A high-throughput system for screening high diacetyl-producing lactic acid bacteria in fermented milk in 96-well microplates

  • HuaiXiang Tian
  • BenJie Yu
  • LianZhong Ai
  • HaiYan Yu
  • Chen ChenEmail author
Original Paper


Lactic acid bacteria play an important role in the food industry and can produce many aromatic compounds during the fermentation process, including diacetyl, an important compound with a buttery flavor. However, existing methods for screening strains that produce high levels of diacetyl are time-consuming and inefficient. An integrated strategy for high-throughput screening of diacetyl-producing strains was therefore developed in this study. This strategy uses 96-deep-well plates for microbial strain cultivation and milk fermentation and trichloroacetic acid for protein precipitation. A colorimetric method based on the reaction of diacetyl with O-phenylenediamine was then developed to determine the diacetyl concentrations in the wells. Good agreement was observed between high-throughput cultivation and traditional method, confirming that the deep-well microplates could replace beaker flasks to scale down cell cultivation and fermentation. The diacetyl concentrations measured with a microplate reader were comparable to those determined via gas chromatography (R2 = 0.9927) and ultraviolet spectrophotometry (R2 = 0.9943), and a high recovery level (94.88%) was obtained. Overall, this strategy increased the screening efficiency by 5- to 30-fold compared with traditional methods. Next, this strategy was used successfully to screen two strains, 1-16 (37.05 ± 1.32 mg/L) and CN-7 (32.35 ± 0.54 mg/L), and the diacetyl yields were verified in a 5-L reactor. Our findings suggest that this newly developed high-throughput strategy can accelerate screening efficiency at a low cost and with a high level of accuracy. This strategy has great potential for targeted screening of strains that produce high levels of diacetyl in fermented milk.


High-throughput screening 96-well microplates Diacetyl Microplate reader 



This work was sponsored by the “Shu Guang” Project (No. 16SG50), which was supported by the Shanghai Municipal Education Commission, Shanghai Education Development Foundation, and Shanghai Rising-Star Program (No. 17QB1404200), “Lvyangjinfeng” Outstanding Doctor Project (No. yzlyjfjh2016YB107).

Compliance with ethical standards

Conflict of interest

The authors declare that there are no conflicts of interest.


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Food Science and TechnologyShanghai Institute of TechnologyShanghaiChina
  2. 2.Shanghai Engineering Research Center of Food Microbiology, School of Medical Instrument and Food EngineeringUniversity of Shanghai for Science and TechnologyShanghaiChina
  3. 3.Shanghai Research Institute of Fragrance & Food FlavorShanghaiChina
  4. 4.Yangzhou University Healthy Source Dairy Co., LtdYangzhouChina

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