微生物發(fā)酵配制培養(yǎng)基的原則
發(fā)布時間：
2022-11-16
作者：
國內(nèi)培養(yǎng)基環(huán)測碟/平板生產(chǎn)廠家
微生物發(fā)酵配制培養(yǎng)基的原則
1. 選擇適宜的營養(yǎng)物質(zhì)
總體而言,所有微生物生長繁殖均需要培養(yǎng)基含有碳源、氮源、無機鹽、生長因子、水及能源,但由于微生物營養(yǎng)類型復(fù)雜,不同微生物對營養(yǎng)物質(zhì)的需求是不一樣的,因此首先要根據(jù)不同微生物的營養(yǎng)需求配制針對性強的培養(yǎng)基。自養(yǎng)型微生物能從簡單的無機物合成自身需要的糖、脂類、蛋白質(zhì)、核酸、維生素等復(fù)雜的有機物,因此培養(yǎng)自養(yǎng)型微生物的培養(yǎng)基完全可以(或應(yīng)該)由簡單的無機物組成。例如,培養(yǎng)化能自養(yǎng)型的氧化硫硫桿菌 ，在該培養(yǎng)基配制過程中并未專門加入其他碳源物質(zhì),而是依靠空氣中和溶于水中的CO2為氧化硫硫桿菌提供碳源。
培養(yǎng)其他化能自養(yǎng)型微生物與上述培養(yǎng)基成分基本類似,只是能源物質(zhì)有所改變。對光能自養(yǎng)型微生物而言,除需要各類營養(yǎng)物質(zhì)外,還需光照提供能源。培養(yǎng)異養(yǎng)型微生物需要在培養(yǎng)基中添加有機物,而且不同類型異養(yǎng)型微生物的營養(yǎng)要求差別很大,因此其培養(yǎng)基組成也相差很遠。例如,培養(yǎng)大腸桿菌的培養(yǎng)基組成比較簡單,而有些異養(yǎng)型微生物的培養(yǎng)基的成份非常復(fù)雜,如腸膜明串珠菌需要生長因子,若配制培養(yǎng)它的合成培養(yǎng)基時,需要在培養(yǎng)基中添加的生長因子多達33種,因此通常采用天然有機物來為它提供生長所需的生長因子。
就微生物主要類型而言,有細菌、放線菌、酵母菌、霉菌、原生動物、藻類及病毒之分,培養(yǎng)它們所需的培養(yǎng)基各不相同。在實驗室中常用牛肉膏蛋白胨培養(yǎng)基（或簡稱普通肉湯培養(yǎng)基）培養(yǎng)細菌;用高氏1號合成培養(yǎng)基培養(yǎng)放線菌;培養(yǎng)酵母菌一般用麥芽汁培養(yǎng)基,它是將麥芽粉與4倍水混勻,在58~65℃條件下保溫3~4小時至完全糖化,調(diào)整糖液濃度為10о巴林, 煮沸后用紗布過濾,調(diào)pH為6.0配制而成。麥芽粉組成復(fù)雜,能為酵母菌提供足夠的營養(yǎng)物質(zhì);培養(yǎng)霉菌則一般用查氏合成培養(yǎng)基。
原生動物也可用培養(yǎng)基培養(yǎng),有的原生動物需要較多的營養(yǎng)物質(zhì),例如梨型四膜蟲(Tetrahymena pyriformis)的培養(yǎng)基含有10種氨基酸、7種維生素、鳥嘌呤、尿嘧啶及一些無機鹽等,而有些變形蟲可在較簡單的蛋白胨肉湯(peptone broth)中生長。大多數(shù)藻類可以利用光能,只需要CO2、水和一些無機鹽就可生長,而某些藻類如眼蟲藻(Euglena)中的一些種可在黑暗條件下利用有機物質(zhì)生長。有些藻類需要在培養(yǎng)基中補加土壤浸液,培養(yǎng)海洋藻類時可直接利用海水,但如果在特殊情況下需要用合成培養(yǎng)基培養(yǎng)海洋藻類時,則必需在培養(yǎng)基中加入海水中含有的各種鹽。
2.營養(yǎng)物質(zhì)濃度及配比合適
培養(yǎng)基中營養(yǎng)物質(zhì)濃度合適時微生物才能生長良好,營養(yǎng)物質(zhì)濃度過低時不能滿足微生物正常生長所需,濃度過高時則可能對微生物生長起抑制作用,例如高濃度糖類物質(zhì)、無機鹽、重金屬離子等不僅不能維持和促進微生物的生長,反而起到抑制或殺菌作用。另外，培養(yǎng)基中各營養(yǎng)物質(zhì)之間的濃度配比也直接影響微生物的生長繁殖和(或)代謝產(chǎn)物的形成和積累,其中碳氮比(C/N)的影響較大。嚴(yán)格地講，碳氮比指培養(yǎng)基中碳元素與氮元素的摩爾數(shù)比值,有時也指培養(yǎng)基中還原糖與粗蛋白之比。例如，在利用微生物發(fā)酵生產(chǎn)谷氨酸的過程中,培養(yǎng)基碳氮比為4/1時,菌體大量繁殖,谷氨酸積累少;當(dāng)培養(yǎng)基碳氮比為3/1時,菌體繁殖受到抑制,谷氨酸產(chǎn)量則大量增加。再如,在抗生素發(fā)酵生產(chǎn)過程中,可以通過控制培養(yǎng)基中速效氮(或碳)源與遲效氮(或碳)源之間的比例來控制菌體生長與抗生素的合成。
3.控制pH條件
培養(yǎng)基的pH必須控制在一定的范圍內(nèi),以滿足不同類型微生物的生長繁殖或產(chǎn)生代謝產(chǎn)物。各類微生物生長繁殖或產(chǎn)生代謝產(chǎn)物的最適pH條件各不相同,一般來講,細菌與放線菌適于在pH7~7.5范圍內(nèi)生長,酵母菌和霉菌通常在pH4.5~6范圍內(nèi)生長。值得注意的是,在微生物生長繁殖和代謝過程中,由于營養(yǎng)物質(zhì)被分解利用和代謝產(chǎn)物的形成與積累,會導(dǎo)致培養(yǎng)基pH發(fā)生變化,若不對培養(yǎng)基pH條件進行控制,往往導(dǎo)致微生物生長速度或(和)代謝產(chǎn)物產(chǎn)量降低。因此,為了維持培養(yǎng)基pH的相對恒定,通常在培養(yǎng)基中加入pH緩沖劑,常用的緩沖劑是一氫和二氫磷酸鹽(如K2HPO4和KH2PO4)組成的混合物。K2HPO4溶液呈堿性, KH2PO4溶液呈酸性,兩種物質(zhì)的等克分子混合溶液的pH值為6.8。當(dāng)培養(yǎng)基中酸性物質(zhì)積累導(dǎo)致H 濃度增加時, H 與弱堿性鹽結(jié)合形成弱酸性化合物,培養(yǎng)基pH不會過度降低;如果培養(yǎng)基中OH-濃度增加, OH-則與弱酸性鹽結(jié)合形成弱堿性化合物,培養(yǎng)基pH也不會過度升高。
但K2HPO4/KH2PO4緩沖系統(tǒng)只能在一定的pH范圍（pH6.4~7.2）內(nèi)起調(diào)節(jié)作用。有些微生物，如乳酸菌能大量產(chǎn)酸,上述緩沖系統(tǒng)就難以起到緩沖作用,此時可在培養(yǎng)基中添加難溶的碳酸鹽(如CaCO3)來進行調(diào)節(jié), CaCO3難溶于水,不會使培養(yǎng)基pH過度升高,但它可以不斷中和微生物產(chǎn)生的酸,同時釋放出CO2,將培養(yǎng)基pH控制在一定范圍內(nèi)。
青島日水微生物培養(yǎng)基
在培養(yǎng)基中還存在一些天然的緩沖系統(tǒng),如氨基酸、肽、蛋白質(zhì)都屬于兩性電解質(zhì),也可起到緩沖劑的作用。
The principle of microbial fermentation preparation culture medium.
Choose the right nutrients.
In general, all microorganisms grow need medium containing carbon source, nitrogen source, inorganic salt, growth factors, water and energy, due to the complex microbial nutrition type, different microorganisms on nutrient requirements is different, so first of all, according to the requirements of different microbial preparation pertinence of medium. Autotrophic microbes can from simple inorganic synthesis of their needs sugar, lipid, protein, nucleic acids, vitamins and other complex organic compounds, thus cultivating autotrophic microbe culture medium can (or should) is composed of simple inorganic matter. For example, sulfur oxide can cultivate the autotrophic thiobacillus, in the process of the culture medium preparation did not specifically to join the other carbon source material, but rely on air and CO2 dissolved in the water for sulfur oxide sulfur bacillus carbon source.
The cultivation of other autotrophic microorganisms is basically similar to that of the above medium, except that the energy material has changed. In addition to all kinds of nutrients, photoautotrophic microorganisms need light to provide energy. The cultivation of heterotrophic microorganisms requires the addition of organic matter in the medium, and the nutritional requirements of different types of heterotrophic microorganisms are very different, so the composition of the culture medium is far away. Culture medium composition of e. coli, for example, is simpler, and some heterotrophic microbial culture medium composition is very complicated, such as intestinal membrane bright beads bacterium need growth factor, if the synthetic medium of preparation training it, need to add as many as 33 kinds of growth factors in the culture medium, so often need to grow and provide it with using natural organic growth factor.
Bacteria, actinomycetes, yeasts, fungi, protozoa, algae and viruses are the main types of microorganisms, and the cultures they need are different. In the laboratory, common beef paste peptone medium (or common broth medium) is used to cultivate bacteria; The culture of actinomycetes was cultured with the no.1 synthetic culture medium. General with malt juice culture yeast culture medium, it is 4 times malt powder and water blending, under the condition of 58 to 65 ℃ heat preservation 3 ~ 4 hours to fully saccharification, adjust the concentration of sugar solution for 10 о bahrain, boil with gauze filter, made from the pH 6.0. The composition of malt powder is complex, which can provide sufficient nutrients for yeast. The cultivation of mildew is generally used in the synthesis culture medium.
Protozoa could also be a medium, some native animals need more nutrients, such as four pear type membrane worm (Tetrahymena pyriformis) culture medium containing 10 kinds of amino acids, 7 kinds of vitamin, guanine, uracil and some inorganic salts, etc., and some of the amoeba can be simpler peptone broth (peptone broth). Most algae use light energy, which requires only CO2, water and some inorganic salts to grow, while some of the algae, such as Euglena, use organic matter to grow in dark conditions. Some algae need adding soil immersion in culture, cultivating Marine algae can be directly used in the water of the sea, but if in special circumstances need to use synthetic medium to develop Marine algae, are required to join in the seawater in culture contains a variety of salt.
2. The nutrient concentration and proportion are suitable.
Medium nutrients concentration of microorganisms can grow well when appropriate, cannot satisfy microorganisms at low nutrient concentration required for normal growth, when the concentration is too high may inhibitory effect on microorganism growth, such as high concentration of sugar matter, inorganic salt, heavy metal ions such as not only unable to maintain and promote the growth of microorganisms, but inhibit or sterilization effect. In addition, the concentration ratio of various nutrients in the medium also directly influences the growth and reproduction of microorganisms and the formation and accumulation of metabolic products, among which the carbon nitrogen ratio (C/N) has a greater influence. Strictly speaking, carbon nitrogen ratio refers to the ratio of the number of moles of carbon to nitrogen in medium, and sometimes the ratio of raw sugar to crude protein in the medium. For example, in the process of using microbial fermentation to produce glutamate, when the medium carbon nitrogen ratio is 4/1, the bacteria multiply and the glutamate accumulation is small. When the carbon nitrogen ratio of the medium was 3/1, the growth of the bacteria was inhibited and the yield of glutamate increased substantially. Again, such as, in the antibiotic fermentation process, can control the medium available nitrogen or carbon source and delayed the ratio between the nitrogen or carbon source to control the bacteria growth and the synthesis of antibiotics.
3. Control pH condition
The pH of the culture medium must be controlled within a certain range to meet the growth and reproduction of different types of microorganisms or to produce metabolites. All kinds of microorganisms grow or produce metabolites optimum pH conditions each are not identical, in general, applicable for bacteria and actinomycetes pH7 ~ 7.5 within the scope of growth, yeast and mold usually grow in pH4.5 ~ 6 range. It is important to note that in the process of microbial growth, reproduction and metabolism, as nutrients are broken down to use and formation and accumulation of metabolites, medium pH changes, if it is not medium to control the pH condition, often leads to microbial growth or (and) metabolites production decrease. Therefore, in order to maintain the relatively constant pH of the medium, the pH buffer is usually added in the medium, and the commonly used buffer is a mixture of hydrogen and dihydrophosphate (such as K2HPO4 and KH2PO4). K2HPO4 solution is alkaline, and KH2PO4 solution is acidic. The pH value of the isogram mixed solution of two substances is 6.8. When the accumulation of acid in the medium resulted in the increase of H concentration, H combined with weak alkaline salt to form a weak acid compound, and the pH of the medium was not excessively reduced; If the concentration of OH- concentration in the medium increased, OH- then combined with weakly acidic salt to form a weakly alkaline compound, the medium pH would not be excessively elevated.
However, the K2HPO4/KH2PO4 buffer system can only be adjusted in a certain pH range (pH6.4~7.2). Some microorganisms, such as lactic acid bacteria can produce more acid, the buffer system, it will be difficult to play a buffer action at this point can be added in the culture medium of refractory carbonate (CaCO3) to adjust, CaCO3 poorly soluble in water, does not make the medium pH rising excessively, but it can counteract acidity of microbes, releases CO2 at the same time, the culture medium pH control within a certain range.
There are also some natural buffer systems in the medium, such as amino acids, peptides and proteins, which are both ampholytes, which can also act as a buffer.