为了确定槐糖脂生物表面活性剂生产工艺的性能,重要的是要有准确和特定的分析技术。最受欢迎的是蒽酮测定法,重量分析(己烷:乙酸乙酯萃取)和高效液相色谱(HPLC)。分析工具的选择因成本而异,可用性和易用性,然而,这些技术从未直接相互比较。在这项工作中,用三种技术全面测试了75种具有不同产物/底物浓度的发酵液,并进行了比较。HPLC-UV检测(198nm)能够定量C18:1亚末端羟基二乙酰化的乳酸槐糖脂至0.3g/L的下限,具有低的变异性(<3.21%)。液体后的肉汤重量定量:与HPLC相比,用己烷和乙酸乙酯进行的液体萃取显示出一定的线性(R2=0.658),但不能定量低于11.06g/L,即使在样品中没有检测到槐糖脂,强调了该方法在最终重量测量中共同提取非槐糖脂成分的非特异性。蒽酮分析显示没有线性(R2=0.129),并且发现与培养基成分(菜籽油,玉米浆酒,葡萄糖),导致一致的过高估计的槐糖脂浓度。注意到在离心样品制备过程中生物质分离不良的出现,并通过使用纯乙醇的新型样品制备方法解决。探索了最常见的槐糖脂定量技术的广泛分析和比较,并强调了局限性/优点。这些发现为科学家提供了一个指导,让他们对满足他们需求的合适的量化工具做出明智的决定,从收获开始探索分析过程的各个方面,样品制备和分析。
To determine the performance of a
sophorolipid biosurfactant production process, it is important to have accurate and specific analytical techniques in place. Among the most popular are the anthrone assay, gravimetric quantification (hexane:ethyl acetate extraction), and high-performance liquid chromatography (HPLC). The choice of analytical tool varies depending on cost, availability, and ease of use; however, these techniques have never been compared directly against one another. In this work, 75 fermentation broths with varying product/substrate concentrations were comprehensively tested with the 3 techniques and compared. HPLC-ultraviolet detection (198 nm) was capable of quantifying C18:1 subterminal hydroxyl diacetylated lactonic
sophorolipid down to a lower limit of 0.3 g/L with low variability (<3.21%). Gravimetric quantification of the broths following liquid:liquid extraction with hexane and ethyl acetate showed some linearity (R2 = .658) when compared to HPLC but could not quantify lower than 11.06 g/L, even when no sophorolipids were detected in the sample, highlighting the non-specificity of the method to co-extract non-
sophorolipid components in the final gravimetric measure. The anthrone assay showed no linearity (R2 = .129) and was found to cross-react with media components (rapeseed oil, corn steep liquor, glucose), leading to consistent overestimation of
sophorolipid concentration. The appearance of poor biomass separation during sample preparation with centrifugation was noted and resolved with a novel sample preparation method with pure ethanol. Extensive analysis and comparisons of the most common
sophorolipid quantification techniques are explored and the limitations/advantages are highlighted. The findings provide a guide for scientists to make an informed decision on the suitable quantification tool that meets their needs, exploring all aspects of the analysis process from harvest, sample preparation, and analysis.