Determination Of Trehalose In Saccharomyces Cerevisiae With Near Infre-Red Reflectance Spectrometry After Extraction And Purification

Trehalose has been attracted by its unique biological characteristics, it has becomed the researching and developing hotspot currently. This paper mainly based on Saccharomyces cerevisiae which contained high trehalose,can reach 20% of dry weight. We studied on screening, extracting and testing trehalose of yeast, establishing the yeast fermentation dynamics control model, establishing of near infrared spectroscopy determination trehalose. And mainly got some results as below:(1) Screened Saccharomyces cerevisiae PF2 is the best strain to produce trehalose during 24 strains. We also established PF2 growing dynamics model, generating dynamics model of trehalose , consumption dynamic model of substrate.(2) Established the oxygenated sulfuric acid colorimetric and high performance liquid chromatograpHic method to detect intracellular trehalose in s.cerevisiae. Among them, the oxygenated sulfuric acid colorimetric method is easy to operate and suitable for qualitative determination;The results of high-performance liquid chromatograpHic method is preciser, reproducibility is better, was suitable for the accurate quantitative.(3) Using Plackett–Burman, Box - Behnken Design and Design-Expert software, we determined the high accumulation trehalose culture conditions in trehalose accumulation quantity and biomass index: initial PH was 5.4, inoculum concentration was 15%, temperature was 40℃, rotate speed was 150r/min, fermentation time was 20h. We could get more biomass and trehalose under this condition. Also we got highly medium formula to accumulate trehalose: glucose was 40g/L, yeast extract powder was 15 g/L, (NH4)2SO4 was 4.5 g/L, peptone was 20g/L, CuSO4 was 31.5 mg/L, lactoflavin was 0.17 mg/L, tocopHerol was 0.8 mg/L, MgSO4 was 400 mg/L, PABA was 0.6 mg/L, CaSO4 was 140 mg/L, and Nicl2·6H2O was 36 mg/L. Trehalose accumulation could amount to 0.23 g/L under optimized conditions, was 54.34% higher than before which was 0.125g/L.(4) Using high performance liquid chromatograpHy (HPLC) technology to monitor and test trehalose online during s.cerevisiae fermentation process. The tests found that: There was a regular pattern with the yeast fermentation time changes, when fermentation time reaching 20h, we could get most trehalose, after 20h it kept stability; Yeast growth also has the same rule, It could explain that 20h is best time for both trehalose accumulating and yeast growing; Exocellular trehalose generation time earlier than the intracellular trehalose and has certain regularity and yield more, can achieve inside the cell, the discovery of 50% for exocellular trehalose further research and exploration to provide the basis.(5) Using near infrared spectral analysis technology could achieve intracellular trehalose’s quantitative determination. By using QUNAT-2 OPUS 5.5 analysis program, choosing partial least-squares regression method to establish the calibration model to detect intracellular trehalose during yeast fermentation process, then using validation samples to test this model, the results was accurate and reliable.