Potential of fermentation profiling via rapid measurement of amino acid metabolism by liquid chromatography–tandem mass spectrometry (original) (raw)
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Analytical Biochemistry, 2004
Anion-exchange chromatography with integrated pulsed amperometric detection (AE-IPAD) separates and directly detects amino acids, carbohydrates, alditols, and glycols in the same injection without pre-or post-column derivatization. These separations use a combination of NaOH and NaOH/sodium acetate eluents.We previously published the successful use of this technique, also known as AAA-Direct, to determine free amino acids in cell culture and fermentation broth media. We showed that retention of carbohydrates varies with eluent NaOH concentration differently than amino acids, and thus separations can be optimized by varying the initial NaOH concentration and its duration. Unfortunately, some amino acids eluting in the acetate gradient portion of the method were not completely resolved from system-related peaks and from unknown peaks in complex cell culture and fermentation media. In this article, we present changes in method that improve amino acid resolution and system ruggedness. The success of these changes and their compatibility with the separations previously designed for fermentation and cell culture are demonstrated with yeast extract-peptone-dextrose broth, M199, Dulbecco's modified Eagle's (with F-12), L-15 (Leibovitz), and McCoy's 5A cell culture media.
Biotechnology and Bioengineering, 1991
We investigated the relationship of dissolved oxygen and culture redox potential (CRP) on amino acid production. Corynebacterium glutamicum ATCC 14296 was used for all experiments. The fermentation can be divided into a growth phase and a production phase. Our results indicate that in order to get higher amino acid production, a lower oxygen supply during the exponential phase is favored. A higher oxygen supply rate appears to be necessary during the production phase. Culture redox potential (CRP) was used to monitor the fermentation. CRP readings were observed to drop to a characteristic minimum value as the metabolic state changed from a growth to production phase. This was evidenced by the commencement of amino acid production and a simultaneous uptake of lactate. Upon lactate exhaustion, the CRP increased abruptly. At the same time, maximal amino acid yields were observed. By the use of minimun CRP as an indication of metabolic phase changes, the agitation rate was changed to increase oxygen supply during the production phase. This significantly increased amino acid production. These results show that culture redox potential measurements can be used to monitor and optimize amino acid production by process manipulation.
Analytical Chemistry, 2007
A method has been developed for rapid quantification of organic acids using ultraperformance liquid chromatography/electrospray-tandem mass spectrometry (UPLC/ ESI-MS-MS) to monitor the metabolism of 10 organic acids during microbial fermentation. Because comprehensive chromatographic separation is not required, analysis time is less than traditional ion chromatography assays, with complete organic acid analyses by UPLC/ ESI-MS-MS being achieved in less than 3 min. Quantification is accomplished using nine isotopically labeled organic acids as internal standards. Intrasample precisions for organic acid measurements in fermentation supernatants using this method average 8.9% (RSD). Calibration curves are linear over the range of 0.06-100 µg/mL, and detection limits are estimated at 0.06-1 µg/ mL. This method has the potential to demonstrate correlation of organic acid consumption and production by microorganisms with observed growth profiles, novel media formulations, and cellular growth events. Data visualization software has been used to profile organic acid levels during fermentation and correlate these profiles to nutrient supplementation protocols employed during microbial production. The potential use of this capability in computational modeling and simulation of microbial metabolism to accelerate the bioprocess development cycle is recognized.
Monitoring Alcoholic Fermentation: An Untargeted Approach
Journal of Agricultural and Food Chemistry, 2014
This work describes the utility and efficiency of a metabolic profiling pipeline that relies on an unsupervised and untargeted approach applied to a HS-SPME/GC-MS data. This noninvasive and high throughput methodology enables "real time" monitoring of the metabolic changes inherent to the biochemical dynamics of a perturbed complex biological system and the extraction of molecular candidates that are latter validated on its biochemical context. To evaluate the efficiency of the pipeline five different fermentations, carried on a synthetic media and whose perturbation was the nitrogen source, were performed in 5 and 500 mL. The smaller volume fermentations were monitored online by HS-SPME/GC-MS, allowing to obtain metabolic profiles and molecular candidates time expression. Nontarget analysis was applied using MS data in two ways: (i) one dimension (1D), where the total ion chromatogram per sample was used, (ii) two dimensions (2D), where the integrity time vs m/z per sample was used. Results indicate that the 2D procedure captured the relevant information more efficiently than the 1D. It was also seen that although there were differences in the fermentation performance in different scales, the metabolic pathways responsible for production of metabolites that impact the quality of the volatile fraction was unaffected, so the proposed pipeline is suitable for the study of different fermentation systems that can undergo subsequent sensory validation on a larger scale.
Advances in microbiology, 2014
This study focused on defining the differences in L. plantarum gene expression levels in different media and in different growth phases using an easy and cost-efficient monitoring of gene expression. A macroarray based on a group of selected L. plantarum genes, 178 genes belonging to 18 main groups, printed onto a nitrocellulose filter was designed in this work. Using the macrofilters designed, the expression of a selected set of L. plantarum genes was assayed in synthetic MRS medium and in extracted carrot juice. To compare the potential differences of starter gene expression in hygienic and contaminated cultivation media, the L. plantarum strain was cultivated in both sterile and contaminated (yeast and Escherichia coli) MRS and carrot juice. The number of genes found to be regulated as a function of growth was clearly higher in MRS-based growth medium than in carrot juice, In carrot juice, expression of the gene encoding malolactic enzyme (MLE), which makes L. plantarum an advantageous microbe in e.g. wine making, was found to be upregulated in logarithmic phase of growth. The current study demonstrated that macroarrays printed on nitrocellulose filters with simple robotic systems can be analyzed by standard laboratory equipment and methods usually available in molecular laboratories. Using this technology, rapid and cost-efficient analysis of genome function of L. plantarum can be carried out e.g. in developing regions, where lactic acid fermentation of food and feed matrices is a common practice.
Advances in Microbiology, 2014
This study focused on defining the differences in L. plantarum gene expression levels in different media and in different growth phases using an easy and cost-efficient monitoring of gene expression. A macroarray based on a group of selected L. plantarum genes, 178 genes belonging to 18 main groups, printed onto a nitrocellulose filter was designed in this work. Using the macrofilters designed, the expression of a selected set of L. plantarum genes was assayed in synthetic MRS medium and in extracted carrot juice. To compare the potential differences of starter gene expression in hygienic and contaminated cultivation media, the L. plantarum strain was cultivated in both sterile and contaminated (yeast and Escherichia coli) MRS and carrot juice. The number of genes found to be regulated as a function of growth was clearly higher in MRS-based growth medium than in carrot juice, In carrot juice, expression of the gene encoding malolactic enzyme (MLE), which makes L. plantarum an advantageous microbe in e.g. wine making, was found to be upregulated in logarithmic phase of growth. The current study demonstrated that macroarrays printed on nitrocellulose filters with simple robotic systems can be analyzed by standard laboratory equipment and methods usually available in molecular laboratories. Using this technology, rapid and cost-efficient analysis of genome function of L. plantarum can be carried out e.g. in developing regions, where lactic acid fermentation of food and feed matrices is a common practice.
The emergence of food metabolomics, otherwise known as foodomics, has opened new frontiers and possibilities for scientists to characterize and simultaneously determine and obtain the comprehensive profile of the food metabolome. Qualitative and quantitative determinations of this metabolome offer insights into the underlying processes involved and details about the content of the food analytes. This had seemed technically challenging and impossible over time, but can now be done due to the advent of sophisticated analytical equipment and chemometric tools. The application of this technique offers enormous opportunities to obtain detailed information that can be correlated to various properties, functionalities and potentials in fermented foods. This chapter thus evaluated and documented studies presented in the literature on the food metabolomics study of fermented foods, with a view of appraising its prospects, applications and subsequent utilization in the study of fermented foods.
Food Research International, 2013
Metabolomics, also called metabonomics or metabolic profiling, deals with the simultaneous determination and quantitative analysis of intracellular metabolites or lowmolecular-mass molecules. The metabolomics field, which has begun a little more than ten years ago thanks to the development of technologies such as nuclear magnetic resonance (NMR) and mass spectrometry (MS), has been successfully applied in different areas of food science. This review deals with the recent achievements of metabolomics in the comprehensive analysis of fermented foods predominated by lactic acid bacteria, the fermentative capacity of these microorganisms and the beneficial effects of functional foods and probiotics. Content 2.2. Cheeses 2.3. Wines 2.4. Cocoa bean fermentation
A Method to Detect Anti-metabolic Factors in Fermentations
Journal of the Institute of Brewing, 2010
Premature yeast flocculation (PYF) has been described as the rapid settling of yeast cells during fermentation despite the presence of sufficient nutrients. PYF can cause negative impacts on beer quality and thus can be quite costly to brewers and maltsters. To investigate the causative agent of PYF, small-scale fermentations were undertaken in both test tubes and cuvettes (15 and 3.5 mL respectively) using worts prepared from PYF-positive and PYF-negative malt samples. Fermentations were carried out using six malts, for up to seven days. Turbidity and extract values were monitored for all samples. The small scale (test tube) assay exhibited clear yeast cell flocculation differences between malts. In the cuvette assay the wort fermented, but the yeast cells settled out of suspension rapidly. While this property made the cuvette assay unsuitable for detecting PYF malt, it did allow for measurement of impaired sugar uptake by the yeast independent of yeast in suspension effects. All wort samples fermented in the cuvette assay showed a similar decline in apparent extract (p > 0.05), indicating that (at least in the samples studied) premature yeast flocculation was not caused by a decline in yeast activity. We believe the simple cuvette assay reported here could have application in the measurement of antimetabolic factors in fermenting media.
Systems biology for industrial strains and fermentation processes—Example: Amino acids
Journal of Biotechnology, 2007
Systems biology is attracting significant interest finding applications not only in pharmaceutical development but also for basic studies on microbial systems. The latter often concentrate on the quantitative understanding of global regulation phenomena. So far, these activities are dominated by academic groups basically mirroring the necessity to prepare the sound scientific understanding first, before industrial applications can be derived later. However, this short-term view may not be sufficient because systems biology already offers numerous benefits for industrial applications, provided that special constraints are considered. This contribution indicates some of the constraints worth noticing when industrial systems biology projects are carried out. Consequently, differences in project structure and goals between purely academic and industrial systems biology projects are outlined.