An alternative approach for quantification of glyceraldehyde and dihydroxyacetone as trimethylsilyl derivatives by GC-FID.

A method for quantification of glyceraldehyde (GA), dihydroxyacetone (DHA) and glycerol (GLY) by gas chromatography coupled to a flame ionization detector (GC-FID) involving one-step derivatization into trimethylsilyl ethers is presented. In pyridine, DHA and GA showed predominant peaks assigned to dimeric structures and smaller peaks corresponding to the monomers. The later were identified by GC-MS as their completely derivatized molecules and were useful for construction of calibration curves with high linear correlation. On the other hand, DHA dimers were completely dissociated in water but GA dimers remained whereas with both, intermediates peaks arose which were associated to hydrated trymethyil silyl species. A calibration approach involving the sum of areas of most relevant peaks associated to aqueous solutions of GA and DHA was developed. Replicates measurements of a problem solution were in accordance with the results obtained by a well stablished HPLC technique. The coefficient of variation was below 5% for GLY and below 12% for GA and DHA. Compared with the HPLC method, the new GC-FID method presented a similar limit of quantification in the case of GA whereas for GLY and DHA a one-order-of-magnitude increase of sensitivity was achieved. TMS derivatives of GA and DHA without prior oximation enable a useful technique to study the equilibrium of the different tautomeric forms in solution.

Influence of environmental and genetic factors on 3-hydoxypropionaldehyde production by Lactobacillus reuteri.

The influence of environmental factors such as glycerol concentration, time of production, presence of Escherichia coli, and two different strains of Lactobacillus reuteri (ATCC 55730 and ATCC 53608) on 3-hydroxypropionaldehyde (3-HPA) production was analyzed. Additionally, the influence of those factors on gene expression in the 3-HPA production pathway was evaluated. The genes studied were GldC, cbiP, and Lreu_1734. The results of this study showed that the principal environmental factors that influence 3-HPA production are glycerol concentration and Lactobacillus reuteri strain. As glycerol concentration increased, 3-HPA content increased. The greatest 3-HPA concentration (56.6 mM ±5.99) was achieved by L. reuteri ATCC 55730. Gene expression was also affected by environmental factors. Factor that showed the greatest influence were also strain and glycerol concentration. The genes cbiP, GldC, and Lreu_1734 had basal gene expression in glycerol absence; however, glycerol regulated its expression. Glycerol induced overexpression of cbiP and GldC genes (Strain ATCC 53608), probably to ensure its efficient utilization. On the contrary, glycerol concentration suppressed Lre_1734 expression in both analyzed strains, as a mechanism for 3-HPA accumulation. Down-regulation was observed in all the genes tested in strain ATCC 55730, probably due to feedback inhibition by 3-HPA.

Production of reuterin in a fermented milk product by Lactobacillus reuteri: Inhibition of pathogens, spoilage microorganisms, and lactic acid bacteria.

We assessed the antimicrobial activity of reuterin produced in vitro in glycerol aqueous solutions in situ by Lactobacillus reuteri ATCC 53608 as part of a fermented milk product against starter (Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus), spoilage (Penicillium expansum), pathogenic (Staphylococcus aureus Salmonella enterica ssp. enterica, and Listeria monocytogenes), and pathogen surrogate (Escherichia coli DH5α) microorganisms. We also assayed the influence of cold storage (28 d at 4°C) and reuterin on the color and rheology of the fermented milk product. We obtained maximum reuterin concentrations of 107.5 and 33.97 mM in glycerol aqueous solution and fermented milk product, respectively. Reuterin was stable throughout its refrigerated shelf life. Gram-positive microorganisms were more resistant to reuterin than gram-negative microorganisms. Penicillium expansum and Lactobacillus reuteri ATCC 53608 survived at concentrations up to 10 and 8.5 mM, respectively. Escherichia coli DH5α was the most sensitive to reuterin (0.9 mM). The presence of reuterin did not cause relevant changes in the quality parameters of the fermented milk product, including pH, acidity, soluble solids, color, and rheological aspects (storage and loss moduli and viscosity). This study demonstrated the viability of using Lactobacillus reuteri ATCC 53608 as a biopreservative in a fermented milk product through reuterin synthesis, without drastically modifying its quality parameters.

Crosslinked, cryostructured Lactobacillus reuteri monoliths for production of 3-hydroxypropionaldehyde, 3-hydroxypropionic acid and 1,3-propanediol from glycerol.

Crosslinked, cryostructured monoliths prepared from Lactobacillus reuteri cells were evaluated as potential immobilized whole-cell biocatalyst for conversion of glycerol, to potentially important chemicals for the biobased industry, i.e. 3-hydroxypropionaldehyde (3HPA), 3-hydroxypropionic acid (3HP) and 1,3-propanediol (1,3PDO). Glutaraldehyde, oxidized dextran and activated polyethyleneimine/modified polyvinyl alcohol (PEI/PVA) were evaluated as crosslinkers; the latter gave highly stable preparations with maintained viability and biocatalytic activity. Scanning electron microscopy of the PEI/PVA monoliths showed high density of crosslinked cells with wide channels allowing liquid flow through. Flux analysis of the propanediol-utilization pathway, incorporating glycerol/diol dehydratase, propionaldehyde dehydrogenase, 1,3PDO oxidoreductase, phosphotransacylase, and propionate kinase, for conversion of glycerol to the three chemicals showed that the maximum specific reaction rates were -562.6, 281.4, 62.4 and 50.5mg/gCDWh for glycerol consumption, and 3HPA (extracellular), 3HP and 1,3PDO production, respectively. Under optimal conditions using monolith operated as continuous plug flow reactor, 19.7g/L 3HPA was produced as complex with carbohydrazide at a rate of 9.1g/Lh and a yield of 77mol%. Using fed-batch operation, 1,3PDO and 3HP were co-produced in equimolar amounts with a yield of 91mol%. The monoliths embedded in plastic carriers showed high mechanical stability under different modes in a miniaturized plug flow reactor.

Clinical and microbiological effects of probiotic lozenges in the treatment of chronic periodontitis: a 1-year follow-up study.

AIM: The objective of this study was to evaluate the effects of lozenges containing L. reuteri as an adjuvant treatment to initial periodontal therapy for chronic periodontitis patients and to detect the level of L. reuteri colonization in the periodontal pockets of treated patients. MATERIAL AND METHODS: A total of 40 patients were selected and randomly divided into two groups. Each patient had at least two teeth with one approximal site each with a probing depth (PD) of 5-7 mm and gingival index (GI) of ≥2 in each quadrant. Group I received scaling and root planing (SRP) plus L. reuteri-containing lozenges, and Group II received SRP plus placebo. The plaque index (PI), GI, bleeding on probing (BoP), PD and relative attachment level were measured. Microbiological sampling was performed at baseline and on days 21, 90, 180 and 360 and were analysed by culturing. The Bonferroni-corrected paired sample t-test, Bonferroni-corrected Wilcoxon signed rank test and paired sample t-test were used to evaluate intra-group differences. The Bonferroni-corrected Student's t-test and the Mann-Whitney U-tests were used to evaluate inter-group differences. RESULTS: After treatment, the measured PI, GI, BoP and PD were significantly (p < 0.05) lower in Group I compared with Group II at all time points. Similar observations were made for the total viable cell counts and the proportions of obligate anaerobes with the exception of day 360. In Group I, significantly fewer patients required surgery on ≥3 sites. CONCLUSION: L. reuteri-containing lozenges may be a useful adjuvant agent to slow re-colonization and improve clinical outcomes of chronic periodontitis. Further studies are required to clarify the optimal dose of the lozenges.

Elevated 3-hydroxypropionaldehyde production from glycerol using a Citrobacter freundii mutant.

A mutant strain of Citrobacter freundii capable of elevated 3-hydroxypropionaldehyde production from glycerol was isolated using chemical mutagenesis and a screening protocol. The protocol involved screening mutagenized bacterial cells on solid minimal medium containing 5 % (v/v) glycerol. Colonies were picked onto duplicate solid minimal medium plates and one plate was stained with 1 % (w/v) phloroglucinol. Those colonies staining red were further screened and a mutant, HPAO-1, was identified. The mutant strain produced a several-fold higher 3-hydroxypropionaldehyde concentration than did the parent strain when grown on 5 % (v/v) glycerol. The ratio of culture volume to flask volume influenced 3-hydroxypropionaldehyde production by the mutant cells compared to the parent cells. Aldehyde production was highest when the mutant strain was grown on 5 % (v/v) glycerol at a ratio of culture volume to flask volume of 1:3 or 1:12.5.

Non-enzymatic glycation of melamine with sugars and sugar like compounds.

Melamine (1,3,5-triazine-2,4,6-triamine) is employed in the manufacture of plastics, laminates and glues, yet, it has been found sometimes added illegally to dairy products to artificially inflate foods' protein content. In 2008, dairy products adulterated with melamine were blamed for the death of several infants in China, a situation that forced Beijing to introduce stricter food safety measures. The objectives of this study were threefold: (1) to investigate the susceptibility of the amine groups of melamine to glycation with D-galactose, D-glucose and lactose, sugars commonly found in milk, (2) to study the rate and extent of melamine's glycation with methylglyoxal, glyoxal and DL-glyceraldehyde, three highly reactive metabolites of D-galactose, D-glucose and lactose, and (3) to characterize, using mass spectrometry, the Advanced Glycation Endproducts (AGEs) of melamine with sugars found commonly in milk and their metabolites. Incubation of D-galactose, D-glucose and lactose with melamine revealed that D-galactose was the most potent glycator of melamine, followed by D-glucose, then lactose. Methylglyoxal, glyoxal, and DL-glyceraldehyde glycated melamine more extensively than D-galactose, with each yielding a broader range of AGEs. The non-enzymatic modification of melamine by sugars and sugar-like compounds warrants further investigation, as this process may influence melamine's toxicity in vivo.

Acrolein in wine: importance of 3-hydroxypropionaldehyde and derivatives in production and detection.

Certain lactic acid bacteria strains belonging to the genus Lactobacillus have been implicated in the accumulation of 3-hydroxypropionaldehyde (3-HPA) during anaerobic glycerol fermentation. In aqueous solution 3-HPA undergoes reversible dimerization and hydration, resulting in an equilibrium state between different derivatives. Wine quality may be compromised by the presence of 3-HPA due to the potential for spontaneous conversion into acrolein under winemaking conditions. Acrolein is highly toxic and has been implicated in the development of bitterness in wine. Interconversion between 3-HPA derivatives and acrolein is a complex and highly dynamic process driven by hydration and dehydration reactions. Acrolein is furthermore highly reactive and its steady-state concentration in complex systems very low. As a result, analytical detection and quantification in solution is problematic. This paper reviews the biochemical and environmental conditions leading to accumulation of its precursor, 3-HPA. Recent advances in analytical detection are summarized, and the roles played by natural chemical derivatives are highlighted.

Glycerol metabolism and bitterness producing lactic acid bacteria in cidermaking.

Several lactic acid bacteria were isolated from bitter tasting ciders in which glycerol was partially removed. The degradation of glycerol via glycerol dehydratase pathway was found in 22 out of 67 isolates. The confirmation of glycerol degradation by this pathway was twofold: showing their glycerol dehydratase activity and detecting the presence of the corresponding gene by a PCR method. 1,3-propanediol (1,3-PDL) and 3-hydroxypropionic acid (3-HP) were the metabolic end-products of glycerol utilization, and the accumulation of the acrolein precursor 3-hydroxypropionaldehyde (3-HPA) was also detected in most of them. The strain identification by PCR-DGGE rpoB showed that Lactobacillus collinoides was the predominant species and only 2 belonged to Lactobacillus diolivorans. Environmental conditions conducting to 3-HPA accumulation in cidermaking were studied by varying the fructose concentration, pH and incubation temperature in L. collinoides 17. This strain failed to grow with glycerol as sole carbon source and the addition of fructose enhanced both growth and glycerol degradation. Regarding end-products of glycerol metabolism, 1,3-PDL was always the main end-product in all environmental conditions assayed, the only exception being the culture with 5.55 mM fructose, where equimolar amounts of 1,3-PDL and 3-HP were found. The 3-HPA was transitorily accumulated in the culture medium under almost all culture conditions, the degradation rate being notably slower at 15 degrees C. However, no disappearance of 3-HPA was found at pH 3.6, a usual value in cider making. After sugar exhaustion, L. collinoides 17 oxidated lactic acid and/or mannitol to obtain energy and these oxidations were accompanied by the removal of the toxic 3-HPA increasing the 1,3-PDL, 3-HP and acetic acid contents.

Formation mechanisms of melanoidins and fluorescent pyridinium compounds as advanced glycation end products.

The formation mechanisms of melanoidins as advanced glycation end products (AGEs) have not been resolved. Blue and red pigments generated in the D-xylose-glycine reaction system are postulated to be intermediate oligomers in the generation of melanoidins. A novel blue pigment, designated blue-M5, was identified as a similar structure to blue-M1 except for the side chain of two dihydroxypropyl groups. Blue pigments were also generated in the D-glucose-glycine and D-xylose-beta-alanine reaction systems as well as in the D-xylose-glycine reaction system. Blue pigments by the Maillard reaction might be formed by the decarboxylation of two molecules of pyrrolopyrrole-2-carbaldehydes (PPA). PPA, composed of a side chain of a dihydroxypropyl group, was identified as a precursor of blue pigments. In fact, blue-M5 was generated by the incubation of PPA alone. Blue pigments, which involved pyrrolopyrrole structures, were readily changed to brown polymers. Glyceraldehyde-derived pyridinium (GLAP) compound, a glyceraldehyde-derived fluorescent AGE, and lysyl-pyrropyridine, a 3-deoxyglucosone-derived fluorescent AGE, were detected at higher levels in the plasma proteins and the tail tendon collagen of streptozotocin-induced diabetic rats compared to normal rats. GLAP and lysyl-pyrropyridine, therefore, might be related to the progression of diabetic complications.

Production of 1,3-Propanediol by Clostridium butyricum VPI 3266 in continuous cultures with high yield and productivity.

The effects of dilution rate and substrate feed concentration on continuous glycerol fermentation by Clostridium butyricum VPI 3266, a natural 1,3-propanediol producer, were evaluated in this work. A high and constant 1,3-propanediol yield (around 0.65 mol/mol), close to the theoretical value, was obtained irrespective of substrate feed concentration or dilution rate. Improvement of 1,3-propanediol volumetric productivity was achieved by increasing the dilution rate, at a fixed feed substrate concentration of 30, 60 or 70 g l(-1). Higher 1,3-propanediol final concentrations and volumetric productivities were also obtained when glycerol feed concentration was increased from 30 to 60 g l(-1), at D=0.05-0.3 h(-1), and from 60-70 g l(-1), at D=0.05 and 0.1 h(-1).30 g l(-1) of 1,3-propanediol and the highest reported value of productivity, 10.3 g l(-1) h(-1), was achieved at D=0.30 h(-1) and 60 g l(-1) of feed glycerol. A switch to an acetate/butyrate ratio higher than one was observed for 60 g l(-1) of feed glycerol and a dilution rate higher than 0.10 h(-1); moreover, at D=0.30 h(-1) 3-hydroxypropionaldehyde accumulation was observed for the first time in the fermentation broth of C. butyricum.

Characterization of a reuterin-producing Lactobacillus coryniformis strain isolated from a goat's milk cheese.

Lactobacillus coryniformis CECT 5711, a strain isolated from a goat's milk cheese, displayed a broad-spectrum antimicrobial activity; as a consequence, its ability to produce the antagonistic compounds associated to lactic acid bacteria, including bacteriocins, hydrogen peroxide, lactic acid, acetic acid, and reuterin (3-hydroxypropionaldehyde, 3-HPA) was investigated. Production of bacteriocins or hydrogen peroxide by this strain could not be detected. However, in addition to lactic acid and acetic acid, it produced reuterin and cobalamin, a cofactor required for conversion of glycerol to 3-HPA through a glycerol dehydratase. The gene encoding a glycerol dehydratase subunit was detected by PCR and the corresponding amplicon was sequenced. This strain showed a high survival after exposition to conditions simulating those existing in the gastrointestinal tract as well as a notable ability to adhere to intestinal cells, which suggests that its reuterin-producing ability may be used for the host benefit. In addition, the strain showed a strong beta-galactosidase activity. Production of biogenic amines and degradation of mucin could not be detected.

Studies on the reactions between daptomycin and glyceraldehyde.

The objectives of this project were to determine the reaction pathways of daptomycin in the presence of glyceraldehyde in acidic solutions, and to quantitate the kinetics of the major pathways. In the presence of glyceraldehyde (pH range 1-7 at 25 to 60 degrees C), daptomycin formed two major products separable by RP-HPLC. The products were identified using UV spectroscopy, fluorimetry, mass spectrometry, and 2D-1H NMR. The reaction scheme involved the reversible formation of imine and anilide derivatives. Carbinolamine was believed to be a common intermediate in formation pathways of both products. The carbinolamine intermediate underwent either acid catalyzed dehydration resulting in imine formation or intramolecular hydrogen bonding and bond cleavage giving rise to anilide formation. In mild acid conditions, both products reversed to daptomycin. The reaction between daptomycin and glyceraldehyde was first-order with respect to both reactants. In a pH range of 1-7, the imine formation rate was pH dependent with a maximum rate at approximate pH values of 3-4. The observed pH dependence was consistent with the pH dependence of typical amine-aldehyde reactions.

Glyceraldehyde phosphate at the reducing terminus of Salmonella Q haptens. Salmonella montevideo.

The O antigen polysaccharide of Salmonella montevideo was isolated from a core-defective mutant by the phenol/water procedure, and was suspected to contain phosphomonester and cyclic phosphodiester at its reducing end in anology to the O hapten from Salmonella typhimurium (Kent and Obsborn, 1968. Therefore, it was chromatographed on a DEAE-cellulose column. Whereas one part eluted with water the other part of the polysaccharide could only be eluted with buffer. Both fractions were further purified on Sephadex G100 and contained mannose, glucose, N-acetylglucosamine and phosphate in a molar ratio of 4:1:1: less than 0.1. In order to specifically label the reducing end phosphate was removed enzymatically, or the presumed cyclic diester was cleaved by mild hydrolysis, and the fractions were reduced with sodium horo[3H]hydride. Both fractions yield mainly [3H]glycerol after hydrolysis and paper chromatogaphy. In addition, [3H]mannitol and [H]monohydroxyacetone could be identified by paper chromatography and were concluded to be the result of phosphate migration and beta-elimination reactions taking place during the isolation procedure and the various treatments prior to sodium boro[3H]hydride reduction. These findings in addition to periodate oxidation studies indicated that the O antigen polysaccharide of Salmonella montevideo had glyceraldehyde phosphate at its reducing end. From the incorporation of 3H into the polysaccharide the O antigen was calculated to consist of about 19 repeating units of 6 sugar residues each.