[Objective] Based on GB 5009.211-2022,key experimental conditions including inoculum preparation method,culture time and pretreatment methods were optimized and refined to established an accurate and stable microtiter plate microbiological method for the determination of folic acid content in milk powder. [Method] The effects of different inoculum preparation methods（centrifugation-washing and consumption）,McFarland turbidity of inoculum（0.5 MCF,1.0 MCF,1.5 MCF）,culture time（22 h,32 h,40 h）on the growth standard curve of Lactobacillus rhamnosus were analyzed,and the folic acid content measured by the standard reference material（SRM 1869）was combined to optimize the test conditions. SRM 1869,QC-IP-737 and 20 commercial milk powder were treated with sodium hydroxide ethanol solution by ultrasound and water bath, respectively. The determination results of folic acid content were compared,and t-test was conducted at 95% confidence level to analyze the differences between the two pretreatment methods. [Result] The results showed that under the same McFarland turbidity,the inoculum prepared by centrifugation-washing was better than consumption, and adjusting the McFarland turbidity to 1.0 MCF and incubating at 36 ℃ for 40 h was recommended. At 95% confidence level,the P-values of the t-test results of SRM 1869,QC-IP-737 and 20 commercial milk powder were > 0.05,indicating that the two pretreatment methods had no significant difference in the influence of the determination results. [Conclusion] This study optimized the preparation method of inoculum,refined the culture time and the pretreatment method of milk powder in GB 5009.211-2022. The established microtiter plate microbiological method could provide data support for the subsequent revision of national standards. The optimized experimental conditions could provide scientific reference for other laboratories to carry out this work.

folic acid  /  microtiter plate  /  microbiological method  /  milk powder  /  pretreatment