摘要
肉毒梭状芽孢杆菌(Clostridium botulinum,简称肉毒梭菌)在厌氧环境
下可产生一系列毒性极强烈的外毒素——肉毒毒素(botulinum toxin,BTX)。
目前,肉毒毒素已广泛应用于眼科、神经科、康复科、消化科及皮肤科(多汗症、
美容)等领域的病症治疗,并在美容抗皱方面有极好的效果。此外,肉毒毒素也
可作为一种新的生物灭鼠农药。因而,肉毒毒素具有重要的应用价值。
本论文对肉毒毒素的产生菌——肉毒梭菌的筛选方法、分离纯化及检测手段
进行了初步的研究,得出了以下结果:
1.从豆制品、肉制品、水产品等食品加工场所采集了50份样品,结果表明
芽孢杆菌以在豆制品加工场所的土壤样品中为多。
2.针对芽孢杆菌的筛选,样品预处理采用了两种方法——加热处理法和乙醇
处理法。试验结果表明,乙醇处理法的效果优于加热处理。
3.经富集培养、分离培养及多次分离纯化后,最终从豆腐坊附近土壤样品中
获得一株具有肉毒梭菌典型特征的菌株。
4.对筛选得到的菌株进行了初步的鉴定,该菌株革兰氏染色呈阳性,为粗大
杆菌,两侧平行,两端钝园,直杆状或稍弯曲,芽孢为卵圆形,位于次极端,呈
网球拍形;乳糖卵黄牛奶平板上,其菌落下培养基为乳浊,菌落表面及周围形成
彩虹薄层,与肉毒梭菌的典型形态特征相符。生理生化试验结果表明该菌株属于
分解蛋白质的肉毒梭菌B、F组中的一型。毒性试验结果说明筛选得到的菌株属
于B型肉毒梭菌。免疫学鉴定表明菌株所产毒素与B型抗血清出现沉淀反应,确
定该菌株属于B型肉毒梭菌。依据毒性试验及免疫学鉴定结果基本确认该菌株即
B型肉毒梭菌。
5.毒性试验中,大体判定该菌株发酵液中毒素的毒力为1000~10000MLD/ml,
毒力较高。
Clostridium botulinum (C. botulinum) in the anaerobic condition can produce botulinum toxin (BTX), a neurotoxin,which can inhibit acetylcholine 's release after injected into muscles .At present, Botulinum toxin has been used to therapy some diseases. Botulinum toxin is widely applied to clinical therapy, medicinal hairdressing, and new pattern biological pesticide.
This assay preliminarily studied screening condition and separation method to achieve a strain of C. botulinum, which can produce stable and abundant botulinum toxin. The results were as follows:
1.50 samples were respectively collected from the possessing places of leguminous food workshop, meaty food plant and aquatic products. We finally screened a strain of C. botulinum from soil of leguminous food workshop.
2.Countering the screening of bacilli, samples were pre-treated by two methods —thermal disposal and alcohol disposal .The results declared that alcohol method was prior to thermal disposal.
3.After proliferate cultivation, disport cultivation and purification, a strain, which had the same typical characteristic with C.botulinum , was obtained from soil surrounding leguminous food workshop.
The identification of the strain included microstructure, cultural characteristic, physiological and biochemical feature. Comparison with the identification characteristic of C. botulinum, the strain belonged to serotype B or F of C. botulinum.
Toxicity test is the most important evidence that identifies a certain serotype of C. botulinum. The result of this experiment indicated that the strain was serotype B of C. botulinum. Immune-serum reaction also obtained the same result. On the basis of the results of these two tests, the strain was identified as serotype B of C. botulinum.
4.The toxicity of fermentative liquor was about 1000~10000MLD/ml.
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