日水培養(yǎng)基：凝結(jié)芽孢桿菌BC01培養(yǎng)基成分及培養(yǎng)條件
發(fā)布時間：
2022-12-27
作者：
凝結(jié)芽孢桿菌BC01培養(yǎng)基成分及培養(yǎng)條件
摘要：在凝結(jié)芽孢桿菌芽孢的形成過程中，碳源、氮源都能作為限制性生長底物，其中碳源對芽孢的形成有重要的影響。碳源缺乏可誘導(dǎo)芽孢形成；而碳源充足有利于營養(yǎng)菌體的生長，不利于芽孢的形成，且菌體易發(fā)生自溶。
在氮源基礎(chǔ)培養(yǎng)基中分別加入酵母浸出粉、蛋白胨、黃豆餅粉、酵母膏、氯化銨和硫酸銨 6 種氮源進(jìn)行考察，結(jié)果如表 8 所示，凝結(jié)芽孢桿菌 BC01 對這 5 種氮源均可以利用，其中以蛋白胨和酵母浸出粉為氮源時其產(chǎn)孢率較高，分別達(dá)到了 37.5%和 59.5%；其他有機(jī)氮源產(chǎn)孢率偏低，而以無機(jī)氮源硫酸銨作為氮源時產(chǎn)孢率最低?？赡苁怯袡C(jī)氮源是天然的營養(yǎng)物，富含多種營養(yǎng)成分及未知生長因子，能促進(jìn)菌體生長。無機(jī)氮源相對營養(yǎng)成分單一，因此導(dǎo)致凝結(jié)芽孢桿菌將其作為氮源時的產(chǎn)孢率不高。綜上所述，選擇酵母浸出粉作為氮源。
Mn2+是微生物生長和芽孢形成所需的一種微量元素，可作為超氧化物歧化酶、黃嘌呤氧化酶和 L-阿拉伯糖異構(gòu)酶等酶催化過程中重要的輔助因子，因此考察了不同錳離子濃度對活菌數(shù)及芽孢數(shù)量的影響。表 9 中的實(shí)驗(yàn)結(jié)果顯示 Mn2+ 濃度對凝結(jié)芽孢桿菌芽孢的形成有顯著影響，當(dāng) Mn2+濃度為 10 mg/L 時，芽孢數(shù)量達(dá)到最高 (2.5×108 CFU/mL)，是 5 mg/L Mn2+濃度下芽孢數(shù)的 3 倍，且產(chǎn)孢率可達(dá) 71.4%；此后隨著 Mn2+濃度的增加芽孢數(shù)量呈下降趨勢，當(dāng) Mn2+濃度達(dá)到 30 mg/L 時芽孢率僅為 21.4%。由此可見，Mn2+最適濃度為 10 mg/L。
根據(jù)上述實(shí)驗(yàn)結(jié)果選擇糖蜜、酵母浸出粉、硫酸錳進(jìn)行 3 因素 3 水平的正交試驗(yàn)(表 10)，正交試驗(yàn)結(jié)果見表 11。
正交試驗(yàn)結(jié)果表明，培養(yǎng)基各組分對芽孢產(chǎn)率的影響主次為 A>B>C，最優(yōu)組合為 A2B2C2。通過單因素試驗(yàn)及正交試驗(yàn)得出的最佳培養(yǎng)基成分為：糖蜜 10.0 g/L，酵母浸出粉 20.0 g/L，NaCl 5.0 g/L，K2HPO4 5.0 g/L，MnSO4 10.0 mg/L。
培養(yǎng)條件對產(chǎn)孢率影響的結(jié)果如圖 3 所示。當(dāng)接種量增加到 4%時，其產(chǎn)孢率達(dá)到最高(65.1%)，而進(jìn)一步提高接種量后，產(chǎn)孢率不斷下降(圖 3A)，可能是由于菌體生長過快導(dǎo)致代謝產(chǎn)物大量積累，使培養(yǎng)基 pH 急劇降低所致。培養(yǎng)時間對產(chǎn)孢率的影響表現(xiàn)為：前 36 h 其產(chǎn)孢率呈上升趨勢， 36 h 時達(dá)到最高(69.1%)；36 h 后由于營養(yǎng)物質(zhì)缺乏、培養(yǎng)條件惡劣，菌體大量死亡，其產(chǎn)孢率也逐漸降低(圖 3B)。初始 pH 值為中性時，其產(chǎn)孢率達(dá)到最高 64.2%，如圖 3C 所示。由于凝結(jié)芽孢桿菌 BC01 為高溫篩選所得，因此最適培養(yǎng)溫度高于一般細(xì)菌，可達(dá)到45 °C，在該培養(yǎng)溫度下菌株BC01 長勢良好，產(chǎn)孢率最高可達(dá) 65.4%。綜合以上結(jié)果，單因素最佳條件為接種量4%，溫度45 °C，初始 pH 7.0，培養(yǎng)時間 36 h。
按照搖瓶實(shí)驗(yàn)優(yōu)化獲得的最佳培養(yǎng)基成分及培養(yǎng)條件，于 20 L 發(fā)酵罐中進(jìn)行擴(kuò)大培養(yǎng)，考察菌體生長、活菌數(shù)及芽孢個數(shù)，為進(jìn)一步大規(guī)模工業(yè)化生產(chǎn)提供基礎(chǔ)。按實(shí)驗(yàn)獲得的最佳培養(yǎng)基組分配制培養(yǎng)基，即：糖蜜 10.0 g/L，酵母浸出粉 20.0 g/L，NaCl 5.0 g/L，K2HPO4 5.0 g/L，MnSO4 10.0 mg/L。初始 pH 為 7.0，培養(yǎng)溫度為 45 °C，起始通氣量為 10 L/min，轉(zhuǎn)速為 200 r/min，通過調(diào)節(jié)轉(zhuǎn)速及通氣量控制發(fā)酵過程中溶氧水平在 40%以上，培養(yǎng)時間為 48 h。發(fā)酵過程中取樣測活菌數(shù)及芽孢數(shù)，并計(jì)算產(chǎn)孢率，結(jié)果如圖 4 所示。
從圖4可以看出，凝結(jié)芽孢桿菌BC01的芽孢接入發(fā)酵培養(yǎng)基后，0–4 h 為菌體生長的延滯期，4 h 后菌體生長迅速，活菌數(shù)快速上升，在 36 h 達(dá)到最高，為 6.7×109 CFU/mL，隨后由于培養(yǎng)基營養(yǎng)物質(zhì)的耗竭及代謝產(chǎn)物的積累，周圍環(huán)境不利于菌體生長，菌體進(jìn)入衰亡期并開始自溶，表現(xiàn)為活菌數(shù)逐漸降低。芽孢數(shù)在 12 h 時開始逐漸增加，產(chǎn)孢率隨之上升；培養(yǎng) 30 h 后鏡檢可見芽孢成熟脫落，40 h 時達(dá)到最高，為 5.8×109 CFU/mL，相應(yīng)產(chǎn)孢率最高達(dá)到 89.2%；40 h 后芽孢數(shù)逐漸降低，可能是因?yàn)椴糠盅挎邚?fù)蘇為菌體，從而導(dǎo)致芽孢數(shù)量的減少。實(shí)驗(yàn)結(jié)果表明，凝結(jié)芽孢桿菌 BC01 在 20 L 自動發(fā)酵罐中擴(kuò)大培養(yǎng)效果良好，上述實(shí)驗(yàn)獲得的最佳培養(yǎng)條件及培養(yǎng)基成分為進(jìn)一步應(yīng)用于工業(yè)化大規(guī)模生產(chǎn)奠定基礎(chǔ)。
本實(shí)驗(yàn)根據(jù)凝結(jié)芽孢桿菌的相關(guān)特性，通過選擇性培養(yǎng)基在特定培養(yǎng)條件下篩選并鑒定得到抑菌能力較強(qiáng)的凝結(jié)芽孢桿菌，并對其產(chǎn)孢條件進(jìn)行優(yōu)化，得到高產(chǎn)芽孢的發(fā)酵工藝，為其實(shí)現(xiàn)工業(yè)化生產(chǎn)提供參考依據(jù)。本研究通過營養(yǎng)限量和高溫誘導(dǎo)的方式提高凝結(jié)芽孢桿菌 BC01 的芽孢率，這與徐世榮等和路程等的研究結(jié)果大致相同。
在實(shí)際生產(chǎn)中需要選擇合適的碳、氮源，并注意限量添加。很多研究表明 Mn2+ 可以提高菌株的芽孢形成率，本研究的實(shí)驗(yàn)結(jié)果也符合上述結(jié)論。本研究雖然篩選到可用于微生態(tài)制劑的凝結(jié)芽孢桿菌，但是其功能性如何還需要后期通過動物實(shí)驗(yàn)來驗(yàn)證。
日水培養(yǎng)基技術(shù)資料 http://m.ricoportland.com/jishu/newsCategoryId=12.html
The composition and conditions of culture medium of bacillus clotting BC01
Abstract: carbon and nitrogen sources can be used as the limiting growth substrate in the formation of bacillus cerevisiae spores, among which carbon sources have an important influence on the formation of spores. Lack of carbon can induce spore formation. However, sufficient carbon source is conducive to the growth of vegetative bacteria, the formation of spores, and the bacteria are easy to occur autolysis.
Based on the source of nitrogen leaching medium respectively to join yeast powder, peptone, yellow bean cake powder, yeast extract, ammonium chloride and ammonium sulfate, 6 kinds of nitrogen source, the results as shown in table 8, condensation bacillus BC01 of these 5 kinds of nitrogen source can use, including leaching peptone and yeast powder as nitrogen source in the spore production rate is higher, at 37.5% and 37.5% respectively; The spore-producing rate of other organic nitrogen sources was low, while that of inorganic nitrogen source ammonium sulfate was the lowest. It may be that organic nitrogen source is a natural nutrient, rich in a variety of nutrients and unknown growth factors, which can promote the growth of bacteria. The inorganic nitrogen source is relatively simple in nutrient composition, so the spore-producing rate of bacillus clotting is not high when it is used as nitrogen source. In conclusion, yeast extract powder was selected as nitrogen source.
Mn2 + is needed for the formation of microbial growth and spores in a kind of trace elements, can be used as superoxide dismutase (sod), xanthine oxidase and L - Arab sugar in the process of enzyme catalysis of enzymes in the heterogeneous important cofactor, thus examines the different manganese ion concentration on the number of living bacterium and the influence of the number of spores. The experimental results show that the concentration of Mn2 + in table 9 for condensation bacillus spores has remarkable effect on the formation of when the concentration of Mn2 + 10 mg/L, the spore number reached the highest (2.5 x 108 CFU/mL), is 5 mg/L Mn2 + concentrations spore number three times, and the spore production rate could reach 71.4%; After that, the number of spores decreased with the increase of Mn2+ concentration. When the concentration of Mn2+ reached 30 mg/L, the spore rate was only 21.4 percent. Therefore, the optimal concentration of Mn2+ is 10 mg/L.
According to the above experimental results, molasses, yeast leaching powder and manganese sulfate were selected for orthogonal test at the level of 3 factors (table 10). The orthogonal test results were shown in table 11.
The orthogonal test results show that the main effect of each component of culture medium on spore yield is A>B>C, and the optimal combination is A2B2C2. The optimal media composition obtained by single factor test and orthogonal test was: molasses 10.0 g/L, yeast extract powder 20.0 g/L, NaCl 5.0g /L, K2HPO4 5.0g /L, MnSO4 10.0 mg/L.
The effect of culture conditions on spore-producing rate is shown in FIG. 3. When the inoculation amount increased to 4%, the spore production rate reached the highest (65.1%), and further improve the inoculation quantity, spore production rate decline (figure 3 a), may be caused by the bacteria to grow too fast metabolite accumulation in great quantities, make the medium pH caused by reduced sharply. The effect of culture time on spore-producing rate was as follows: the spore-producing rate showed an upward trend in the first 36 hours, and reached the highest level at 36 hours (69.1%). After 36 h, due to lack of nutrients and poor conditions of culture, a large number of bacteria died, and its spore-producing rate gradually decreased (FIG. 3B). When the initial pH value is neutral, its spore-producing rate reaches up to 64.2%, as shown in figure 3C. Due to condensation bacillus BC01 for high temperature screening, so the optimal temperature is higher than general bacteria, can reach 45 ° C, under the culture temperature strain BC01 grew well, spore production rate of up to 65.4%. Comprehensive the above results, the optimum condition for the single factor for inoculation 4%, 45 ° C temperature, initial pH 7.0, 36 h of incubation time.
According to the shaker experimental optimization to obtain the best culture medium and culture conditions, expand training in 20 L fermentation tank, bacteria growth, the number of living bacterium and spore number, in order to further provide the basis for large-scale industrial production. According to the optimal medium group obtained in the experiment, the medium was prepared, i.e., molasses 10.0g /L, yeast extract powder 20.0g /L, NaCl 5.0g /L, K2HPO4 5.0g /L, and MnSO4 10.0mg /L. Initial pH 7.0, raises the temperature of 45 ° C, starting ventilation of 10 L/min, the speed of 200 r/min, by adjusting the rotational speed and ventilation control in the fermentation process of dissolved oxygen level in more than 40%, incubation time for 48 h. The number of viable bacteria and spores were sampled during the fermentation process, and the spore-producing rate was calculated, as shown in figure 4.
Can be seen from the figure 4, condensation spores of bacillus BC01 access after fermentation medium, 0-4 h demurrage of the growth of bacteria, bacteria grow quickly after 4 h, rapidly rising number of living bacterium, reached the highest in 36 h 6.7 x 109 CFU/mL, then due to the culture medium of nutrient depletion and the accumulation of metabolites, the environment is not conducive to bacteria growth, bacteria enter the decline phase and start autolyzed, show the number of living bacterium reduce gradually. The number of spores increased gradually at 12 h, and the spore-producing rate increased accordingly. After 30 h of culture, microscopic examination showed that spores were mature and shed, and reached the highest at 40 h, which was 5.8 * 109 CFU/mL, and the corresponding spore-producing rate was up to 89.2%. The number of spores decreased gradually after 40 h, possibly because some spores recovered to be bacteria, resulting in the decrease of spores. The experimental results show that the condensation bacillus BC01 in 20 L fermentor automatically expand the training effect is good, the experiment to obtain the best culture conditions and medium composition lay a foundation for the further applied to the industrialized mass production.
This experiment based on relevant characteristics of condensation bacillus, through selective medium in a particular culture under the condition of screening and identification of bacteriostatic ability stronger condensation bacillus, and optimize the conditions for its spores, spore get high yield of fermentation process, provide a reference basis for the industrialization production. In this study, the spore rate of coagulative bacillus BC01 was increased by means of nutrition limitation and high temperature induction, which was roughly the same as the results of xu shirong et al., et al.
It is necessary to select suitable carbon and nitrogen sources in actual production, and pay attention to the limited addition. Many studies have shown that Mn2+ can improve the spore formation rate of the strain, and the experimental results of this study are consistent with the above conclusions. Although this study screened bacillus clostriae which can be used in microecological preparation, its function needs to be verified by animal experiments later.