Fermentation conditions of serine/alkaline milk-clotting enzyme production by newly isolated Bacillus licheniformis BL312

  • Yao Zhang
  • Yongjun Xia
  • Phoency F.-H. Lai
  • Xiaofeng Liu
  • Zhiqiang Xiong
  • Jichao Liu
  • Lianzhong AiEmail author
Original Article



This study was conducted to find a microbial milk-clotting enzyme (MCE) with a high and stable milk-clotting activity (MCA) to proteolytic activity (PA) ratio suitable for the cheese industry.


Microbial strains were isolated from soil suspensions cultured in solid casein medium. 16S rDNA of representative isolates were sequenced to identify the microbial species. Nutrition and fermentation conditions were systematically examined to optimize MCA of the selected MCE. Protease inhibitors were used to identify the type of MCE. The casein hydrolysis was analyzed through reversed-phase HPLC (RP-HPLC) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).


The Bacillus licheniformis BL312 was identified from 50 bacterial strains. BL312 MCE achieved a maximal MCA (460 ± 15 SU/mL) at 48 h that was 2.7-fold higher than the control, and the MCA/PA ratio (9.0) and pH (6.6) remained stable throughout the fermentation process. Medium containing 30 g/L wheat bran shorts, 5 g/L glucose, and 3 g/L corn steep liquor was sufficient for optimal BL312 MCE production. Fermentation conditions of an inoculum size of 7.0% (v/v), fermentation temperature of 37 °C, agitation speed of 210 rpm, and initial pH 6.6 were required to achieve maximal MCA. BL312 MCE was inhibited by phenylmethanesulfonyl fluoride (PMSF) and high concentrations of ethylenediaminetetraacetic acid (EDTA) (5–25 mM). The αs-casein (αs-CN) and β-casein (β-CN) hydrolysates generated by BL312 MCE and calf rennet were different.


BL312 MCE is a serine/alkaline protease that exhibits high MCA and various hydrolysis for caseins in comparison with calf rennet.


Isolation Fermentation Bacillus licheniformis Milk-clotting enzyme Serine/alkaline protease Casein hydrolysis 



The authors gratefully acknowledge the National Key R&D Program of China (grant no. 2018YFD0502306) and Shanghai Engineering Research Center of Food Microbiology (grant no. 19DZ2281100) for the granted fellowships.

Funding information

This work was supported by the National Key R&D Program of China (grant no. 2018YFD0502306) and Shanghai Engineering Research Center of Food Microbiology (grant no. 19DZ2281100).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Informed consent

This manuscript is approved by all authors for publication.


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Copyright information

© Università degli studi di Milano 2019

Authors and Affiliations

  • Yao Zhang
    • 1
  • Yongjun Xia
    • 1
  • Phoency F.-H. Lai
    • 1
  • Xiaofeng Liu
    • 1
  • Zhiqiang Xiong
    • 1
  • Jichao Liu
    • 2
  • Lianzhong Ai
    • 1
    Email author
  1. 1.Shanghai Engineering Research Center of Food Microbiology, School of Medical Instrument and Food EngineeringUniversity of Shanghai for Science and TechnologyShanghaiChina
  2. 2.Beijing Sanyuan Foods Co., LtdBeijingChina

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