1、1Outline of a fermentation processRaw MaterialsProduction microorganismFermentationProduct purificationProductEffluent wasteChapter 3 Strain Breeding and scale-up Five Common Characteristics of Microorganisms1.Small volume,large surface area2.Fast absorption and conversion3.Rapid duplication and gro
2、wth4.Strong adaptability5.Widespread distribution and diversified speciesWhat types of microorganisms cause fermentation to occur?nBacterianYeastnMoldnActinomycetes Bacteria Bacteria are typically one-celled organisms that multiply by simple division and can be seen only with a microscope.1.Lactic a
3、cid bacteriaLactobacillusyoghourt Streptococcusyoghourt Pediococcus sour picklesBifidobacteriumprobioticsLactobacillusStreptococcusPediococcusBifidobacterium2.Acetic acid bacteria AcetobactervinegarnCorynebacterium glutamicum4.BacillusBacillus subtilis-protease,amylase Bacillus licheniformis-proteas
4、e,amylase Yeast Yeasts are unicellular(single-celled)fungi and that ordinarily reproduce by budding.Happy YeastSaccharomyces cerevisiae 1.Saccharomyces Saccharomyces cerevisiaebeerSaccharomyces ellipsoideuswine 2.CandidaMould Fungi that grow in the form of multicellular filaments,called hyphae.In co
5、ntrast,microscopic fungi that grow as single cells are called yeasts.1.2.Rhizopus R.oryzoe amylase3.Aspergillus A.nigercitric acid,glucoamylase FungiPlant or animals ActinomycetesBacteriaActinomycetesMicrobes related to antibiotic productingStreptomyces-streptomycin About SCP,which is correct?A.Is a
6、 kind of protein which extracted from microbial cells.B.Is microbial cell which produced by fermentation.C.Is antibiotics secreted by microbial cells.D.Could not be regarded as food.Can antibiotic producting microbes be used in the amino acids production?Choosing Microorganismsfor Industrial Microbi
7、ology 1.The nutritional characteristics of the organism.2.The productivity of the organism,3.The stability of the organism and its amenability to genetic manipulation.4.The ease of product recovery from the culture.Strain SelectionPurchase from Culture CollectionsScreening of nature circumstances Mu
8、tationsGenetic engineering International Culture CollectionsAbbreviationNameLocationATCCAmerican Type Culture CollectionUnited StatesIAM Institute of Applied MicrobiologyUniversity of Tokyo,JapanNCTCNational Collection of Type CulturesLondon,United kingdomChina Culture CollectionsCGMCCChina General
9、Microbiological Culture Collection CenterBeijingCulture Collections原种斜面初筛，复筛，再复筛采样设计实验方案调查研究及查阅充分的资料 增殖培养平板分离分离单株纯种分离分离菌种鉴定Steps of new species separation and screening Screening of nature circumstances Most major sources of microbes for use in industrial microbiology are natural materialsMicrobial
10、strain collection 1.soil slurry 2.Enrichment Special media Control condition Inhibit unwanted strain3.Isolation cultivation Streak plate method Pour plate method Spread plate method Streak plate method nBacteria are“streaked”over the surface of an agar plate so as to obtain single colonies.nIt can a
11、lso highlight the presence of contaminating micro-organisms.This is an example of a good streak for isolation using the four corners method.Performing a Plate Streak IPerforming a Plate Streak IIPour plate method Bacteria are mixed with melted agar.Poured into an empty plate and allowed to solidify.
12、Spread plate method Small samples of the diluted bacteria are pipetted onto the surface of agar plates.A sterile,bent-glass rod is then used to spread the bacteria evenly over the entire agar surface.稀释的稀释的4.Pure cultivationGrow only one kind of microbeMust use aseptic technique to avoid contaminati
13、ng the cultureTransfer a single colony from agar plate to liquid medium5.Productivity measurement Primary screening：quantity Secondary screening：qualityIf I couldnt find an ideal strain from nature,how would I do?q Spontaneous(natural)mutationq Induced mutationmutationTechnique for inducing mutation
14、 Physical mutagens e.g.X-rays,g g-rays,UV Chemical mutagens e.g.base analogs,nitrous acid,alkylating agents碱基类似物碱基类似物亚硝酸亚硝酸烷化剂烷化剂Steps:Steps:分离和筛选出发菌株的选择处理菌悬液的制备诱变处理中间培养 How are mutants detected by scientists?nVisiblenNutritional(auxotrophic)nWhat is replica plating,and how is it used to detect auxo
15、trophic mutants?nResistance mutations(plate on media containing the chemical)营养缺陷型营养缺陷型影印培养法影印培养法降解纤维素产生透明圈摇床复筛Replica-plating technique to screen for mutant strains of a colony-forming microorganism.Q:How would you select for protease(amylase,celluase or lase)-producing mutant strain?Cell Biology T
16、echniquesnProtoplastnRemoving the cell wall with lytic enzymes in the presence of osmotic stabilizers.nIn the presence of fusogenic agent such as polyethylene glycol(PEG),protoplasts are induced to fuse and form transient hybrids or diploids.nRegeneration of viable cells from the fused protoplasts.融
17、合剂融合剂二倍体二倍体Genetic EngineeringVarious high value added products have been produced fromgenetic engineeringmethods.Culture preservation International Culture CollectionsAbbreviationNameLocationATCCAmerican Type Culture CollectionUnited StatesIAM Institute of Applied MicrobiologyUniversity of Tokyo,Ja
18、panNCTCNational Collection of Type CulturesLondon,United kingdomChina Culture CollectionsCGMCCChina General Microbiological Culture Collection CenterBeijingdryoxygen deficit low temperaureCulture preservationOligotrophe菌种保藏的方法很多，一般有下面几种：A：斜面冰箱保藏法B：沙土管保藏法 C：石蜡油封存法 D：真空冷冻干燥保藏法E：液氮超低温保藏法 If fermentatio
19、n in a large tank receive only one loop of inoculum,a prolonged period would result.energy consumingcontamination Necessity of inoculum developmentInoculum Development The preparation of a population of microorganisms from a dormant stock culture to an active state of growth that is suitable for ino
20、culation in the final production stage.休眠的休眠的 So the inoculum volume has to be quite largeA seed fermenter is usually required to produce the inoculum volumeThe seed fermenters purpose is not to produce product but to prepare inoculumAeration:通风,通气 In inoculum developmentInoculum level:approximately
21、 0.5 to 5 percent inoculum by volume from the preceding step.Incubation period:short,in log growth phase,little fermentation productInoculum media:balanced for rapid cell growth and not for product formation.The process of inoculum development实验室阶段实验室阶段1、培养物选择的原则、培养物选择的原则目的：种子扩培到一定的量和质，根据菌种的特点最终的目的：
22、种子扩培到一定的量和质，根据菌种的特点最终的培养物可分为两类：培养物可分为两类：q 对于不产孢子和芽胞的微生物对于不产孢子和芽胞的微生物 获得一定数量和质量的菌体获得一定数量和质量的菌体对于不产芽孢和孢子的微生物，实验室阶段的种子扩培最对于不产芽孢和孢子的微生物，实验室阶段的种子扩培最终是获得一定数量和质量的菌体，如谷氨酸的种子培养。终是获得一定数量和质量的菌体，如谷氨酸的种子培养。获得一定数量和质量的孢子获得一定数量和质量的孢子菌丝体培养步骤少，因而更容易获得量和质稳定的种菌丝体培养步骤少，因而更容易获得量和质稳定的种子，但操作繁琐。子，但操作繁琐。q 对于产孢子的微生物对于产孢子的微生物
23、获得一定数量和质量的菌丝体获得一定数量和质量的菌丝体便于操作，但需要更仔细的控制。便于操作，但需要更仔细的控制。2、培养基选择的原则、培养基选择的原则培养基的选择应该是有利于菌体的生长，对孢子培养基培养基的选择应该是有利于菌体的生长，对孢子培养基应该是有利于孢子的生长应该是有利于孢子的生长。在原料方面，实验室种子培养阶段，规模一般比较小，在原料方面，实验室种子培养阶段，规模一般比较小，因此为了保证培养基的质量，培养基的原料一般都比较因此为了保证培养基的质量，培养基的原料一般都比较精细。精细。3、起始接种物的传代问题、起始接种物的传代问题q 细菌细菌保藏斜面保藏斜面 活化斜面活化斜面q 产孢子产
24、孢子保藏保藏 母斜面母斜面 子斜面子斜面目的目的：使菌种的传代次数尽可能的少。：使菌种的传代次数尽可能的少。母瓶母瓶：活化、纯化，使保藏菌种生长，并去除变异株。：活化、纯化，使保藏菌种生长，并去除变异株。所以接种时要稀一点、便于纯化生长到单菌落。所以接种时要稀一点、便于纯化生长到单菌落。子瓶子瓶:大量繁殖，得到大量孢子。大量繁殖，得到大量孢子。接种：从母斜面上点接种，选取生长好的接种：从母斜面上点接种，选取生长好的 单菌落单菌落 接种时密一点，得到大量的孢子。接种时密一点，得到大量的孢子。孢子培养时注意湿度，子斜面使用一般不超过孢子培养时注意湿度，子斜面使用一般不超过1个月个月孢子培养生产车间
25、阶段生产车间阶段1、培养物的选择原则、培养物的选择原则在生产车间阶段，最终一般都是获得一定数量的菌丝体。在生产车间阶段，最终一般都是获得一定数量的菌丝体。q 缩短发酵时间缩短发酵时间q 有利于获得好的发酵结果有利于获得好的发酵结果菌丝体比孢子要有利：菌丝体比孢子要有利：Inoculation of a Fermenter from another Seed TankSeedTankFermenterA BTrap TrapSteamCJDIHEFG2、培养基选择的原则、培养基选择的原则目的：获得一定数量和质量的菌体，因此培养基的选择应目的：获得一定数量和质量的菌体，因此培养基的选择应首先考虑的
26、是有利于孢子的发育和菌体的生长首先考虑的是有利于孢子的发育和菌体的生长.在原料方面：不如实验室阶段那么精细，而是基本接近于在原料方面：不如实验室阶段那么精细，而是基本接近于发酵培养基，这有两个方面的原因发酵培养基，这有两个方面的原因:一是成本一是成本 二是驯化二是驯化q 薯干粉薯干粉成分成分 斜面斜面 种子罐种子罐 发酵发酵麦芽汁麦芽汁 麦汁麦汁 薯干粉薯干粉 10%22-28%(NH4)2SO4 0.5%琼脂琼脂 2%3、发酵级数的确定、发酵级数的确定谷氨酸：三级发酵谷氨酸：三级发酵一级种子（摇瓶）一级种子（摇瓶）二级种子二级种子（小罐）（小罐）发酵发酵青霉素：三级发酵青霉素：三级发酵一级种
27、子一级种子（小罐）（小罐）二级种子（中罐）二级种子（中罐）发酵发酵 一般由菌丝体培养开始计算发酵级数，但有时，一般由菌丝体培养开始计算发酵级数，但有时，工厂从第一级种子罐开始计算发酵级数工厂从第一级种子罐开始计算发酵级数发酵级数确定的依据发酵级数确定的依据 q 级数受发酵规模、菌体生长特性、接种量的影响级数受发酵规模、菌体生长特性、接种量的影响q 级数大，难控制、易染菌、易变异，管理困难，一级数大，难控制、易染菌、易变异，管理困难，一 般般2-4级。级。q 在发酵产品的放大中，反应级数的确定是非常重要在发酵产品的放大中，反应级数的确定是非常重要 的一个方面的一个方面Aeration:通风,通气
28、 Industrial Fermentation Setting Review Questions1.Cite some micro-organisms used widely in food industry and their corresponding products.2.How to get an ideal strain for fermentation industry?3.Whats the purpose of inoculum development?Cite the process of it.3.Why can not the micro-organisms product excessive metabolites spontaneously?By which methods can we get the excessive metabolites?4.How to reduce the concentration of end products in fermentation?5.How to improve the permeability of cell membrane?
