微生物发酵培养基配制时的关键点

发布时间:

2022-12-27

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微生物发酵培养基配制时的关键点
 
注意营养物质的浓度比和C/N比,如:糖分含量要适合微生物生长才能良好,糖分过多则抑制微生物生长。一般微生物适宜C/N是25:1[  元素C/N的比值,也指培养基中还原糖的含量与粗蛋白质含量的比值 ]。
 
微生物发酵培养基配制关键点
 
第一,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,反而起到抑菌或杀菌作用。
 
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物质的量比值,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/l时,菌体大量繁殖,谷氨酸积累少;当培养基碳氮比为3/l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协调。
微生物在培养基上的形态图
第二,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下降或[ 和 ]代谢产物产量下降。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KH2PO4和K2HPO4 ]组成的混合物。K2HPO4溶液呈碱性,KH2PO4溶液呈酸性,两种物质的等量混合溶液的pH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KH2PO4和K2HPO44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。
 
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。
 
第三,原料来源的选择
 
在配制培養基时应尽量利用廉价且易于获得的原料作为培養基成分,特别是在发酵工业中,培養基用量很大,利用低成本的原料更体现出其经济价值。例如,在微生物单细胞蛋白的工业生产过程中,常常利用糖蜜[ 制糖工业中含有蔗糖的废液 ],乳清[ 乳制品工业中含有乳糖的废液 ],豆制品工业废液及黑废液[ 造纸工业中含有戊糖和己糖的亚硫酸纸浆 ]等都可作为培養基的原料。再如,工业上的甲烷发酵主要利用废水,废渣作原料,而在我国农村,已推广利用人畜粪便及禾草为原料发酵生产甲烷作为燃料。另外,大量的农副产品或制品,如鼓皮,米糠,玉米浆,酵母浸膏,酒糟,豆饼,花生饼,蛋白胨等都是常用的发酵工业原料。
 
第四,灭菌处理
 
要获得微生物纯培养,必须避免杂菌污染,因此对所用器材及工作场所进行消毒与灭菌。对培養基而言,更是要进行严格的灭菌。对培養基一般采取高压蒸汽灭菌,一般培養基用 1.05kg/cm2,121.3摄氏度条件下维持15~30min可达到灭菌目的。在高压蒸汽灭菌过程中,长时间高温会使某些不耐热物质遭到破坏,如使糖类物质形成氨基糖,焦糖,因此含糖培養基常在0.56kg/ cm2,112.6摄氏度15~30min进行灭菌,某些对糖类要求较高的培養基,可先将糖进行过滤除菌或间歇灭菌,再与其他已灭菌的成分混合;长时间高温还会引起磷酸盐,碳酸盐与某些阳离子[ 特别是钙,镁,铁离子 ]结合形成难溶性复合物而产生沉淀,因此,在配制用于观察和定量测定微生物生长状况的合成培養基时,常需在培養基中加入少量螯合剂,避免培養基中产生沉淀,常用的螯合剂为乙二胺四乙酸[ EDTA ]。还可以将含钙,镁,铁等离子的成分与磷酸盐,碳酸盐分别进行灭菌,然后再混合,避免形成沉淀;高压蒸汽灭菌后,培養基pH会发生改变[ 一般使pH降低0.2 ],可根据所培养微生物的要求,在培養基灭菌前后加以调整。
 

 
Key Points of Microbial Fermentation Medium Preparation
Pay attention to the concentration ratio of nutrients and C/N ratio, such as: sugar content to be suitable for microbial growth can be good, too much sugar will inhibit microbial growth. The optimum C/N ratio for general microorganisms is 25:1 [the ratio of C/N elements, also refers to the ratio of reducing sugar content to crude protein content in the medium].
Key points of microbial fermentation medium preparation
First, the nutrient concentration and proportion are suitable
The microorganisms can grow well when the nutrient concentration is suitable in the medium, and the low concentration of nutrients can not satisfy the normal growth of microbes. When the concentration is too high, it may inhibit the growth of microbes, such as high concentration of sugar, inorganic salts, heavy metal ions and so on, not only can not maintain and promote microorganism. Growth, on the contrary, plays a bacteriostasis or bactericidal effect.
In addition, the concentration ratio of various nutrients in the medium also directly affects the growth and reproduction of microorganisms and the formation and accumulation of metabolites, of which carbon and nitrogen ratio [C/N] has a greater impact. Strictly speaking, C/N ratio refers to the ratio of carbon to nitrogen in the medium, and sometimes also refers to the ratio of reducing sugar to crude protein in the medium. For example, in the process of producing glutamic acid by microbial fermentation, when the medium carbon and nitrogen ratio is 4/l, the mycelium is multiplied and the accumulation of glutamic acid is less. When the medium carbon and nitrogen ratio is 3/l, the growth of the bacteria is inhibited and the yield of glutamic acid is greatly increased. In the process of antibiotic fermentation, the growth of the bacteria and the synthesis of antibiotics can be controlled by controlling the ratio of the available nitrogen [or carbon] source to the source of the slow nitrogen [or carbon] in the medium.
Second, control the pH condition
The pH of the medium must be controlled within a certain range to meet the growth and reproduction of different types of microorganisms or to produce metabolites. In general, bacteria and actinomycetes grow in the range of pH7 to 7.5, and the yeast and mould usually grow within the range of pH4.5 to 6, in general, the optimum pH conditions for the growth and reproduction of various microorganisms are different. It is worth noting that in the process of microbial growth and metabolism, the decomposition and utilization of nutrients and the formation and accumulation of the metabolites can lead to the change in the medium pH. If the condition of the medium pH is not controlled, the growth rate of microorganism is often reduced or [and] the yield of metabolites is decreased. Therefore, in order to maintain the relative constant of the medium pH, pH buffer is usually added in the medium, and the commonly used buffer is a mixture of one hydrogen and two hydrogen phosphate, such as KH2PO4 and K2HPO4. The K2HPO4 solution is alkaline, the KH2PO4 solution is acidic, and the pH of the two mixture is 6.8. When the accumulation of acidic substances in the medium leads to the increase of H+ concentration, H+ and weak alkaline salts are combined to form weak acid compounds, and the medium pH will not be over reduced. If the concentration of OH- in the medium is increased, OH- is combined with weak acid salt to form a weak alkali compound, and the culture medium pH will not increase.
However, the KH2PO4 and K2HPO44 buffer systems can only play a regulatory role within a certain pH range [pH6.4 ~ 7.2]. Some microorganisms, such as lactic acid bacteria, can produce a large amount of acid, and the buffer system can not play a buffer role. At this time, it can be adjusted by adding hard soluble carbonate, such as CaCO3, in the medium. The CaCO3 is difficult to dissolve in water, and the medium pH can not be overly raised, but it can neutralize the acid produced by the microorganism and release the CO2 at the same time. The culture medium pH is controlled within a certain range.
There are also some natural buffering systems in the medium, such as amino acids, peptides and proteins, which are both amphoteric electrolyte and buffer.
Third, the selection of source of raw materials
In the preparation of culture medium, we should try to make use of cheap and easy to obtain raw materials as culture medium, especially in the fermentation industry, the amount of culture medium is great, and its economic value is reflected by the low cost raw material. For example, during the industrial production of microbiological single cell proteins, molasses is often used [the waste liquid containing sucrose in the sugar industry], whey [dairy industry containing lactose waste], bean product industrial waste liquid and black waste liquid [bisulfite pulp containing pentose and hexose in the paper industry] as a medium of culture. Material. In addition, the industrial methane fermentation mainly uses waste water and waste residue as raw material. In rural areas of China, methane is produced by fermenting human and livestock manure and grass as raw materials. In addition, a large number of agricultural and sideline products or products, such as drum skin, rice bran, corn pulp, yeast extract, distiller's grains, bean cake, peanut cake, peptone and so on are commonly used fermenting industrial raw materials.
Fourth, sterilization treatment
In order to get pure culture of microorganism, contamination of mixed bacteria must be avoided. Disinfection and sterilization of the equipment and workplace are necessary. For the culture medium, it is necessary to sterilize strictly. Generally, high pressure steam sterilization is applied to the medium, and the general medium can maintain 15 to 30min under the condition of 1.05kg/cm2121.3 degree Celsius, which can achieve the purpose of sterilization. In the process of high pressure steam sterilization, a long time high temperature can cause some heat resistant substances to be destroyed, such as making carbohydrates to form amino sugar and caramel, so the sugar containing medium is usually sterilized at 15 ~ 30min degrees centigrade at 0.56kg/ cm2112.6 degrees Celsius. Bacteria are mixed with other sterilized ingredients; long time high temperatures can also cause phosphate, and carbonates produce precipitates with certain cations, especially calcium, magnesium, and iron ions, and, therefore, are often needed in the preparation of a synthetic medium for observing and quantifying the growth state of microbes. A small amount of chelating agent is added to the base to avoid precipitation in the medium. The commonly used chelating agent is ethylenediamine tetra acetic acid [EDTA]. The calcium, magnesium, and iron plasma can be sterilized with phosphate and carbonate respectively, and then mixed to avoid the formation of precipitation. After autoclave, the culture medium pH will change [generally make pH 0.2], and can be adjusted according to the requirements of the cultured microorganisms before and after the culture of the culture medium.