Abstract: An improved colorimetric method was developed for the determination of lactic acid in fermentation broth. Firstly, the fermentation broth was given a suitable treatment that the protein and glucose were removed, and then the traditional colorimetry method applied for the determination of lactic acid was optimized by orthogonal test. The results showed that the optimum absorbance wavelength is 565 nm. The optimal colorimetric conditions are: 0.05 g Ca(OH) 2, 0.8 ml 20% CuSO4, 6 ml concentrated H2SO4, 0.125 ml 0.5% p-hydroxydiphenyl,reaction time 15 minutes and heating time 5 minutes. Linear range is 15～50μg/ml (A=0.0189C-0.0808, r=0.9989). The average recovery is 102.4%. The color development for determination of lactic acid by colorimetric method is stable within more than two hours, and the RSD is 2.6% (n=12). The method established in this study is quick, accurate and stable.

Key words: lactic acid, determination, p-hydroxybiphenol colorimetry, orthogonal test