南台科技大学
化学工程研究所生物技术组 硕士论文
硫磺菌之液态培养条件及其生理活性研究
The Bioactivities of Laetiporus sulphureus by Investigation of Growth Condition
研 究 生:郑淑蓉
指导教授:陈健祺 陈启桢 博士
中华民国九十三年七月
致
谢
由衷感谢论文指导老师陈健祺博士及陈启桢博士於硕士生涯中悉 心的指导与启迪薰陶,在研究过程中,提供一切之协助与支持,得以完 成本论文.撰稿期间,更於百忙之中就论文架构,格式及内容加以斧正, 使学生论文得以顺利完成. 文稿初成,复蒙林锡璋博士,林正辉博士拨冗斧正,并提供诸多宝 贵的意见,受益匪浅,由衷感谢. 接著,要感谢真绮於实验初期的指导与照顾;感谢育真,57 号同学 在实验上的倾囊相授,不厌其烦地解答我的大小问题;感谢青桦,佩岑, 品儒,必莹,欣怡,致良,琼月,嘉徽,锦珊於实验上的协助;感谢靖 缇,小陆及学妹祉彤,阿贵陪我度过难忘的岁月;也感谢教程的各位伙 伴们,若不是你们平日的帮忙,鱼与熊掌难以兼得.更要感谢 Patrick, Carol,Amy,娟在我最低潮时陪我走过,让我懂得转换角度去思考,从 逆境中走出,更懂得珍惜身边所有. 最后,深深感谢多年来家人对我无尽的关怀与支持,驻足回首,若 非你们的包容与这一路上许多人的帮助与提携,难以成就此篇论文.
摘
要
本研究探讨在不同培养条件下对硫磺菌生长速率之影响,利用固态培养基探 讨最适培养硫磺菌之 pH 值,温度及培养基,再以液态培养探讨最适生长培养基 之碳氮源组成及比例.结果显示,在 24℃,pH 6.0 下以蛋白腖 0.5 % + 麦芽抽 出物 3 % 为培养基,菌丝体生长状况最好. 将液态培养所得之硫磺菌发酵滤液( the filtrate from Laetiporus sulphureus of submerged culture; LSSC )处理肿瘤细胞株,结果显示,随著发酵天数递增,对癌 细胞株的存活率有递减现象产生,亦即液态培养天数越久,抑制癌细胞生长能力 也随之增加.且菌丝体生长状况愈好之发酵滤液有愈佳抑制肿瘤细胞增生能力. 以有最佳抑制肿瘤细胞生长之培养基组合(蛋白腖 0.5 % + 麦芽抽出物 3 %)之第 12 天发酵液处理肿瘤细胞后,细胞存活率如下:HT-1080 10 %,MCF-7 17%, MDA-MB-231 28 %,DU-145 24%,HeLa 15 %,Hep G2 26 %. 以硫磺菌发酵液处理肿瘤细胞后以 Gelatin Zymography analysis 分析后,发 现硫磺菌发酵液对转移蛋白 MMP-9 有抑制效果.以培养基组合(蛋白腖 0.5 % + 麦芽抽出物 5 %)之第 12 天发酵液处理 HT1080 MCF-7 MDA-MB-231 及 DU-145 , , 后,MMP-9 蛋白质表现强度依序为: 12 %,8 %,0 %,0 %.显示硫磺菌发酵液 有强烈的抑制转移蛋白质 MMP-9 能力.另外,本研究亦以相同的样品进行 RT-PCR 分析,检测 MMP-9 mRNA 基因之表现,结果如下:10%,1.2%,16%, 3%. 癌细胞株抗转移试验方面,亦证实第 12 天 P0.5M5 发酵滤液对 HT-1080 细胞 的抑制转移能力具有浓度效应.与上述结果比对,证实了硫磺菌发酵滤液的确有 抑制肿瘤细胞转移能力.
抗生活性方面,以硫磺菌发酵滤液处理 E. coli,Staphylococcus aureus subsp aureus,Pseudomonas aeruginosa 等三株细菌,发现硫磺菌之发酵滤液不具抗生 活性.
I
其他生理活性,如:免疫能力或抗氧化能力,皆随著硫磺菌液态培养天数 的增加,其发酵滤液皆有正面意义之提升效果.另外,实验结果显示以上这些生 理活性与多醣体无关.
关键字:硫磺菌,菌丝体,液态培养,抑癌试验,抗转移试验,免疫能力,抗氧 化能力,DPPH,螯合亚铁离子能力,抗生活性,多醣体
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Abstract
The aim of this thesis selected optimum cultural medial combination of Laetiporus sulphureus on agar. Afterward by modified conditions to study anti-tumor by submerged culture fermentation in 100/250 mL flask. The optimum culture condition was medial components of peptone 0.5% and malt extract 3 % at 24℃ initial on pH 6.0 by 100 rpm shaking for 12 days. The biological activity for anti-tumor was assayed by MTT assay. The cell lines inhibition ability of the broth from submerged culture fermentation (LSSC) had remarkable effect. The result showed that the medial component of peptone 0.5% and malt extract 3% on the 12th day had 10 % survival rate against HT-1080, 17 % against MCF-7, 28 % against MDA-MB-231, 24 % against DU-145, 15 % against HeLa, and 26 % against Hep G2. The secretion of matrix metalloproteinases (MMP-2 / MMP-9) was assayed by Gelatin Zymography analysis. Zymography assay revealed excellent result, which the MMP-9 proteins were obviously suppressed while HT-1080, MCF-7, MDA-MB-231, and DU-145 cells were treated with the 12th day broth from the medial component of peptone 0.5% and malt extract 5%. The results have shown that the inhibition of MMP-9 were 12 %, 8 %, 0 %, and 0 % of control respectively. The mRNA expression level was assayed by RT-PCR assay. It also resulted excellently that the mRNA expression level of tumor cells ( HT-1080, MCF-7, MDA-MB-231, and DU-145 ) were 10 %, 1.2 %, 16%, and 3% of control. The result of invasion assay in HT-1080 (with PMA) treated with the 12th day broth from the medial component of peptone 0.5% and malt extract 3% has also shown remarkable effect. It proved that the LSSC has contributed to the inhibitory

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