第四章灭菌英文

4.1 IntroductionA fermentation product is produced by the culture of a certain organism, or organisms, in a nutrient medium. If the fermentation is invaded by a foreign micro-organism then the following consequences may occur:1.The medium would have to support the growth of both the production organism and the contaminant, resulting in a loss of productivity.2.If the fermentation is a continuous one then the contaminant may outgrow the production organism and displace it from the fermentation.3.The foreign organism may contaminate the final product, e.g. single-cell protein where the cells, separated from the broth, constitute the product.4.The contaminant may produce compounds which make subsequent extraction of the final product difficult.5.The contaminant may degrade the desired product; this is common in bacterial contamination of antibiotic fermentations where the contaminant would have to be resistant to the normal inhibitory effects of the antibiotic and degradation of the antibiotic is a common resistance mechanism, e.g. the degradation of -lactam antibiotics(-内酰胺类抗生素 ) by -lactamase producing bacteria.6. Contamination of a bacterial fermentation with phage could result in the lysis of the culture.Avoidance of contamination may be achieved by:1.Using a pure inoculum to start the fermentation.2.Sterilizing the medium to be employed.3.Sterilizing the fermenter vessel.4.Sterilizing all materials to be added to the fermentation during the process5.Maintaining aseptic condition during the fermentation.The extent to which these procedures are adopted is determined by the likely probability of contamination and the nature of its consequences. Some fermentations are described as protected-that is, the medium may be utilized by only a very limited range of micro-organisms, or the growth of the process organism conditions, such as a reduction in pH. The brewing of beer falls into this category; hop resins tend to inhibit the growth of many micro-organisms and the growth of brewing yeasts tends to decrease the pH of the medium. Thus, brewing worts are boiled, but not necessarily sterilized, and the fermenters are thoroughly cleaned with disinfectant solution but are not necessarily sterile.Also, the precautions used in the development of inoculum for brewing are far less stringen than, for example, in an antibiotic fermentation. However, the vast majority of fermentations are not protected and, if contaminated, would suffer some of the consequences previously listed. The approaches adopted to avoid contamination will now be discussed in more detail.Basic terms sterilization灭菌 disinfection消毒 Antisepsis 防腐 Bacteriostasis： bk,tiristeisis抑菌 Asepsis 无菌 Factors influencing antimicrobial activity the concentration and kind of an agent used the length of exposure to the agent the temperature at which the agent is used the number of microorganisms present the kinds of microorganisms present the existence of organic materials4.2 物理消毒灭菌法Controlling Microorganisms By Physical Agents一、热力灭菌法 (Heat)二、辐射灭菌法 (Radiation)三、滤过除菌法 (Filtration)四、超声灭菌法 (Supertronic)五、低温干燥抑菌法 (Desiccation)六、低温（ Low Temperature ）4.2.1.2 Moist heat 湿热灭菌方法： denature proteins and melt lipids; more effective Autoclaving: 高压蒸汽灭菌 Boiling water煮沸灭菌 Pasteurization巴氏消毒法 Fractional sterilization间歇蒸气灭菌法 Continuous sterilization连续蒸汽灭菌法1) Steam heating to 100 for 30 min 常压 /流动蒸气消毒法 Vegetative cells are destroyed but endospores survive 2) Incubate at 30 -37 overnight Most bacterial endospores germinate 3) Second heat treatment, 100 , 30 min Germinated endospores are killed. 4) Second incubation at 30 -37 overnight Remaining endospores germinate 5) Third heat treatment, 100 , 60 min Last remaining germinated endospores are killed Fractional sterilization间歇蒸气灭菌法 1 :Ionizing Radiation 电离辐射 X-rays and gamma rays more energy and penetrating power than UV used to sterilize pharmaceuticals and disposable medical supplies such as syringes, surgical gloves, catheters导尿管 , and sutures缝合线 used to retard spoilage in sea foods, meats, poultry, and fruits 射线 、高能量的电子束（阴极射线）不升高温度且穿透力强适于忌热物品的灭菌或消毒 Microwave penetrating non-metal materials (glass, plastics, china) Sterilize solutions that may be damaged or denatured by high temperatures or chemical agents 4.2.3 滤过除菌 Filtration l主要用于一些 不耐热 的血清、毒素、抗生素、药液以及空气等除菌。l不能除去病毒、支原体和型细菌 Heavy metals (Hg2+、 Ag+) denaturing proteins and inactivating enzymes 2% mercurochrome（红汞） or 0.1% merthiolate（硫柳汞） skin, mucosa and woundbacteriostatic, ineffective against endospores 1% silver nitrate（硝酸银） eye drops for newborns to prevent gonococcal ophthalmia（淋菌性眼炎）Acids and alkaliesAlter membrane permeability and denature proteins and other moleculesSalts of organic acids: food preservatives Undecylenic acid十一烯酸 : dermatophyte皮肤真菌 infections In virtually all fermentation processes, it is mandatory for a cost-effective operation to have contamination-free seed cultures at all stages, from the preliminary culture to the production fermenter. A bioreactor can be sterilized either by destroying the organisms with some lethal agent such as heat, radiation, or a chemical, or by removing the viable organisms by a physical procedure such as filtration.1、由于杂菌的污染，使生物反应中的基质或产物因杂菌的消耗而损失，造成生产能力的下降； 2、由于杂菌所产生的一些代谢产物，或在染菌后改变了培养液的某些理化性质，使产物 的提取和分离变得困难，造成收率降低或使产品的质量下降；3、杂菌会大量繁殖，会改变反应介质的 PH值，从而使生物反应发生异常变化；4、杂菌可能会分解产物，从而使生产过程失败；5、发生噬菌体污染，微生物细胞被裂解，而使生产失败，等等在发酵生产中，为什么要进行灭菌操作？问题 1：在一定温度下，微生物的受热死亡遵照分子反应速度理论。在灭菌过程中，活菌数逐渐减少，其减少量随残留活菌数的减少而递减，即微生物的死亡速率与任一瞬时残存的活菌数成正比， 4.4.1.1 微生物的死亡速率和理论灭菌时间The process of destroying a population generally follows first-order kinetics. Using the initial number of cell N0/ml, the number of destroyed organisms N at time t(min), and the surviving cells N, the death rate can be calculated as follows:(k is the specific death constant/min)When integrated between N0 at time t=0 and N at time t=t, the following equation is obtained:kt-=InN/N0 OrInN/N0=-ktThe ratio of N/N0 is the inactivation factor, the ratio of N/N0 is the survival factor and the InN/N0 =V is the design criterion, a parameter which encompasses the contamination level of the medium to be sterilized, N0, and the desired sterility level , N.The ideal curve expressing the exponential decline in survivors with time is plotted in follow Figure:This line has a negative slope and in following Figure part2 corredponds to the death curve for vegetative cells of Escherichia coli at 54-60 . This type of killing kinetics is referred to as a logarithmic death curve. In actuality, this curve is not always linear, as shown for the death curve of Bacillus stearothermophilus spore.Batch sterilizationMost nutrient media are presently sterilized in batch volumes in the bioreactor at 121 . Approximate sterilization times can be calculated from the nature of the medium and the size of the fermenter. Not only the nutrient media, but also the fittings, valves and electrodes of the fermenter itself must be sterilized. Therefore, actual sterilization times are significantly longer than calculated ones and must be empirically determined for the specific nutrient solutions in the fermenter. One method of sterilization is to inject steam into the fermenter mantle or interior coils (indirect sterilization). Another method is to inject steam into the nutrient solution itself (direct procedure), in which case pure steam (free of chemical additives) is a prerequisite pri:rekwizit. Many industrial steam supplies contain potentially toxic chemicals de