Branched Amino Acids Valine Research Articles

Optimization of malting conditions for two landraces of West African sorghum and influence of mash bio-acidification on saccharification improvement

Traditional sorghum beer processing based on uncontrolled malting and mashing conditions limits significant saccharification. We optimized the amylase activity of west African sorghum to improve saccharification. Furthermore, we bio-acidified the mash with Lactobacillus plantarum (ND-32, ND-130), Lactobacillus paracasei ND-34, and studied its impact on wort nitrogen, especially the branched amino acid content. The β-amylase content of the red sorghum malt used for traditional beer processing was 261 U/g of dried malt when sorghum seeds were steeped to 42% and germinated at 27 °C for 5 days. Despite the optimization of hydrolases, protein hydrolysis was still limited. Sorghum mash pre-heating and bio-acidification increased the wort free amino nitrogen content up to 27%. The contents of several amino acids, including the branched amino acids valine, leucine, and isoleucine, were highly improved. Our new approach based on mash pre-heating and biological acidification helped to increase the wort branched amino acid content by up to 50%. The branched amino acids valine, isoleucine, and leucine are respective precursors for isobutanol, amyl alcohol, and isoamyl alcohol, all of which are critical components of beer aroma.

Integrated omics analyses reveal the details of metabolic adaptation of Clostridium thermocellum to lignocellulose-derived growth inhibitors released during the deconstruction of switchgrass

BackgroundClostridium thermocellum is capable of solubilizing and converting lignocellulosic biomass into ethanol. Although much of the work-to-date has centered on characterizing this microbe’s growth on model cellulosic substrates, such as cellobiose, Avicel, or filter paper, it is vitally important to understand its metabolism on more complex, lignocellulosic substrates to identify relevant industrial bottlenecks that could undermine efficient biofuel production. To this end, we have examined a time course progression of C. thermocellum grown on switchgrass to assess the metabolic and protein changes that occur during the conversion of plant biomass to ethanol.ResultsThe most striking feature of the metabolome was the observed accumulation of long-chain, branched fatty acids over time, implying an adaptive restructuring of C. thermocellum’s cellular membrane as the culture progresses. This is undoubtedly a response to the gradual accumulation of lignocellulose-derived inhibitory compounds as the organism deconstructs the switchgrass to access the embedded cellulose. Corroborating the metabolomics data, proteomic analysis revealed a corresponding time-dependent increase in various enzymes, including those involved in the interconversion of branched amino acids valine, leucine, and isoleucine to iso- and anteiso-fatty acid precursors. Additionally, the metabolic accumulation of hemicellulose-derived sugars and sugar alcohols concomitant with increased abundance of enzymes involved in C5 sugar metabolism/pentose phosphate pathway indicates that C. thermocellum shifts glycolytic intermediates to alternate pathways to modulate overall carbon flux in response to C5 sugar metabolites that increase during lignocellulose deconstruction.ConclusionsIntegrated omic platforms provided complementary systems biological information that highlight C. thermocellum’s specific response to cytotoxic inhibitors released during the deconstruction and utilization of switchgrass. These additional viewpoints allowed us to fully realize the level to which the organism adapts to an increasingly challenging culture environment—information that will prove critical to C. thermocellum’s industrial efficacy.

1H NMR-based metabolomics studies of urine reveal differences between type 1 diabetic patients with high and low HbAc1 values

The aim of this study was to investigate relation between level of HbAc1 and concentration of metabolites in urine of T1D patients. To test this hypothesis the 1H NMR (proton nuclear magnetic resonance) target analysis of crucial urine metabolites combined with chemometric approach were applied. Urine samples were collected from 30 children and teenagers aged 4–19 with T1D and 12 healthy children, aged 9, as control group. Patients were divided into two groups according to their level of glycated hemoglobin (HbA1c): below (L-T1D) and above 6.5% (H-T1D). The multivariate data analysis (OPLS-DA) was used to explore data and generate the models for selected groups of patients. Two tailed unpaired t-test was used for statistical analysis of quantified metabolites. Comparing to L-T1D patients, H-T1D group exhibited increased levels of alanine, pyruvate and branched amino acid valine that might be related with endogenous glucose production pathway from proteins as well as in the case of T2D. Application of 1H NMR spectroscopy together with target analysis and chemometric tools based on urine metabolite concentration enable to monitor T1D patients. This methodology can be used as supporting tool for marker HbA1c analysis providing comprehensive information about T1D progression and treatment efficiency.

Determination of the essential nutrient requirements of wine-related bacteria from the genera Oenococcus and Lactobacillus

Wine lactic acid bacteria (LAB) are responsible for the malolactic fermentation (MLF) in wine production. Wine LAB have fastidious nutrient requirements but their auxotrophies remain little studied. The ability of specific wine nutrients to meet the nutritional requirements of wine LAB, and thus support MLF, remains unclear. This work investigated the essential growth requirements of four strains of wine LAB from the genera Oenococcus and Lactobacillus using the single omission technique with a suitable chemically defined medium. For the determination of auxotrophies, at least 3 (and up to 15) subcultures in deficient media were made, and intra- and extracellular nutrient carry over was reduced by small inoculation rates and washing cells 3 times between transfers. This careful methodology revealed more auxotrophies than those described for wine LAB in the literature. The essential bacterial nutrient requirements were found to be strain specific. 10 compounds were essential for all wine LAB tested, the carbon and phosphate source, manganese, as well as several amino acids (proline, arginine and the branched amino acids valine, leucine and isoleucine) and vitamins (nicotinic acid and pantothenic acids). Nucleotides were not essential for any of the bacteria studied. The two Oenococcus oeni strains revealed a larger number of auxotrophies (18 and 21) and had a higher degree of nutritional similarity (86%) defined as percentage of common requirements per maximum total requirements. The two Lactobacillus strains only had 11 and 14 auxotrophies and the similarity was 79%, but both were auxotroph for riboflavin, which was not needed by the O. oeni strains. Data on the common requirements may be used to further study the ability of wines or commercial nutrients to support MLF and to consider the microbiological stability of finished wines. The results indicate that absence of riboflavin in oenological nutrient preparations may allow to create a specific advantage for indigenous or inoculated O. oeni, which are generally desired for MLF.

Different toxic effects of the sulfonylurea herbicides metsulfuron methyl, chlorsulfuron and thifensulfuron methyl on fluorescent pseudomonads isolated from an agricultural soil

A total of 77 strains of genetically different fluorescent Pseudomonas strains were isolated from an agricultural soil. In pure culture growth experiments the strains were screened for their ability to grow in various concentrations of the sulfonylurea herbicides metsulfuron methyl, chlorsulfuron and thifensulfuron methyl. We found that the presence of the herbicides resulted in a reduction of the growth of the fluorescent pseudomonads. Metsulfuron methyl was shown to be toxic to a major proportion of the strains in low concentrations. Chlorsulfuron was found to be less toxic in low concentrations but toxic in high concentrations. Thifensulfuron methyl was toxic only to a minority of the strains. Indirectly, the growth-reducing effect of the sulfonylurea herbicides was shown to be caused by an inhibition of the enzyme acetolactate synthase. The enzyme is involved in the synthesis of the branched amino acids valine, leucine and isoleucine, and we demonstrated that the toxic effects of the sulfonylurea herbicides could be neutralized when the strains were grown in the presence of an excess amount of the three amino acids.

Different toxic effects of the sulfonylurea herbicides metsulfuron methyl, chlorsulfuron and thifensulfuron methyl on fluorescent pseudomonads isolated from an agricultural soil

A total of 77 strains of genetically different fluorescent Pseudomonas strains were isolated from an agricultural soil. In pure culture growth experiments the strains were screened for their ability to grow in various concentrations of the sulfonylurea herbicides metsulfuron methyl, chlorsulfuron and thifensulfuron methyl. We found that the presence of the herbicides resulted in a reduction of the growth of the fluorescent pseudomonads. Metsulfuron methyl was shown to be toxic to a major proportion of the strains in low concentrations. Chlorsulfuron was found to be less toxic in low concentrations but toxic in high concentrations. Thifensulfuron methyl was toxic only to a minority of the strains. Indirectly, the growth-reducing effect of the sulfonylurea herbicides was shown to be caused by an inhibition of the enzyme acetolactate synthase. The enzyme is involved in the synthesis of the branched amino acids valine, leucine and isoleucine, and we demonstrated that the toxic effects of the sulfonylurea herbicides could be neutralized when the strains were grown in the presence of an excess amount of the three amino acids.