微生物发酵配制培养基的原则

发布时间:

2022-11-16

作者:

国内培养基环测碟/平板生产厂家


微生物发酵配制培养基的原则

 
 
1. 选择适宜的营养物质 
  总体而言,所有微生物生长繁殖均需要培养基含有碳源、氮源、无机盐、生长因子、水及能源,但由于微生物营养类型复杂,不同微生物对营养物质的需求是不一样的,因此首先要根据不同微生物的营养需求配制针对性强的培养基。自养型微生物能从简单的无机物合成自身需要的糖、脂类、蛋白质、核酸、维生素等复杂的有机物,因此培养自养型微生物的培养基完全可以(或应该)由简单的无机物组成。例如,培养化能自养型的氧化硫硫杆菌 ,在该培养基配制过程中并未专门加入其他碳源物质,而是依靠空气中和溶于水中的CO2为氧化硫硫杆菌提供碳源。
  培养其他化能自养型微生物与上述培养基成分基本类似,只是能源物质有所改变。对光能自养型微生物而言,除需要各类营养物质外,还需光照提供能源。培养异养型微生物需要在培养基中添加有机物,而且不同类型异养型微生物的营养要求差别很大,因此其培养基组成也相差很远。例如,培养大肠杆菌的培养基组成比较简单,而有些异养型微生物的培养基的成份非常复杂,如肠膜明串珠菌需要生长因子,若配制培养它的合成培养基时,需要在培养基中添加的生长因子多达33种,因此通常采用天然有机物来为它提供生长所需的生长因子。 
  就微生物主要类型而言,有细菌、放线菌、酵母菌、霉菌、原生动物、藻类及病毒之分,培养它们所需的培养基各不相同。在实验室中常用牛肉膏蛋白胨培养基(或简称普通肉汤培养基)培养细菌;用高氏1号合成培养基培养放线菌;培养酵母菌一般用麦芽汁培养基,它是将麦芽粉与4倍水混匀,在58~65℃条件下保温3~4小时至完全糖化,调整糖液浓度为10о巴林, 煮沸后用纱布过滤,调pH为6.0配制而成。麦芽粉组成复杂,能为酵母菌提供足够的营养物质;培养霉菌则一般用查氏合成培养基。
  原生动物也可用培养基培养,有的原生动物需要较多的营养物质,例如梨型四膜虫(Tetrahymena pyriformis)的培养基含有10种氨基酸、7种维生素、鸟嘌呤、尿嘧啶及一些无机盐等,而有些变形虫可在较简单的蛋白胨肉汤(peptone broth)中生长。大多数藻类可以利用光能,只需要CO2、水和一些无机盐就可生长,而某些藻类如眼虫藻(Euglena)中的一些种可在黑暗条件下利用有机物质生长。有些藻类需要在培养基中补加土壤浸液,培养海洋藻类时可直接利用海水,但如果在特殊情况下需要用合成培养基培养海洋藻类时,则必需在培养基中加入海水中含有的各种盐。 
  2.营养物质浓度及配比合适
   培养基中营养物质浓度合适时微生物才能生长良好,营养物质浓度过低时不能满足微生物正常生长所需,浓度过高时则可能对微生物生长起抑制作用,例如高浓度糖类物质、无机盐、重金属离子等不仅不能维持和促进微生物的生长,反而起到抑制或杀菌作用。另外,培养基中各营养物质之间的浓度配比也直接影响微生物的生长繁殖和(或)代谢产物的形成和积累,其中碳氮比(C/N)的影响较大。严格地讲,碳氮比指培养基中碳元素与氮元素的摩尔数比值,有时也指培养基中还原糖与粗蛋白之比。例如,在利用微生物发酵生产谷氨酸的过程中,培养基碳氮比为4/1时,菌体大量繁殖,谷氨酸积累少;当培养基碳氮比为3/1时,菌体繁殖受到抑制,谷氨酸产量则大量增加。再如,在抗生素发酵生产过程中,可以通过控制培养基中速效氮(或碳)源与迟效氮(或碳)源之间的比例来控制菌体生长与抗生素的合成。 
  3.控制pH条件
   培养基的pH必须控制在一定的范围内,以满足不同类型微生物的生长繁殖或产生代谢产物。各类微生物生长繁殖或产生代谢产物的最适pH条件各不相同,一般来讲,细菌与放线菌适于在pH7~7.5范围内生长,酵母菌和霉菌通常在pH4.5~6范围内生长。值得注意的是,在微生物生长繁殖和代谢过程中,由于营养物质被分解利用和代谢产物的形成与积累,会导致培养基pH发生变化,若不对培养基pH条件进行控制,往往导致微生物生长速度或(和)代谢产物产量降低。因此,为了维持培养基pH的相对恒定,通常在培养基中加入pH缓冲剂,常用的缓冲剂是一氢和二氢磷酸盐(如K2HPO4和KH2PO4)组成的混合物。K2HPO4溶液呈碱性, KH2PO4溶液呈酸性,两种物质的等克分子混合溶液的pH值为6.8。当培养基中酸性物质积累导致H 浓度增加时, H 与弱碱性盐结合形成弱酸性化合物,培养基pH不会过度降低;如果培养基中OH-浓度增加, OH-则与弱酸性盐结合形成弱碱性化合物,培养基pH也不会过度升高。 
 
  但K2HPO4/KH2PO4缓冲系统只能在一定的pH范围(pH6.4~7.2)内起调节作用。有些微生物,如乳酸菌能大量产酸,上述缓冲系统就难以起到缓冲作用,此时可在培养基中添加难溶的碳酸盐(如CaCO3)来进行调节, CaCO3难溶于水,不会使培养基pH过度升高,但它可以不断中和微生物产生的酸,同时释放出CO2,将培养基pH控制在一定范围内。
  青岛日水微生物培养基
  在培养基中还存在一些天然的缓冲系统,如氨基酸、肽、蛋白质都属于两性电解质,也可起到缓冲剂的作用。
 

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.