Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai, 600036, India.
Appl Microbiol Biotechnol. 2018 Mar;102(6):2659-2669. doi: 10.1007/s00253-018-8816-9. Epub 2018 Feb 13.
Hyaluronic acid (HA) is a high-value polysaccharide with many biomedical applications. Microbial production of HA is now replacing the traditional extraction method from rooster combs. Production of medical-grade HA with defined characteristics requires controlled process conditions because there are many fermentation process parameters that affect the microbial synthesis of HA. This necessitates the development of online tools for monitoring multiple analytes during microbial fermentation. Here, we describe the application of in situ transflectance spectroscopy for online quantification of seven major fermentation analytes, viz. biomass, glucose, lactate, formate, ethanol, acetate and HA in metabolically engineered Lactococcus lactis fermentations. The near-infrared spectral information acquired from synthetic mixtures and untransformed L. lactis fermentations were used to develop chemometric models. Based on principal component analysis and partial least squares regression methods, analyte-specific models were developed for quantification. These models were then independently validated for fermentation analytes from four different recombinant L. lactis strains. The chemometric model developed for HA based on recombinant L. lactis fermentation data and pure HA standards could accurately predict HA concentrations under homolactic conditions. The online estimation of HA was found to be poor under heterolactic conditions due to the overlapping absorbance of acetate produced in these cultures. Alternatively, an independent model based on yield correlation was successfully developed for indirect real-time quantification of HA.
透明质酸（HA）是一种具有多种生物医学应用的高价值多糖。微生物法生产 HA 正在取代传统的鸡冠提取方法。由于有许多发酵工艺参数会影响微生物合成 HA，因此需要生产具有特定特性的医用级 HA，这就需要开发用于在微生物发酵过程中监测多种分析物的在线工具。在这里，我们描述了原位反射透射光谱法在代谢工程乳球菌发酵过程中在线定量分析七种主要发酵分析物（生物量、葡萄糖、乳酸、甲酸、乙醇、乙酸和 HA）的应用。从合成混合物和未经转化的乳球菌发酵中获得的近红外光谱信息用于开发化学计量模型。基于主成分分析和偏最小二乘回归方法，为定量分析开发了针对特定分析物的模型。然后，这些模型独立地对来自四个不同重组乳球菌菌株的发酵分析物进行了验证。基于重组乳球菌发酵数据和纯 HA 标准物开发的 HA 化学计量模型能够准确预测同型乳酸条件下的 HA 浓度。在异型乳酸条件下，由于这些培养物中产生的乙酸的吸收重叠，HA 的在线估计效果不佳。或者，基于产率相关性成功开发了用于间接实时定量 HA 的独立模型。
