微生物發(fā)酵培養(yǎng)基配制時的關(guān)鍵點
發(fā)布時間：
2022-12-27
作者：
微生物發(fā)酵培養(yǎng)基配制時的關(guān)鍵點
注意營養(yǎng)物質(zhì)的濃度比和C/N比，如:糖分含量要適合微生物生長才能良好,糖分過多則抑制微生物生長。一般微生物適宜C/N是25:1[ 元素C/N的比值,也指培養(yǎng)基中還原糖的含量與粗蛋白質(zhì)含量的比值 ]。
微生物發(fā)酵培養(yǎng)基配制關(guān)鍵點
第一，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，反而起到抑菌或殺菌作用。
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物質(zhì)的量比值，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/l時，菌體大量繁殖，谷氨酸積累少；當(dāng)培養(yǎng)基碳氮比為3/l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協(xié)調(diào)。
第二，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(xì)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)致微生物生長速度下降或[ 和 ]代謝產(chǎn)物產(chǎn)量下降。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KH2PO4和K2HPO4 ]組成的混合物。K2HPO4溶液呈堿性，KH2PO4溶液呈酸性，兩種物質(zhì)的等量混合溶液的pH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KH2PO4和K2HPO44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)。
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。
第三，原料來源的選擇
在配制培養(yǎng)基時應(yīng)盡量利用廉價且易于獲得的原料作為培養(yǎng)基成分，特別是在發(fā)酵工業(yè)中，培養(yǎng)基用量很大，利用低成本的原料更體現(xiàn)出其經(jīng)濟價值。例如，在微生物單細(xì)胞蛋白的工業(yè)生產(chǎn)過程中，常常利用糖蜜[ 制糖工業(yè)中含有蔗糖的廢液 ]，乳清[ 乳制品工業(yè)中含有乳糖的廢液 ]，豆制品工業(yè)廢液及黑廢液[ 造紙工業(yè)中含有戊糖和己糖的亞硫酸紙漿 ]等都可作為培養(yǎng)基的原料。再如，工業(yè)上的甲烷發(fā)酵主要利用廢水，廢渣作原料，而在我國農(nóng)村，已推廣利用人畜糞便及禾草為原料發(fā)酵生產(chǎn)甲烷作為燃料。另外，大量的農(nóng)副產(chǎn)品或制品，如鼓皮，米糠，玉米漿，酵母浸膏，酒糟，豆餅，花生餅，蛋白胨等都是常用的發(fā)酵工業(yè)原料。
第四，滅菌處理
要獲得微生物純培養(yǎng)，必須避免雜菌污染，因此對所用器材及工作場所進行消毒與滅菌。對培養(yǎng)基而言，更是要進行嚴(yán)格的滅菌。對培養(yǎng)基一般采取高壓蒸汽滅菌，一般培養(yǎng)基用 1.05kg/cm2，121.3攝氏度條件下維持15～30min可達到滅菌目的。在高壓蒸汽滅菌過程中，長時間高溫會使某些不耐熱物質(zhì)遭到破壞，如使糖類物質(zhì)形成氨基糖，焦糖，因此含糖培養(yǎng)基常在0.56kg/ cm2，112.6攝氏度15～30min進行滅菌，某些對糖類要求較高的培養(yǎng)基，可先將糖進行過濾除菌或間歇滅菌，再與其他已滅菌的成分混合；長時間高溫還會引起磷酸鹽，碳酸鹽與某些陽離子[ 特別是鈣，鎂，鐵離子 ]結(jié)合形成難溶性復(fù)合物而產(chǎn)生沉淀，因此，在配制用于觀察和定量測定微生物生長狀況的合成培養(yǎng)基時，常需在培養(yǎng)基中加入少量螯合劑，避免培養(yǎng)基中產(chǎn)生沉淀，常用的螯合劑為乙二胺四乙酸[ EDTA ]。還可以將含鈣，鎂，鐵等離子的成分與磷酸鹽，碳酸鹽分別進行滅菌，然后再混合，避免形成沉淀；高壓蒸汽滅菌后，培養(yǎng)基pH會發(fā)生改變[ 一般使pH降低0.2 ]，可根據(jù)所培養(yǎng)微生物的要求，在培養(yǎng)基滅菌前后加以調(diào)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.