顶空-气相色谱法测定丁酸梭菌发酵液中 短链脂肪酸含量

短链脂肪酸是丁酸梭菌生长代谢过程中的重要产物,是肠道内发生益生作用的主要物质之一。利用顶空-气相色谱(HS-GC)内标法测定短链脂肪酸,对该方法进行了方法学验证,并测定了丁酸梭菌在48 h发酵过程中短链脂肪酸含量的变化。结果表明:采用HS-GC可以很好地将各短链脂肪酸分离,且选用的内标物2-乙基丁酸与待测组分之间分离度较好;各短链脂肪酸标准品在0. 1～20. 0 mmol/L范围内,线性相关系数均大于0. 999,检出限为0. 01～0. 17 mmol/L,定量限为0. 02～0. 53 mmol/L,3 d内稳定性良好,样品加标回收率在88. 90%～108. 71%之间,符合测定要求;发酵液中检测到乙酸和丁酸两种短链脂肪酸,且含量随发酵时间延长逐渐增加,在40～48 h期间,乙酸和丁酸含量分别稳定在11. 9 mmol/L和14. 1 mmol/L左右。该方法无需复杂的前处理操作,准确度和精密度均符合要求,总分析时间较短,适用于丁酸梭菌发酵液中短链脂肪酸的快速检测。

Determination of short chain fatty acids in Clostridium butyricum fermentation broth by headspace-gas chromatography

Short fatty acids are important products in the growth and metabolism of Clostridium butyricum and are one of the main substances of probiotics in the intestinal tract. The detection method of short fatty acids was established by headspace-gas chromatography (HS-GC) and internal standard method. The method was verified and used to detect the change of short fatty acid content in the fermentation broth of Clostridium butyricum within 48 h fermentation. The results showed that the HS-GC method could separate the short chain fatty acids well, and the selected internal standard of 2-ethylbutyric acid had good separation between the components to be tested. When each short chain fatty acid standard was in the range of 0.1-20.0 mmol/L, the linear correlation coefficient was greater than 0.999, the limit of detection was 0.01-0.17 mmol/L, and the limit of quantification was 0.02-0.53 mmol/L. The stability within three days was good, and the recovery of standard addition ranged from 88.90% to 108.71%, which met the determination requirements. Two short chain fatty acids of acetic acid and butyric acid were detected in the fermentation broth of Clostridium butyricum, and the contents of the two short chain fatty acids increased with the fermentation time prolonging. The acetic acid and butyric acid contents were stable at about 11.9 mmol/L and 14.1 mmol/L respectively when the fermentation time was from 40 h to 48 h. The method did not require complicated pre-treatment operations, the accuracy and precision were in compliance with the requirements, and the total analysis time was short. The method was suitable for the rapid detection of short chain fatty acids in the fermentation broth of Clostridium butyricum.