015年
4 刘艳萍;掷孢酵母Sporobolomyces sp.YB 2013的分离鉴定及其所产色素的检测[D];西南大学;2015年
5 施秋妤;锁掷酵母圆酵母素的分离鉴定及生物活性研究[D];江南大学;2014年
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