盐增强培养对弗氏链霉菌产新霉素的影响

目的: 弗氏链霉菌(Streptomyces fradiae)作为氨基糖苷类抗生素新霉素的主要生产菌株,其新霉素B具有抗菌活性强、抗癌、抗HIV等作用,提高新霉素B的效价具有重要意义。方法: 在满足微生物正常生长所需盐离子的条件下,通过盐增强培养的方式向培养基中添加不同种类、浓度无机盐来改变细胞壁附近的理化特性、渗透压以及培养基中的碳氮比。结果: 不同无机盐对新霉素B效价影响程度由高到低为(NH₄)₂SO₄、NaCl、KCl、K₂SO₄;当添加60 mmol/L(NH₄)₂SO₄时新霉素B的效价达到最高为15 864 U/mL,而NaCl在80 mmol/L浓度时产量最高为7 429.7 U/mL,相比未添加无机盐分别提高3.8倍和0.82倍。在含有80 mmol/L的NaCl的发酵培养基中添加不同浓度的(NH₄)₂SO₄并定时取样检测其中氨基氮、还原糖、pH、TG、菌浓以及新霉素B效价的变化,发现60 mmol/L(NH₄)₂SO₄浓度下氨基氮、还原糖、TG的消耗速率加快,菌体比生长速率μ最大为0.097/h,新霉素B的合成加快且产量提高为17 399 U/mL,同时菌丝呈现出聚集变短的形态且产孢提前。结论: 盐增强培养方式既可以作为一种形态学工程手段,通过改变弗氏链霉菌的微观形态来促进次级代谢产物新霉素B产量的提高,同时也可以作为培养基优化策略来提高新霉素B的产量,这为进一步提高弗氏链霉菌中次级代谢产物产量的研究奠定了基础。

Effect of Salt-enhanced Culture on the Production of Neomycin by Streptomyces fradiae

Objective: Streptomyces fradiae is the main production strain of the aminoglycoside antibiotic neomycin. Its neomycin B has strong antibacterial activity, anti-cancer and anti-HIV effects, and it is of great significance to increase the titer of neomycin B.Methods: We added different kinds and concentrations of inorganic salts to the medium to change the physical and chemical properties near the cell wall, osmotic pressure and C/N ratio of the medium under the condition of satisfying the salt ions required for the normal growth of microorganisms.Results: The degree of influence of different inorganic salts on the titer of neomycin B from high to low is (NH₄)₂SO₄, NaCl, KCl and K₂SO₄ from high to low, and the titer of neomycin B reached the highest value of 15 864 U/mL when 60 mmol/L (NH₄)₂SO₄ was added, the maximum yield of NaCl at 80 mmol/L concentration was 7 429.7 U/mL, which was 3.8 times and 0.82 times higher than that without adding inorganic salts. Then we added different concentrations of (NH₄)₂SO₄ to the culture medium containing 80 mmol/L NaCl and sampled them regularly to detect the changes of amino nitrogen, reducing sugar, pH, TG, bacterial concentration and neomycin B titer, it was found that the consumption rate of amino nitrogen, reducing sugar and TG was increased at the concentration of 60 mmol/L (NH₄)₂SO₄, the maximum specific growth rate of mycelium was 0.097 /h, the synthesis of neomycin B was accelerated and the yield was increased to 17 399 U/mL, at the same time, the mycelium was shortened and the sporulation was advanced.Conclusion: The salt-enhanced culture could be used as a morphological engineering method to improve the yield of neomycin B by changing the microscopic morphology of Streptomyces fradiae. It can also be used as a medium optimization strategy to increase the yield of neomycin B, which laid the foundation for further studies on increasing the yield of Streptomyces secondary metabolite.