近红外光谱技术在生物发酵和酶法制备中的应用
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摘要
过程分析技术(PAT)是使用一系列的工具以保证产品的质量和生产过程的可靠性,提高工作效率。目前最常用的PAT主要是近红外(NIR)光谱技术。NIR区域主要反映了中红外区域含H基团的倍频和组合频吸收,因此,这一波段非常适合有机化合物的理化参数测定。NIR光谱富含物质的结构信息和组成信息,所以可以用来进行定性定量分析。NIR光谱仪拥有多种检测终端,如光纤探头、液体流通池、积分球等,可以采用透射(透反射、漫透射)、反射(漫反射)等检测方法进行测定。NIR测定对样品要求简单,可以直接测定,且测定速度快,结果可靠,非常适合生产中的PAT要求。
生物发酵行业是我国的支柱行业之一。但是目前行业发展存在着诸多问题,如生产技术水平落后,检测水平低下,自动化程度不普及等。发酵过程分析强调实时自动监测各项关键工艺参数,统计分析数据结果并反馈控制工艺过程,确保批间工艺一致,产品质量可靠,减少废品几率,降低劳力成本,减少人为误差,实时产品放行,降低生产周期,利于工艺改进和工艺移交。由于生物过程的复杂性、非线性和时变性,基于物理和化学传感器建立的过程检测与控制系统已不能满足实际生产的需要,很多优化模型及自动控制系统在实际生产中并没有很好地实现。原因之一就是生产过程最直接的控制参数-生物(生化)参数(包括底物、重要中间代谢物和目标产物等)快速检测或在线检测技术的缺乏,这也是整个行业面临的瓶颈技术。
本文首先采用化学物质组学的理念研究了玉米浆的化学物质基础。玉米浆(Corn Steep Liquor, CSL)是玉米湿法工艺生产淀粉过程中得到的副产物,含有多种营养成分,可以作为微生物发酵的氮源和碳源。本研究引入化学物质组学(Chemomics)研究策略,首先建立了高效、快速的用于质量控制的指纹图谱,继而采用色谱-质谱联用技术对其物质基础进行系统的研究。然后应用先进的UPLC/Q-TOF分析技术,结合主成分分析法对CSL的整体化学物质组(Global chemome)进行了深入研究,确定了区别不同来源CSL可能的特征性成分为氨基酸类成分,继而采用柱前衍生化高效液相色谱法对CSL中的氨基酸成分进行定量分析,初步确定了六个氨基酸可能为特征性成分。
本文采用NIR光谱技术首先对玉米浆进行了快速质量分析,测定了玉米浆中的主要成分,包括了干物质、酸度、亚硫酸盐、总糖、总还原糖、总氮等成分,同时针对玉米浆中含有的部分维生素类成分进行了快速测定分析,结果表明,NIR技术可以作为一种快速测定发酵原料主要成分含量的手段。然后对谷氨酸亚适量发酵过程中的谷氨酸和葡萄糖含量进行了快速测定分析,结果表明NIR技术可以作为一种快速测定发酵过程中的流加底物(如葡萄糖、甘油等)和主产物(如谷氨酸等)含量的手段,为发酵行业的在线自动化智能化控制进行了一定的基础研究。
生物转化酶法制备是目前较为常用的氨基酸生产方法,其底物转化率较好,产品纯度好,生产周期快,操作方便,后续处理简单,绿色环保等。但是在转化过程中目前尚没有良好的检测方法来实时检测整个生产过程。本研究采用NIR技术对L-谷氨酸钠酶法制备γ-氨基丁酸(y-aminobutyric acid, GABA)和L-精氨酸为底物酶法转化生产L-瓜氨酸的过程进行监控,以便为实现其生产过程的在线监控及智能调控提供一定的研究基础。本研究所建立的近红外模型(GABA、瓜氨酸等)预测能力较好。结果表明,采用NIR技术快速测定酶法制备过程的主要成分含量是可行的。
通过本项目研究,基本上明确了玉米浆的化学物质基础；并采用近红外光谱技术对玉米浆、发酵过程、酶法制备过程等生物发酵行业的主要阶段进行了快速测定分析,为生物发酵行业的智能化、自动化、全程化控制提供了新的思路和解决手段。
Process Analytical Technology(PAT) is a way to use a range of tools to ensure product quality and the reliability of the production process to improve work efficiency. The most commonly used PAT is near-infrared spectroscopy (NIR) technology. NIR is mainly generated due to the molecular vibration of the non-resonant vibration of the molecule from the ground state to a high energy level transitions, which reflects the absorption of the overtones and combination bands of the hydrogen-containing group X-H (X=C, N, O). NIR spectra are of rich structure and composition information, so it is very suitable for detecting hydrocarbon organic substances such as physical and chemical parameters. NIR spectrometer can be flexible with a transmission, reflection, through reflection, diffuse reflection, and other detectors, to analyze directly on the samples of liquid, solid, powder, extract, and spectral scanning speed of up to milliseconds or second grade without sample pretreatment. So NIR is suitable for the quality of the online production process.
Biomanufacturing technology is rapidly developing. Corn steep liquor (CSL) serves as a nitrogen source in the fermentation industry. The chemical material basis was systemic investigated by the new strategy of "chemomics". A rapid and high-performance fingerprint method was developed and the chromatographic method demonstrated good precision, stability and reproducibility. The CSL quality directly affects the yield and quality of fermentation products. In this work, a fingerprinting technique was used to identify the potential markers of CSL. Forty-two CSL samples from different manufacturers were profiled by UPLC Q/TOF MS. Sixteen compounds were considered as the potential markers. Then, o-phthalaldehyde-9-fluorenylmethyl chloroformate precolumn derivatization by high-performance liquid chromatography was performed to identify the free amino acids in CSL. Principal component analysis (PCA) was used to distinguish among the samples from different manufacturers. The results demonstrated that the fingerprinting technique combined with PCA analysis was a powerful tool for determining the CSL quality.
This study aimed to develop a fast, versatile, cheap, and environmentally safe analytical method of quantifying the total acidity (TA) of CSL as well as its contents of dry matter (DM), total sugars (TS), total reducing sugars (TRS), total free amino acids (TFAA), total nitrogen (TN), and total sulfite (TSu). The near-infrared (NIR) spectroscopy measurements of66samples (22batches) of CSL were analyzed by partial least-square regression using several spectral preprocessing methods. Multivariate models developed in the NIR area showed good predictive abilities for DM, TA, TS, TRS, TFAA, TN, and TSu determination. These results confirm the feasibility of the multivariate spectroscopic approach as a replacement for expensive and time-consuming conventional chemical methods. Thus, a convenient and feasible method for the quality control of fermentation raw materials for food additives and fine chemicals, especially in corn steep liquor, is established.
L-glutamate is one of the most important products of the fermentation industries. In this work, multivariate models were deleloped to quantify glutamate and glucose, which were determined by conventional laboratory methods, for a subabundance process of glutamate fermentation using NIR spectroscopy and PLS, whose results are of critical significance for control of the fermentation process and glutamate production.
The enzymatic preparation is a commonly used method to produce amino acids. It's of many advantages, the substrate conversion rate and the product purity. In this work, multivariate models were deleloped to quantify y-aminobutyric, arginine and citrulline, which were deyermined by conventional laboratory methods, whose results are of critical significance for control of the enzymatic preparation process.
This research demonstrated the clear material basis of CSL. And determiantaion of the main components in CSL, fermentation process and enzymatic preparation process by NIR and PLSR provided a new strategy for the biofermentation industry.
生物发酵行业是我国的支柱行业之一。但是目前行业发展存在着诸多问题,如生产技术水平落后,检测水平低下,自动化程度不普及等。发酵过程分析强调实时自动监测各项关键工艺参数,统计分析数据结果并反馈控制工艺过程,确保批间工艺一致,产品质量可靠,减少废品几率,降低劳力成本,减少人为误差,实时产品放行,降低生产周期,利于工艺改进和工艺移交。由于生物过程的复杂性、非线性和时变性,基于物理和化学传感器建立的过程检测与控制系统已不能满足实际生产的需要,很多优化模型及自动控制系统在实际生产中并没有很好地实现。原因之一就是生产过程最直接的控制参数-生物(生化)参数(包括底物、重要中间代谢物和目标产物等)快速检测或在线检测技术的缺乏,这也是整个行业面临的瓶颈技术。
本文首先采用化学物质组学的理念研究了玉米浆的化学物质基础。玉米浆(Corn Steep Liquor, CSL)是玉米湿法工艺生产淀粉过程中得到的副产物,含有多种营养成分,可以作为微生物发酵的氮源和碳源。本研究引入化学物质组学(Chemomics)研究策略,首先建立了高效、快速的用于质量控制的指纹图谱,继而采用色谱-质谱联用技术对其物质基础进行系统的研究。然后应用先进的UPLC/Q-TOF分析技术,结合主成分分析法对CSL的整体化学物质组(Global chemome)进行了深入研究,确定了区别不同来源CSL可能的特征性成分为氨基酸类成分,继而采用柱前衍生化高效液相色谱法对CSL中的氨基酸成分进行定量分析,初步确定了六个氨基酸可能为特征性成分。
本文采用NIR光谱技术首先对玉米浆进行了快速质量分析,测定了玉米浆中的主要成分,包括了干物质、酸度、亚硫酸盐、总糖、总还原糖、总氮等成分,同时针对玉米浆中含有的部分维生素类成分进行了快速测定分析,结果表明,NIR技术可以作为一种快速测定发酵原料主要成分含量的手段。然后对谷氨酸亚适量发酵过程中的谷氨酸和葡萄糖含量进行了快速测定分析,结果表明NIR技术可以作为一种快速测定发酵过程中的流加底物(如葡萄糖、甘油等)和主产物(如谷氨酸等)含量的手段,为发酵行业的在线自动化智能化控制进行了一定的基础研究。
生物转化酶法制备是目前较为常用的氨基酸生产方法,其底物转化率较好,产品纯度好,生产周期快,操作方便,后续处理简单,绿色环保等。但是在转化过程中目前尚没有良好的检测方法来实时检测整个生产过程。本研究采用NIR技术对L-谷氨酸钠酶法制备γ-氨基丁酸(y-aminobutyric acid, GABA)和L-精氨酸为底物酶法转化生产L-瓜氨酸的过程进行监控,以便为实现其生产过程的在线监控及智能调控提供一定的研究基础。本研究所建立的近红外模型(GABA、瓜氨酸等)预测能力较好。结果表明,采用NIR技术快速测定酶法制备过程的主要成分含量是可行的。
通过本项目研究,基本上明确了玉米浆的化学物质基础；并采用近红外光谱技术对玉米浆、发酵过程、酶法制备过程等生物发酵行业的主要阶段进行了快速测定分析,为生物发酵行业的智能化、自动化、全程化控制提供了新的思路和解决手段。
Process Analytical Technology(PAT) is a way to use a range of tools to ensure product quality and the reliability of the production process to improve work efficiency. The most commonly used PAT is near-infrared spectroscopy (NIR) technology. NIR is mainly generated due to the molecular vibration of the non-resonant vibration of the molecule from the ground state to a high energy level transitions, which reflects the absorption of the overtones and combination bands of the hydrogen-containing group X-H (X=C, N, O). NIR spectra are of rich structure and composition information, so it is very suitable for detecting hydrocarbon organic substances such as physical and chemical parameters. NIR spectrometer can be flexible with a transmission, reflection, through reflection, diffuse reflection, and other detectors, to analyze directly on the samples of liquid, solid, powder, extract, and spectral scanning speed of up to milliseconds or second grade without sample pretreatment. So NIR is suitable for the quality of the online production process.
Biomanufacturing technology is rapidly developing. Corn steep liquor (CSL) serves as a nitrogen source in the fermentation industry. The chemical material basis was systemic investigated by the new strategy of "chemomics". A rapid and high-performance fingerprint method was developed and the chromatographic method demonstrated good precision, stability and reproducibility. The CSL quality directly affects the yield and quality of fermentation products. In this work, a fingerprinting technique was used to identify the potential markers of CSL. Forty-two CSL samples from different manufacturers were profiled by UPLC Q/TOF MS. Sixteen compounds were considered as the potential markers. Then, o-phthalaldehyde-9-fluorenylmethyl chloroformate precolumn derivatization by high-performance liquid chromatography was performed to identify the free amino acids in CSL. Principal component analysis (PCA) was used to distinguish among the samples from different manufacturers. The results demonstrated that the fingerprinting technique combined with PCA analysis was a powerful tool for determining the CSL quality.
This study aimed to develop a fast, versatile, cheap, and environmentally safe analytical method of quantifying the total acidity (TA) of CSL as well as its contents of dry matter (DM), total sugars (TS), total reducing sugars (TRS), total free amino acids (TFAA), total nitrogen (TN), and total sulfite (TSu). The near-infrared (NIR) spectroscopy measurements of66samples (22batches) of CSL were analyzed by partial least-square regression using several spectral preprocessing methods. Multivariate models developed in the NIR area showed good predictive abilities for DM, TA, TS, TRS, TFAA, TN, and TSu determination. These results confirm the feasibility of the multivariate spectroscopic approach as a replacement for expensive and time-consuming conventional chemical methods. Thus, a convenient and feasible method for the quality control of fermentation raw materials for food additives and fine chemicals, especially in corn steep liquor, is established.
L-glutamate is one of the most important products of the fermentation industries. In this work, multivariate models were deleloped to quantify glutamate and glucose, which were determined by conventional laboratory methods, for a subabundance process of glutamate fermentation using NIR spectroscopy and PLS, whose results are of critical significance for control of the fermentation process and glutamate production.
The enzymatic preparation is a commonly used method to produce amino acids. It's of many advantages, the substrate conversion rate and the product purity. In this work, multivariate models were deleloped to quantify y-aminobutyric, arginine and citrulline, which were deyermined by conventional laboratory methods, whose results are of critical significance for control of the enzymatic preparation process.
This research demonstrated the clear material basis of CSL. And determiantaion of the main components in CSL, fermentation process and enzymatic preparation process by NIR and PLSR provided a new strategy for the biofermentation industry.
引文
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