4微生物培養(yǎng)基Culturalmedium

1、14. 微生物培養(yǎng)基微生物培養(yǎng)基 Cultural medianA cultural medium is the substance in which a specific organism lives and grows. It must contain the essential nutrients needed for the microbe to grow.nA good medium is very important to the success of an industrial fermentation. The medium supplies nutrients for gro

2、wth, energy, building of cell substance, and biosynthesis of fermentation products. 24.1 培養(yǎng)基的類型及功能培養(yǎng)基的類型及功能 Classifications of media and their functions n按純度分類按純度分類： -合成培養(yǎng)基合成培養(yǎng)基 chemically defined media: compound identity and concentration of all components are known. Synthetic media -天然培養(yǎng)基天然培養(yǎng)基 che

3、mically undefined media: ingredients include one or more chemically complex substances such as protein hydrolysates and extracts. Crude media 3nThe choice of defined or undefined medium is dependent upon its application. -Chemically defined media are useful in biochemical or metabolic studies of org

4、anisms. -General laboratory growth media and industrial media are often chemically complex. Such as 花花生餅、蛋白胨等。一般不需外加微量元素生餅、蛋白胨等。一般不需外加微量元素 (trace elements)、維生素等。、維生素等。 4n按狀態(tài)分類按狀態(tài)分類： -Solid media：suitable to culture and preserve species and spores; cultivating fungi. -Liquid media: 80%-90%water, indu

5、strial fermentation media. -Semi-solid media: liquid medium + agar 主要用于鑒定細(xì)菌、觀察細(xì)菌運(yùn)動特征等。主要用于鑒定細(xì)菌、觀察細(xì)菌運(yùn)動特征等。5n按用途分類按用途分類 孢子培養(yǎng)基、種子培養(yǎng)基和發(fā)酵培養(yǎng)基孢子培養(yǎng)基、種子培養(yǎng)基和發(fā)酵培養(yǎng)基1.1.孢子培養(yǎng)基孢子培養(yǎng)基 spore media 要求：使菌體迅速生長，產(chǎn)生較多的優(yōu)質(zhì)孢子，要求：使菌體迅速生長，產(chǎn)生較多的優(yōu)質(zhì)孢子，不易引起菌種發(fā)生變異不易引起菌種發(fā)生變異 (variation)。 基本配制要求：基本配制要求： not rich in nutrition, especia

6、lly organic nitrogen soure. optimal mineral salt concentration optimal pH and humidity 6n生產(chǎn)上常用孢子培養(yǎng)基生產(chǎn)上常用孢子培養(yǎng)基： 麩皮、大米、小米培養(yǎng)基；由葡萄糖、蛋白麩皮、大米、小米培養(yǎng)基；由葡萄糖、蛋白胨、牛肉膏和食鹽等配制的瓊脂斜面培養(yǎng)基。胨、牛肉膏和食鹽等配制的瓊脂斜面培養(yǎng)基。大米和小米常用作霉菌孢子培養(yǎng)基，因它們大米和小米常用作霉菌孢子培養(yǎng)基，因它們含氮少、疏松、表面積大，是較好的孢子培含氮少、疏松、表面積大，是較好的孢子培養(yǎng)基。水分控制在養(yǎng)基。水分控制在21212525。7n種子培養(yǎng)基種子培

7、養(yǎng)基 inoculum media: 種子培養(yǎng)基種子培養(yǎng)基是供孢子發(fā)芽、生長和大量繁是供孢子發(fā)芽、生長和大量繁殖菌絲體，并使菌絲體長得粗壯，成為活殖菌絲體，并使菌絲體長得粗壯，成為活力強(qiáng)的力強(qiáng)的種子種子。n Inoculum media are usually less nutritious than are production media, and they usually contain a considerably lower level of the main nutritive carbon source.8n一般一般種子培養(yǎng)基常包括有機(jī)氮源和無機(jī)氮源種子培養(yǎng)基常包括有機(jī)氮源和無機(jī)

8、氮源n最后一級的種子培養(yǎng)基的成分最好接近發(fā)酵培最后一級的種子培養(yǎng)基的成分最好接近發(fā)酵培養(yǎng)基。養(yǎng)基。n發(fā)酵培養(yǎng)基發(fā)酵培養(yǎng)基 production media 發(fā)酵培養(yǎng)基發(fā)酵培養(yǎng)基是供菌體生長、繁殖和合成產(chǎn)物之是供菌體生長、繁殖和合成產(chǎn)物之用。用。 要求：既要使種子接種后能迅速生長，達(dá)到一要求：既要使種子接種后能迅速生長，達(dá)到一定的菌絲濃度，又要使長好的菌體迅速合成所定的菌絲濃度，又要使長好的菌體迅速合成所需產(chǎn)物。需產(chǎn)物。9n配制要求：其組成除有菌體生長所必需配制要求：其組成除有菌體生長所必需的元素和化合物外，還需有合成產(chǎn)物所的元素和化合物外，還需有合成產(chǎn)物所需的特定元素、前體和促進(jìn)劑等。需的特定

9、元素、前體和促進(jìn)劑等。nIn addition to the carbon and nitrogen compounds, a medium contains inorganic salts, vitamins, growth factors, precursors of fermentation products.10nMicrobial growth under industrial fermentation conditions usually utilizes a “l(fā)uxury metabolism” of the organisms. Thus, good yields of fe

10、rmentation products occur only if relatively large amounts of carbon, and possibly nitrogen, are channeled through the metabolic pathways of the microorganism. 114.2 發(fā)酵培養(yǎng)基的成分及來源發(fā)酵培養(yǎng)基的成分及來源Composition of media and their sourcesnThe particular composition of a fermentation medium can be simple to comp

11、lex depending on the particular microorganism and its fermentation. nAutotrophic(自養(yǎng)的自養(yǎng)的) microorganisms require only the simplest of inor-ganic media.nHeterotrophic (異養(yǎng)的異養(yǎng)的) microorganisms lack the ability to synthesize many of their sustenance and growth requirements.124.2.1 碳源碳源 carbon sources nCa

12、rbon source is one of main composition of media.nThe functions are: providing carbons and energy; the components for synthesis of products. nThese can include simple sugars, complex carbohydrates, alcohols, amino and other organic acids, and short-chain lipids. 常用的碳源有糖類、油脂、有機(jī)酸和低碳醇。蛋白質(zhì)、氨基酸也可用作碳源。134.

13、2.1.1 糖類糖類 sugarsnSugars used mainly: glucose, molasses (糖蜜糖蜜), dextrin (糊精糊精).nGlucose: most microbes can utilize it. 速效碳源、速效碳源、 high concentrationspeed respiration upDO no enoughintermediates (中間產(chǎn)物）中間產(chǎn)物）accumulation inhibiting growth14nmolasses (糖蜜糖蜜)nBeet(甜菜甜菜) and cane molasses are by-products o

14、f the sugar industry. ncontaining 50% -75% fermentable sugars, mainly sucrose. Also containing nitrogenous compounds, vitamins and minerals. nCheaper 15Starch and dextrin (糊精糊精)nStarch: -widely used in fermentation industry- starch hydrolyze using microbial amylase dextrin glucose-Most common used s

15、tarches: corn, wheat, potato starches. Also using corn flour. -advantages: 緩效碳源、緩效碳源、cheaper than glucose16油和脂肪油和脂肪 oils and fatsnOil and fats are also used as carbon sources because some microbes contain lipase (脂肪酶脂肪酶)nFat glycerol(甘油甘油) and fatty acids oxidize CO2+ water, releasing much energynPr

16、oviding enough O2 is important.n常用的油：豆油、菜子油、豬油、魚油、棉子常用的油：豆油、菜子油、豬油、魚油、棉子油等。油等。17有機(jī)酸有機(jī)酸 organic acidsnSome microbes can use organic acids as carbon sources, such as lactic acid, citric acid, acetic acid. npH of cultural medium will increase. CH3COONa + O2 2CO2 + H2O + NaOH18烴和醇類烴和醇類 hydrocarbon and e

17、thanol n石油及裂解產(chǎn)物如正烷烴也可用作石油及裂解產(chǎn)物如正烷烴也可用作 carbon sources.n自然界中能同化乙醇的微生物和能同化糖質(zhì)的自然界中能同化乙醇的微生物和能同化糖質(zhì)的微生物一樣普遍。微生物一樣普遍。194.2.2 氮源氮源 nitrogen sourcesn氮源主要用于構(gòu)成細(xì)胞物質(zhì)（氨基酸、蛋白質(zhì)、氮源主要用于構(gòu)成細(xì)胞物質(zhì)（氨基酸、蛋白質(zhì)、核酸等）和含氮代謝物。核酸等）和含氮代謝物。n有機(jī)氮源有機(jī)氮源 organic nitrogen sources 常用的常用的: 花生餅、棉子餅、玉米漿、玉米蛋白粉、花生餅、棉子餅、玉米漿、玉米蛋白粉、蛋白胨、酵母粉、魚粉、蠶蛹粉、尿

18、素、廢菌蛋白胨、酵母粉、魚粉、蠶蛹粉、尿素、廢菌絲體和酒糟等。絲體和酒糟等。 organic nitrogen sources microbial proteinase amino acids metabolized by microbes.20norganic nitrogen sources contain rich proteins, peptides, amino acids, vitamins and growth factors.n在含有機(jī)碳源的培養(yǎng)基中，微生物常表現(xiàn)出生在含有機(jī)碳源的培養(yǎng)基中，微生物常表現(xiàn)出生長旺盛、菌絲或細(xì)胞濃度增長迅速的特點(diǎn)。長旺盛、菌絲或細(xì)胞濃度增長迅速的特

19、點(diǎn)。n有些微生物對氨基酸有特殊的需求。如纈氨酸有些微生物對氨基酸有特殊的需求。如纈氨酸可提高紅霉素的發(fā)酵單位?？商岣呒t霉素的發(fā)酵單位。21玉米漿玉米漿 Cornsteep LiquornCornsteep liquor is the water extract by-product resulting from the steeping (浸泡浸泡) of corn during the commercial production of corn starch and other corn products.nOf the 50% solids of cornsteep liquor, nea

20、rly half is lactic acid. The rest includes amino acids, glucose and other reducing sugars, salts, vitamins, and precursors (前體前體)such as those for the penicillin molecule. 22nThe high lactic acid content of cornsteep liquor results from the growth of lactic acid bacteria during its manufacture.nThis

21、 variation in composition, at times, can lead to poor reproducibility of an industrial fermentation. 23n尿素尿素 ureanUrea is also a commonly used nitrogen source.nIts feature: -single component, no nutritional feature of complex nitrogen sources; -cheaper, often used in production of penicillin and glu

22、tamic acid. 24nyeast extractnpeptonesn有機(jī)氮源除了作為菌體生長繁殖的營養(yǎng)外，有有機(jī)氮源除了作為菌體生長繁殖的營養(yǎng)外，有的還是產(chǎn)物的前體。的還是產(chǎn)物的前體。n纈氨酸、半胱氨酸和纈氨酸、半胱氨酸和 -是合成青霉素和頭孢菌是合成青霉素和頭孢菌素的主要前體；甘氨酸可作為素的主要前體；甘氨酸可作為L-絲氨酸的前體絲氨酸的前體等。等。25無機(jī)氮源無機(jī)氮源 inorganic nitrogen sourcen常用的常用的：氨水：氨水(ammonia)、銨鹽、銨鹽(ammonium)和和硝酸鹽硝酸鹽(nitrate)nMicrobes can utilize it fas

23、ter.nAfter it is utilized, the pH of medium will be changed. (NH4)2SO4 2NH3 + H2SO4 NaNO3 + 4H2 NH3 + 2H2O +NaOH26n生理酸性物質(zhì)生理酸性物質(zhì)：NH4 酸性物，硫酸銨酸性物，硫酸銨n生理堿性物質(zhì)：生理堿性物質(zhì)： NH4 堿性物，硝酸銨堿性物，硝酸銨n正確使用正確使用生理酸堿性物質(zhì)生理酸堿性物質(zhì)nExcept using as a nitrogen source, ammonia is often used to adjust pH of media. Stirring is nece

24、ssary. 274.2.3 無機(jī)鹽及微量元素?zé)o機(jī)鹽及微量元素 mineral salts and trace elementsnMineral salts and trace elements are needed when microbial cells grow and reproduce. nMost common mineral salts and trace elements: 磷磷(phosphorus)、鎂、鎂 (magnesium)、硫、硫 (sulphur)、鉀鉀(potassium)、鈉、鈉(sodium)、鐵、鐵(iron)、氯、氯(chlorine)、錳、錳(mang

25、anese)、鋅、鋅(zinc)、鈷、鈷(cobalt)、 鈣等鈣等28n作為微生物細(xì)胞生理活性物質(zhì)組成或生理活性作為微生物細(xì)胞生理活性物質(zhì)組成或生理活性作用的調(diào)節(jié)物。作用的調(diào)節(jié)物。nWhen their concentration is low, they show a stimulation function for cell grow and product synthesis. When their concentration is high, they will inhibit the cell growth. nTheir optimal concentrations depend

26、 on the species and strains. 29無機(jī)鹽成分一般所用的濃度范圍無機(jī)鹽成分一般所用的濃度范圍成分成分濃度濃度g/L成分成分濃度濃度g/LKH2PO4MgSO47H2OKClCaCO3FeSO4 4H2O1.04.00.25 3.00.5 12.05 170.01 0.1ZnSO4 8H2OMnSO4 H2OCuSO4 5H2ONa2MoO4 2H2O0.1 1.00.01 0.10.003 0.010.01 0.130nThese elements are added into cultural media in the form of their salts. nE

27、xcept synthetic media, cobalt鈷鈷, copper, iron, manganese錳錳, zinc, molybdenum鉬鉬 are not added into crude media. 31nPhosphorus element is one of nucleic acids and proteins composition, one component of ATP, energy transfer. nPhosphorus can stimulate microbial growth and metabolism. But it inhibits cel

28、l growth when its concentration is high. n許多許多次級代謝次級代謝過程對磷酸鹽濃度的承受限度比過程對磷酸鹽濃度的承受限度比生長繁殖過程低，故必須嚴(yán)格控制。生長繁殖過程低，故必須嚴(yán)格控制。32nMagnesium does not involve the cell institution. But Mg is the activator for many important enzymes.如己糖磷酸化酶、檸檬酸脫氫酶等如己糖磷酸化酶、檸檬酸脫氫酶等n鎂離子可提高抗生素產(chǎn)生菌的抗生素耐受能力，鎂離子可提高抗生素產(chǎn)生菌的抗生素耐受能力，如鏈霉素、卡那霉素等

29、產(chǎn)生菌。如鏈霉素、卡那霉素等產(chǎn)生菌。n常以常以硫酸鎂硫酸鎂的形式加入培養(yǎng)基。的形式加入培養(yǎng)基。33n硫存在于細(xì)胞的蛋白質(zhì)中，硫存在于細(xì)胞的蛋白質(zhì)中， 是含硫氨基酸的是含硫氨基酸的組分和某些輔酶的活性基，如輔酶組分和某些輔酶的活性基，如輔酶 A (coenzyme A), 谷胱甘肽等谷胱甘肽等。n硫是某些產(chǎn)物如青霉素、頭孢菌素等分子的硫是某些產(chǎn)物如青霉素、頭孢菌素等分子的組成部分，在培養(yǎng)基中加入組成部分，在培養(yǎng)基中加入Na2SO4等含硫化等含硫化合物作硫源。合物作硫源。34n鐵鐵(iron)是細(xì)胞色素、細(xì)胞色素氧化酶和過氧是細(xì)胞色素、細(xì)胞色素氧化酶和過氧化氫酶的成分，因此鐵是菌體有氧氧化必不可化

30、氫酶的成分，因此鐵是菌體有氧氧化必不可少的元素。少的元素。nIn iron-made fermentor, generally iron ion concentration can be 30 g/ml. So iron containing compounds are not necessary for industrial media.35nChlorine does not have nutritional functions for most microorganisms, but it is necessary for cell growth of halophile.n一些產(chǎn)含氯代

31、謝物如金霉素和灰黃霉素等的發(fā)一些產(chǎn)含氯代謝物如金霉素和灰黃霉素等的發(fā)酵中，除天然含有外，通常還需加入酵中，除天然含有外，通常還需加入0.1%氯化氯化鉀。鉀。n在啤酒生產(chǎn)中，在啤酒生產(chǎn)中，2060mg/ml 的的氯對酶和酵母氯對酶和酵母有一定的促進(jìn)作用。有一定的促進(jìn)作用。 36nSodium, potassium, calcium are not components of cell constitution, they are still necessary composition for cultural media. n鈉、鉀離子與維持細(xì)胞的滲透壓有關(guān)。鉀離子鈉、鉀離子與維持細(xì)胞的滲透壓有

32、關(guān)。鉀離子是許多酶的激活劑，能促進(jìn)糖代謝。是許多酶的激活劑，能促進(jìn)糖代謝。n鈣是某些酶鈣是某些酶(如蛋白酶如蛋白酶)的激活劑，還參與細(xì)胞的激活劑，還參與細(xì)胞膜通透性的調(diào)節(jié)。膜通透性的調(diào)節(jié)。培養(yǎng)基中鈣鹽過多時，會形成磷酸鈣沉淀。培養(yǎng)基中鈣鹽過多時，會形成磷酸鈣沉淀。37 鋅鋅、鎂鎂、鈷鈷、錳錳等是某些酶的輔基或激活劑。等是某些酶的輔基或激活劑。鎂離子還可提高抗生素生產(chǎn)菌對自己所產(chǎn)生抗鎂離子還可提高抗生素生產(chǎn)菌對自己所產(chǎn)生抗生素的耐性；鈷既是一些酶的生素的耐性；鈷既是一些酶的激活劑激活劑，又是，又是VB12的組成元素，發(fā)酵中加入一定量的鈷鹽，的組成元素，發(fā)酵中加入一定量的鈷鹽，能使能使VB12的產(chǎn)

33、量提高數(shù)倍；錳對于羧化作用是的產(chǎn)量提高數(shù)倍；錳對于羧化作用是必需的，糖代謝中許多酶的活性都與錳有關(guān)。必需的，糖代謝中許多酶的活性都與錳有關(guān)。384.2.4 Water nMain composition of medianFunctions: -join directly some metabolisms -solvents -providing a physiological environments for cell growth and synthesis of products.nEffect of water quality on fermentation394.2.5 生長因子、前

34、體、產(chǎn)物促進(jìn)劑生長因子、前體、產(chǎn)物促進(jìn)劑n(1) 生長因子生長因子 growth factorsnDefinition: small amount of organic compounds necessary for microbial growth. nFeatures: no synthesis by itself. nExamples：amino acids, purines, vitaminsnSources: normally organic nitrogen source. Cornsteep liquor. 40(2) 前體前體 precursorsnDefinition: su

35、bstances added prior to or simultaneously with the fermentation, which are incorporated without any major change into the molecule of the fermentation product, and which generally serve to increase the yield or improve the quality of the product.41n前體是指某些化合物加入到發(fā)酵培養(yǎng)基中，前體是指某些化合物加入到發(fā)酵培養(yǎng)基中，能直接被微生物在生物合成過

36、程中結(jié)合到產(chǎn)能直接被微生物在生物合成過程中結(jié)合到產(chǎn)物分子中去，而其自身的結(jié)構(gòu)并沒有多大變物分子中去，而其自身的結(jié)構(gòu)并沒有多大變化，但是產(chǎn)物的產(chǎn)量卻因加入前體而有較大化，但是產(chǎn)物的產(chǎn)量卻因加入前體而有較大的提高的提高。nExamples：苯乙酸：苯乙酸青霉素青霉素G 苯氧乙酸苯氧乙酸青霉素青霉素V42發(fā)酵過程中所用的一些前體物質(zhì)發(fā)酵過程中所用的一些前體物質(zhì)產(chǎn)品產(chǎn)品前體前體產(chǎn)品產(chǎn)品前體前體青霉素青霉素G青霉素青霉素V金霉素金霉素灰黃霉素灰黃霉素紅霉素紅霉素苯乙酸苯乙酸苯氧乙酸苯氧乙酸氯化物氯化物氯化物氯化物正丙醇正丙醇核黃素核黃素類胡蘿卜素類胡蘿卜素L-異亮氨酸異亮氨酸L-色氨酸色氨酸L-絲氨酸絲

37、氨酸丙酸鹽丙酸鹽-紫羅酮紫羅酮 -氨基丁酸氨基丁酸鄰氨基苯甲酸鄰氨基苯甲酸甘氨酸甘氨酸43(3) 產(chǎn)物促進(jìn)劑產(chǎn)物促進(jìn)劑n產(chǎn)物促進(jìn)劑產(chǎn)物促進(jìn)劑 product stimulator Definition: substances are neither nutrients nor precursors, and increase product yield after adding them. nExamples: Tween (0.1%) amylase(淀粉酶淀粉酶), cellulase(纖維素酶纖維素酶) 大豆酒精提取物大豆酒精提取物 cellulase44nMechanisms: not

38、 very clearnPossible reasons: -enzyme inducers 誘導(dǎo)劑誘導(dǎo)劑 -improvement of cell permeability 滲透性滲透性 -protection on enzyme activity454.3 培養(yǎng)基的設(shè)計(jì)及優(yōu)化培養(yǎng)基的設(shè)計(jì)及優(yōu)化media design and its optimizationnImportance of media designnCurrent situations of media design: many factors empirical 經(jīng)驗(yàn)經(jīng)驗(yàn) + scientific n4.3.1 培養(yǎng)基成分選

39、擇的原則培養(yǎng)基成分選擇的原則 (1) 菌體的同化能力： large molecules + enzymes small molecules46 Selection of carbon sources: glucose, starches不同糖化工藝所得糖液質(zhì)量的比較不同糖化工藝所得糖液質(zhì)量的比較項(xiàng)目項(xiàng)目酸法酸法酸酶法酸酶法雙酶法雙酶法葡萄糖值葡萄糖值(DE值值)葡萄糖含量葡萄糖含量灰分灰分色度色度羥甲基糠氨羥甲基糠氨葡萄糖收率葡萄糖收率91861.610.00.3095930.40.30.008較酸法高較酸法高598970.10.20.003較酸法高較酸法高1047nSelection of

40、nitrogen sources: -If the microbe produces proteolytic enzymes, a variety of crude nitrogen sources such as soybean meal can be used. -If the microbe lack of proteases, the organic nitrogen source should be hydrolyzed. 豆餅粉豆餅粉water + hydrochloric acid 鹽酸鹽酸, pH1.0 100C amino acids. 48n(2) 代謝物的阻遏和誘導(dǎo)代謝物的阻遏和誘導(dǎo) -葡萄糖效應(yīng)葡萄糖效應(yīng) - combination of organic and inorganic nitrogen/carbon sources