4微生物培養(yǎng)基Culturalmedium

1、4. 微生物培養(yǎng)基 Cultural mediaA 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.A good medium is very important to the success of an industrial fermentation. The medium supplies nutrients for growth, ener

2、gy, building of cell substance, and biosynthesis of fermentation products. 4.1 培養(yǎng)基的類型及功能 Classifications of media and their functions 按純度分類： -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 chemically undefined media: ingredient

3、s include one or more chemically complex substances such as protein hydrolysates and extracts. Crude media The choice of defined or undefined medium is dependent upon its application. -Chemically defined media are useful in biochemical or metabolic studies of organisms. -General laboratory growth me

4、dia and industrial media are often chemically complex. Such as 花生餅、蛋白胨等。一般不需外加微量元素 (trace elements)、維生素等。 按狀態(tài)分類： -Solid media：suitable to culture and preserve species and spores; cultivating fungi. -Liquid media: 80%-90%water, industrial fermentation media. -Semi-solid media: liquid medium + agar 主要

5、用于鑒定細(xì)菌、觀察細(xì)菌運動特征等。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1.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，不易引起菌種發(fā)生變異 (variation)。 基本配制要求： not rich in nutrition, especially organic nitrogen soure. optimal mineral salt concentration optimal pH and humidity 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

6、含氮少、疏松、表面積大，是較好的孢子培養(yǎng)基。水分控制在2125。種子培養(yǎng)基 inoculum media: 種子培養(yǎng)基是供孢子發(fā)芽、生長和大量繁殖菌絲體，并使菌絲體長得粗壯，成為活力強的種子。 Inoculum media are usually less nutritious than are production media, and they usually contain a considerably lower level of the main nutritive carbon source.一般種子培養(yǎng)基常包括有機氮源和無機氮源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。發(fā)酵培

7、養(yǎng)基 production media 發(fā)酵培養(yǎng)基是供菌體生長、繁殖和合成產(chǎn)物之用。 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需產(chǎn)物。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)物所需的特定元素、前體和促進(jìn)劑等。In addition to the carbon and nitrogen compounds, a medium contains inorganic salts, vitamins, growth factors, precursors of fermentation products.Microbial growth

8、under industrial fermentation conditions usually utilizes a “l(fā)uxury metabolism” of the organisms. Thus, good yields of fermentation products occur only if relatively large amounts of carbon, and possibly nitrogen, are channeled through the metabolic pathways of the microorganism. 4.2 發(fā)酵培養(yǎng)基的成分及來源Comp

9、osition of media and their sourcesThe particular composition of a fermentation medium can be simple to complex depending on the particular microorganism and its fermentation. Autotrophic(自養(yǎng)的) microorganisms require only the simplest of inor-ganic media.Heterotrophic (異養(yǎng)的) microorganisms lack the abi

10、lity to synthesize many of their sustenance and growth requirements.4.2.1 碳源 carbon sources Carbon source is one of main composition of media.The functions are: providing carbons and energy; the components for synthesis of products. These can include simple sugars, complex carbohydrates, alcohols, a

11、mino and other organic acids, and short-chain lipids. 常用的碳源有糖類、油脂、有機酸和低碳醇。蛋白質(zhì)、氨基酸也可用作碳源。 糖類 sugarsSugars used mainly: glucose, molasses (糖蜜), dextrin (糊精).Glucose: most microbes can utilize it. 速效碳源、 high concentrationspeed respiration upDO no enoughintermediates (中間產(chǎn)物）accumulation inhibiting

12、 growthmolasses (糖蜜)Beet(甜菜) and cane molasses are by-products of the sugar industry. containing 50% -75% fermentable sugars, mainly sucrose. Also containing nitrogenous compounds, vitamins and minerals. Cheaper Starch and dextrin (糊精)Starch: -widely used in fermentation industry- starch hydrolyze u

13、sing microbial amylase dextrin glucose-Most common used starches: corn, wheat, potato starches. Also using corn flour. -advantages: 緩效碳源、cheaper than glucose油和脂肪 oils and fatsOil and fats are also used as carbon sources because some microbes contain lipase (脂肪酶)Fat glycerol(甘油) and fatty acids oxidi

14、ze CO2+ water, releasing much energyProviding enough O2 is important.常用的油：豆油、菜子油、豬油、魚油、棉子油等。有機酸 organic acidsSome microbes can use organic acids as carbon sources, such as lactic acid, citric acid, acetic acid. pH of cultural medium will increase. CH3COONa + O2 2CO2 + H2O + NaOH烴和醇類 hydrocarbon and

15、ethanol 石油及裂解產(chǎn)物如正烷烴也可用作 carbon sources.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。4.2.2 氮源 nitrogen sources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。有機氮源 organic nitrogen sources 常用的: 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、尿素、廢菌絲體和酒糟等。 organic nitrogen sources microbial proteinase amino acids metabolized by anic nitro

16、gen sources contain rich proteins, peptides, amino acids, vitamins and growth factors.在含有機碳源的培養(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。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。玉米漿 Cornsteep LiquorCornsteep liquor is the water extract by-product resulting from the steeping (浸泡) of corn during the commercial production

17、of corn starch and other corn products.Of the 50% solids of cornsteep liquor, nearly half is lactic acid. The rest includes amino acids, glucose and other reducing sugars, salts, vitamins, and precursors (前體)such as those for the penicillin molecule. The high lactic acid content of cornsteep liquor

18、results from the growth of lactic acid bacteria during its manufacture.This variation in composition, at times, can lead to poor reproducibility of an industrial fermentation. 尿素 ureaUrea is also a commonly used nitrogen source.Its feature: -single component, no nutritional feature of complex nitrog

19、en sources; -cheaper, often used in production of penicillin and glutamic acid. yeast extractpeptones有機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。纈氨酸、半胱氨酸和-是合成青霉素和頭孢菌素的主要前體；甘氨酸可作為L-絲氨酸的前體等。無機氮源 inorganic nitrogen source常用的：氨水(ammonia)、銨鹽(ammonium)和硝酸鹽(nitrate)Microbes can utilize it faster.After it is utilized, the

20、 pH of medium will be changed. (NH4)2SO4 2NH3 + H2SO4 NaNO3 + 4H2 NH3 + 2H2O +NaOH生理酸性物質(zhì)：NH4 酸性物，硫酸銨生理堿性物質(zhì)： NH4 堿性物，硝酸銨正確使用生理酸堿性物質(zhì)Except using as a nitrogen source, ammonia is often used to adjust pH of media. Stirring is necessary. 4.2.3 無機鹽及微量元素 mineral salts and trace elementsMineral salts and tr

21、ace elements are needed when microbial cells grow and reproduce. Most common mineral salts and trace elements: 磷(phosphorus)、鎂 (magnesium)、硫 (sulphur)、鉀(potassium)、鈉(sodium)、鐵(iron)、氯(chlorine)、錳(manganese)、鋅(zinc)、鈷(cobalt)、 鈣等作為微生物細(xì)胞生理活性物質(zhì)組成或生理活性作用的調(diào)節(jié)物。When their concentration is low, they show a

22、stimulation function for cell grow and product synthesis. When their concentration is high, they will inhibit the cell growth. Their optimal concentrations depend on the species and strains. 無機鹽成分一般所用的濃度范圍成分濃度g/L成分濃度g/LKH2PO4MgSO47H2OKClCaCO3FeSO4 4H2O1.04.00.25 3.00.5 12.05 170.01 0.1ZnSO4 8H2OMnSO

23、4 H2OCuSO4 5H2ONa2MoO4 2H2O0.1 1.00.01 0.10.003 0.010.01 0.1These elements are added into cultural media in the form of their salts. Except synthetic media, cobalt鈷, copper, iron, manganese錳, zinc, molybdenum鉬 are not added into crude media. Phosphorus element is one of nucleic acids and proteins co

24、mposition, one component of ATP, energy transfer. Phosphorus can stimulate microbial growth and metabolism. But it inhibits cell growth when its concentration is high. 許多次級代謝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。Magnesium does not involve the cell institution. But Mg is the activator for many important enzy

25、mes.如己糖磷酸化酶、檸檬酸脫氫酶等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、卡那霉素等產(chǎn)生菌。常以硫酸鎂的形式加入培養(yǎng)基。硫存在于細(xì)胞的蛋白質(zhì)中， 是含硫氨基酸的組分和某些輔酶的活性基，如輔酶 A (coenzyme A), 谷胱甘肽等。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Na2SO4等含硫化合物作硫源。鐵(iron)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少的元素。In iron-made fermentor, generally iron ion concentration can be 30 g/ml. So iro

26、n containing compounds are not necessary for industrial media.Chlorine does not have nutritional functions for most microorganisms, but it is necessary for cell growth of halophile.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0.1%氯化鉀。在啤酒生產(chǎn)中，2060mg/ml 的氯對酶和酵母有一定的促進(jìn)作用。 Sodium, potassium, calcium are not compon

27、ents of cell constitution, they are still necessary composition for cultural media. 鈉、鉀離子與維持細(xì)胞的滲透壓有關(guān)。鉀離子是許多酶的激活劑，能促進(jìn)糖代謝。鈣是某些酶(如蛋白酶)的激活劑，還參與細(xì)胞膜通透性的調(diào)節(jié)。培養(yǎng)基中鈣鹽過多時，會形成磷酸鈣沉淀。 鋅、鎂、鈷、錳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)生抗生素的耐性；鈷既是一些酶的激活劑，又是VB12的組成元素，發(fā)酵中加入一定量的鈷鹽，能使VB12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)。4.2.4 Wa

28、ter Main composition of mediaFunctions: -join directly some metabolisms -solvents -providing a physiological environments for cell growth and synthesis of products.Effect of water quality on fermentation4.2.5 生長因子、前體、產(chǎn)物促進(jìn)劑(1) 生長因子 growth factorsDefinition: small amount of organic compounds necessary

29、 for microbial growth. Features: no synthesis by itself. Examples：amino acids, purines, vitaminsSources: normally organic nitrogen source. Cornsteep liquor. (2) 前體 precursorsDefinition: substances added prior to or simultaneously with the fermentation, which are incorporated without any major change

30、 into the molecule of the fermentation product, and which generally serve to increase the yield or improve the quality of the product.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)并沒有多大變化，但是產(chǎn)物的產(chǎn)量卻因加入前體而有較大的提高。Examples：苯乙酸青霉素G 苯氧乙酸青霉素V發(fā)酵過程中所用的一些前體物質(zhì)產(chǎn)品前體產(chǎn)品前體青霉素G青霉素V金霉素灰黃霉素紅霉素苯乙酸苯氧乙酸氯化物氯化物正丙醇核黃素類胡

31、蘿卜素L-異亮氨酸L-色氨酸L-絲氨酸丙酸鹽-紫羅酮-氨基丁酸鄰氨基苯甲酸甘氨酸(3) 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. Examples: Tween (0.1%) amylase(淀粉酶), cellulase(纖維素酶) 大豆酒精提取物 cellulaseMechanisms: not very clearPossible reasons: -enzy

32、me inducers 誘導(dǎo)劑 -improvement of cell permeability 滲透性 -protection on enzyme activity4.3 培養(yǎng)基的設(shè)計及優(yōu)化media design and its optimizationImportance of media designCurrent situations of media design: many factors empirical 經(jīng)驗 + scientific 4.3.1 培養(yǎng)基成分選擇的原則 (1) 菌體的同化能力： large molecules + enzymes small molecul

33、es Selection of carbon sources: glucose, starches不同糖化工藝所得糖液質(zhì)量的比較項目酸法酸酶法雙酶法葡萄糖值(DE值)葡萄糖含量灰分色度羥甲基糠氨葡萄糖收率91861.610.00.3095908較酸法高598903較酸法高10Selection of 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water + hydrochloric acid 鹽酸, pH1.0 100C amino acids. (2) 代謝物的阻遏和誘導(dǎo) -葡萄糖效應(yīng) - combination of organic and inorganic nitrogen/carbon sources -enzyme agent production: For