Menaquinone-7 production from maize meal hydrolysate by Bacillus isolates with diphenylamine and analogue resistance

Abstract

A menaquinone-7 (MK-7) high-producing strain needs to be isolated to increase MK-7 production, in order to meet a requirement of MK-7 given the low MK-7 content in food products. This article focuses on developing MK-7 high-producing strains via screening and mutagenesis by an atmospheric and room temperature plasma (ARTP) mutation breeding system. We isolated an MK-7-producing strain Y-2 and identified it as Bacillus amyloliquefaciens, which produced (7.1±0.5) mg/L of MK-7 with maize meal hydrolysate as carbon source. Then, an MK-7 highproducing strain B. amyloliquefaciens H.β.D.R.-5 with resistance to 1-hydroxy-2-naphthoic acid, β-2-thienylalanine, and diphenylamine was obtained from the mutation of the strain Y-2 using an ARTP mutation breeding system. Using strain H.β.D.R.-5, efficient production of MK-7 was achieved ((30.2±2.7) mg/L). In addition, the effects of nitrogen sources, prenyl alcohols, and MgSO4 on MK-7 production were investigated, suggesting that soymeal extract combined with yeast extract, isopentenol, and MgSO₄ was beneficial. Under the optimized condition, the MK-7 production and biomass-specific yield reached (61.3±5.2) mg/L and 2.59 mg/L per OD₆₀₀ unit respectively in a 7-L fermenter. These results demonstrated that strain H.β.D.R.-5 has the capacity to produce MK-7 from maize meal hydrolysate, which could reduce the substrate cost.

摘要

目的

通过从自然界中筛选和传统诱变育种相结合的方 法,获得一株以玉米水解液为底物且能高效合成 甲萘醌-7(MK-7)的芽孢杆菌突变菌株。

创新点

首次在中国的发酵豆制品——豆豉中分离得到一 株能以玉米水解液为底物合成MK-7 的解淀粉芽 孢杆菌Y-2(Bacillus amyloliquefaciens Y-2),并 通过传统诱变育种获得一株带有二苯胺和结构 类似物抗性的、以玉米水解液为底物的、高产 MK-7 的菌株B. amyloliquefaciens H.β.D.R.-5。

方法

以来自中国不同省市地区的豆豉为分离样品,筛 选高产纳豆激酶的菌株,再从中挑选出高产 MK-7 的菌株,并通过16S rDNA 分析对其种属进 行鉴定。采用常压室温等离子体(ARTP)系统, 对分离到的高产MK-7 菌株进行诱变处理,获得 解除3-脱氧-D-阿拉伯庚酮糖-7-磷酸合成酶(即 结构类似物抗性)和聚丙烯焦磷酸合成酶(即二 苯胺抗性)反馈调节的菌株。最后,考察不同氮 源、乙戊烯醇和镁离子(Mg²⁺)对突变菌株合成 MK-7 的影响,并分析在7 L 发酵罐中合成MK-7 的区别。

结论

从中国豆豉中分离到了一株以玉米水解液为底物 合成MK-7 的菌株,经16S rDNA 分析比对,鉴 定为Bacillus amyloliquefaciens(图1)。通过比 较MK-7 产量,发现利用ARTP 可以有效获得解 除反馈调节作用的且高产MK-7 的突变菌株 H.β.D.R.-5(表1)。以大豆水解液和酵母水解液 为氮源, 异戊醇和MgSO₄ 有利于突变菌株 H.β.D.R.-5 合成MK-7(图2、表2 和表3)。综 上所述,利用ARTP 处理从中国豆豉中分离到的 以玉米水解液为底物的合成MK-7 的菌株,可获 得高产的MK-7 菌株,该方法对选育工业化合成 MK-7 的菌株有重要参考价值。