烟草内生放线菌Y_（12）鉴定、培养及其发酵产物抑菌活性的初步研究
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摘要
奉论文以湖南中医药大学谭周进教授课题组分离得到的烟草内生放线菌Y12为研究对象,分别对该菌株的分类地位,菌株发酵产物抑菌活性,发酵条件优化,发酵产物稳定性进行了研究,所得结果如下：
1通过该菌的形态特征、培养性状、生理生化特征及16SrRNA测序进行综合实验分析,鉴定该菌与白浅灰链霉菌(streptomyces albogriseolus)最为接近。
2对Y12发酵培养基的筛选、组成和发酵条件进行了优化,最终确定最佳发酵培养基为：黄豆粉1.5%,蛋白胨1.5%,葡萄糖1.5%,可溶性淀粉1.5%,硫酸镁0.05%,硫酸铵0.25%,氯化钠0.4%,磷酸二氢钾0.1%,碳酸钙0.5%。最佳发酵条件为：培养基初始pH值为7.5,发酵温度30℃,发酵时间72 h。经过优化发酵液的抑菌圈直径增加了44.9%。
3抑菌试验结果表明,该菌发酵液对大肠杆菌(Escherichia coli)、金黄色葡萄球菌(Staphylococcus aureus)、青枯雷尔氏菌(Ralstonia solanacoarum)和枯草芽孢杆菌(Bacillus subtil is)的抑菌圈直径分别为28.1mm、29.2mm、22.8mm、20.3mm。连续培养l0代后,其发酵产物对以上几种菌的抑菌活性没有明显变化,有很好的遗传稳定性。
4烟草内生放线菌Y12发酵产物的稳定性实验结果表明,该发酵产物的热稳定性较好,发酵产物在60℃温度水浴稳定性较好,30min后抑菌活性比率只下降了3.01%,90℃水浴30min后其活性比率下降了11.7%。100℃下水浴1h活性比率下降21.5%。在紫外光照射稳定性好,距离30cm在紫外灯下7h后其活性比率下降9.2%。该发酵产物在酸性条件下稳定性好,在pH为4时活性比率下降9.08%,在碱性环境下其活性下降比较明显,在pH为11时已经失去了抑菌活性。在常温下对时间的稳定性比较差,常温下8d后其活性比率为59.7%。研究结果显示,该菌发酵产物对酸、紫外线、热稳定性较好,常温下储存时间对该菌的稳定性有较大的影响。
With the endophytic actinomycetes Y12 separated by professor Tan Zhoujin's Study Group from Hunan University of Chinese Medicine as the study subject, the author make a series of experiments on strain identification, optimal fermentation condition and the fermentation product's antimicrobial activity and stability. The results are as follows:
1. According to morphological, cultural characteristics, physiological and biochemical characteristics and 16SrRNA sequence analysis, the strain Y12 was similar to streptomyces albogriseolus.
2. Through a series of orthogonal experiments on Y12 including the selection and components of the fermentation medium and the optimal fermentation conditions, it is determined that the optimal fermentation medium was:1.5% soybean flour,1.5% peptone,1.5% glucose,1.5% soluble starch,0.05% MgSO4·7H2O,0.25%(NH4)SO4, 0.4% NaCl,0.1% KH2PO4 and 0.5% CaCO3. The optimal fermentation conditions were:optimal pH was 7.5, optimal fermentation temperature was 30℃, optimal fermentation time was 72h. It was observed from the experiments that the diameters of inhibition zones increased by 44.9% under such optimized conditions.
3. It is indicated in the inhibition tests that the fermentation broth of strain Y12 against Escherichia coli, Staphylococcus aureus, Ralstonia solanacoarum, and Bacillus subtilis had obvious inhibitive effect, the diameters of inhibition zones were 28.1mm,29.2mm,22.8mm and 20.3mm respectively. The antimicrobial activity of the fermentation broth had no obvious changes when strain Y12 was sucessively cultivated 10 generations; and is stable to generations.
4. The results of stability study on Y12's fermentation product suggested that it is stable to heat, with the activity ratio only decreasing by 3.01%,11.7%, and 21.5% when heated by 60℃,90℃and 100℃for 30 minutes respectively; The fermentation product is also stable to ultraviolet radiations and acid environment. The activity ratio keeps at 90.8% when radiated by the ultraviolet for 7 hours from the 30cm distance and declines by 9.08% with the pH at 4; however, when situated under the alkaline environments, its activity ratio will decrease evidently, with no activity when the pH reaching 11. Besides, its stability will also decrease with time going on under room temperature, with the activity ratio keeping at only 59.7% after 8 days. It is concluded heat, ultraviolet radiations and acid environment have little effect on the stability of Y12's fermentation broth while the storage time under room temperature influences the stability considerably.
1通过该菌的形态特征、培养性状、生理生化特征及16SrRNA测序进行综合实验分析,鉴定该菌与白浅灰链霉菌(streptomyces albogriseolus)最为接近。
2对Y12发酵培养基的筛选、组成和发酵条件进行了优化,最终确定最佳发酵培养基为：黄豆粉1.5%,蛋白胨1.5%,葡萄糖1.5%,可溶性淀粉1.5%,硫酸镁0.05%,硫酸铵0.25%,氯化钠0.4%,磷酸二氢钾0.1%,碳酸钙0.5%。最佳发酵条件为：培养基初始pH值为7.5,发酵温度30℃,发酵时间72 h。经过优化发酵液的抑菌圈直径增加了44.9%。
3抑菌试验结果表明,该菌发酵液对大肠杆菌(Escherichia coli)、金黄色葡萄球菌(Staphylococcus aureus)、青枯雷尔氏菌(Ralstonia solanacoarum)和枯草芽孢杆菌(Bacillus subtil is)的抑菌圈直径分别为28.1mm、29.2mm、22.8mm、20.3mm。连续培养l0代后,其发酵产物对以上几种菌的抑菌活性没有明显变化,有很好的遗传稳定性。
4烟草内生放线菌Y12发酵产物的稳定性实验结果表明,该发酵产物的热稳定性较好,发酵产物在60℃温度水浴稳定性较好,30min后抑菌活性比率只下降了3.01%,90℃水浴30min后其活性比率下降了11.7%。100℃下水浴1h活性比率下降21.5%。在紫外光照射稳定性好,距离30cm在紫外灯下7h后其活性比率下降9.2%。该发酵产物在酸性条件下稳定性好,在pH为4时活性比率下降9.08%,在碱性环境下其活性下降比较明显,在pH为11时已经失去了抑菌活性。在常温下对时间的稳定性比较差,常温下8d后其活性比率为59.7%。研究结果显示,该菌发酵产物对酸、紫外线、热稳定性较好,常温下储存时间对该菌的稳定性有较大的影响。
With the endophytic actinomycetes Y12 separated by professor Tan Zhoujin's Study Group from Hunan University of Chinese Medicine as the study subject, the author make a series of experiments on strain identification, optimal fermentation condition and the fermentation product's antimicrobial activity and stability. The results are as follows:
1. According to morphological, cultural characteristics, physiological and biochemical characteristics and 16SrRNA sequence analysis, the strain Y12 was similar to streptomyces albogriseolus.
2. Through a series of orthogonal experiments on Y12 including the selection and components of the fermentation medium and the optimal fermentation conditions, it is determined that the optimal fermentation medium was:1.5% soybean flour,1.5% peptone,1.5% glucose,1.5% soluble starch,0.05% MgSO4·7H2O,0.25%(NH4)SO4, 0.4% NaCl,0.1% KH2PO4 and 0.5% CaCO3. The optimal fermentation conditions were:optimal pH was 7.5, optimal fermentation temperature was 30℃, optimal fermentation time was 72h. It was observed from the experiments that the diameters of inhibition zones increased by 44.9% under such optimized conditions.
3. It is indicated in the inhibition tests that the fermentation broth of strain Y12 against Escherichia coli, Staphylococcus aureus, Ralstonia solanacoarum, and Bacillus subtilis had obvious inhibitive effect, the diameters of inhibition zones were 28.1mm,29.2mm,22.8mm and 20.3mm respectively. The antimicrobial activity of the fermentation broth had no obvious changes when strain Y12 was sucessively cultivated 10 generations; and is stable to generations.
4. The results of stability study on Y12's fermentation product suggested that it is stable to heat, with the activity ratio only decreasing by 3.01%,11.7%, and 21.5% when heated by 60℃,90℃and 100℃for 30 minutes respectively; The fermentation product is also stable to ultraviolet radiations and acid environment. The activity ratio keeps at 90.8% when radiated by the ultraviolet for 7 hours from the 30cm distance and declines by 9.08% with the pH at 4; however, when situated under the alkaline environments, its activity ratio will decrease evidently, with no activity when the pH reaching 11. Besides, its stability will also decrease with time going on under room temperature, with the activity ratio keeping at only 59.7% after 8 days. It is concluded heat, ultraviolet radiations and acid environment have little effect on the stability of Y12's fermentation broth while the storage time under room temperature influences the stability considerably.
引文
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