Decarboxylation Of Amino Acids Research Articles

HPLC quantification of biogenic amines in cheeses: correlation with PCR-detection of tyramine-producing microorganisms

The consumption of food and beverages containing high amounts of biogenic amines (BA) can have toxicological effects. BA found in foods and beverages are synthesized by the microbial decarboxylation of certain amino acids. This paper reports the concentrations of BAs in a number of commercial cheeses, as determined by HPLC. The cheeses studied were made from raw and pasteurized milk of different origin, and were subjected to different ripening periods. BA concentrations were lower in short ripening period than in long ripening period cheeses, and higher in cheeses made from raw milk than in those made from pasteurized milk. The highest BA concentrations were recorded in blue cheeses made from raw milk. Tyramine was the most commonly recorded and abundant BA. The presence of tyramine-producing bacteria was determined by PCR, and a good correlation obtained between the results of this method and tyramine detection by HPLC. These methods could be used to complement one another in the detection and quantification of tyramine in cheese prevention of tyramine accumulation in cheese.

A Bacterial Arginine-Agmatine Exchange Transporter Involved in Extreme Acid Resistance

The arginine-dependent extreme acid resistance response of Escherichia coli operates by decarboxylating arginine. AdiC, a membrane antiporter, catalyzes arginine influx coupled to efflux of the decarboxylation product agmatine, effectively exporting a proton in each turnover. Using the adiC coding sequence under control of a tetracycline promoter in an E. coli vector, we expressed and purified the transport-protein with a yield of approximately 10 mg/liter bacterial culture. Glutaraldehyde cross-linking experiments indicate that the protein is a homodimer in detergent micelles and lipid membranes. Purified AdiC reconstituted into liposomes exchanges arginine and agmatine in a strictly coupled, electrogenic fashion. Kinetic analysis yields K(m) approximately 80 microm for Arg, in the same range as its dissociation constant determined by isothermal titration calorimetry.

The ability of biogenic amines and ammonia production by single bacterial cultures

The amino acid decarboxylating activity and production of biogenic amines, trimethylamine and ammonia by Morganella morganii (two strains), Klebsiella pneumoniae (three strains), Hafnia alvei (two strains), Enterococcus faecalis, Photobacterium phosphoreum, Micrococcus sp., Psychrobacter immobilis, Corynebacterium sp., Vibrio fischeri, Vibrio harveyi and Pseudomonas putida were investigated using a rapid HPLC method. In a laboratory medium containing amino acid (histidine, ornithine, lysine, tyrosine and arginine), not all bacterial strains produced the biogenic amines but most of them produced histamine, putrescine, cadaverine and ammonia. Cadaverine production by Klebsiella pneumoniae (8152), Klebsiella pneumoniae (673), Klebsiella pneumoniae (2122), Hafnia alvei (6578), Hafnia alvei (11999), Vibrio fischeri (25) Vibrio harveyi (42) and Pseudomonas putida (10936) was 531, 422, 532, 485, 472, 343, 547 and 343 mg/l, respectively in lysine decarboxylase broth. Tyramine was produced in highest concentration (526 mg/l) by Enterococcus faecalis (775). Agmatine was not produced apart from Psychrobacter immobilis (100) in an arginine decarboxylase broth.

Plasmid Profile Patterns and Properties of Pediococci Isolated from Caper Fermentations

A collection comprising 14 isolates of Pediococcus pentosaceus and one Pediococcus acidilactici from the fermentation of caper fruits was studied. All isolates showed very similar fermentation profiles and produced a limited number of exoenzymes. All isolates carried large plasmids of diverse sizes between 20 and 55 kb, while some also contained smaller plasmids between 10 and 16 kb. Cluster analysis of plasmid profiles revealed four main groups with various degrees of similarities. All amino acid decarboxylation tests were negative, suggesting that pediococci are not involved in generation of biogenic amines. None of the isolates showed hemolytic activity. Antimicrobial resistance tests revealed that all isolates were sensitive to 11 different antimicrobials while being resistant to ciprofloxacin (MIC ≥2 mg/liter) and intrinsically resistant to vancomycin (MIC ≥16 mg/liter) and teicoplanin (MIC ≥16 mg/liter).

Determination of biogenic amines in cheese using HPLC technique and direct derivatization of acid extract

Cheeses are among those high-protein-containing foodstuffs in which enzymatic and microbial activities cause the formation of biogenic amines from amino acids decarboxylation. Most of the methods for amine determination in this products involve acid extraction followed by a liquid–liquid purification step to selectively separate amines and amino acids. In this work a simple direct derivatization of the acidic extract with dansyl chloride (DCl) was applied. After an extraction step with diethyl ether, the amines were separated on a Kromasil C18 column using a water–acetonitrile elution gradient. Taking into consideration the recovery and repeatability data we can conclude that, when free amino acid determination is not of concern, the direct DCl derivatization procedure can be applied also for cheese samples with high free amino acid contents.

Evaluation of two different extraction methods for chromatographic determination of bioactive amines in tomato products

Bioactive amines are organic bases originating from corresponding amino acid which have undergone decarboxylation by putrefactive bacteria or lactic acid bacteria. When formed by microbial enzymatic decarboxylation of amino acids, they are called “ biogenic” and can produce detrimental effects on human health. Many techniques have been developed for extraction and/or clean up of bioactive amines in food, including acidic or organic extraction as well as solid phase extraction. This study deals with the comparison of two different extraction methods, homogenizing and matrix solid phase dispersion, for the chromatographic determination of eight non-volatile bioactive amines in tomato-based products (mashed tomato, biological mashed tomato, concentrated tomato pasta and ketchup) very popular in Italian alimentary habits. In both cases, perchloric acid has been used for analytes extraction and the influence of different parameters affecting amine recoveries have been evaluated. After a derivatization step with dansyl-chloride, samples were analyzed for amines quantitative determination using 1,7-diaminoheptane as internal standard on a C 18-RP-HPLC-UV system. Method performances were tested and good results of linearity, repeatability and recovery were obtained for all the considered amines. The collected data have shown that ketchup contains the highest levels of amines followed by concentrated tomato pasta, biological mashed tomato and common mashed tomato. Moreover, it has been found that in all samples, putrescine is the most abundant amine followed by tyramine, spermidine and tryptamine.

A proteomic approach to studying biogenic amine producing lactic acid bacteria

All fermented foods are subject to the risk of biogenic amine contamination. Histamine and tyramine are among the most toxic amines for consumers' health, exerting undesirable effects on the central nervous and vascular systems, but putrescine and cadaverine can also compromise the organoleptic properties of contaminated foods. These compounds are produced by fermenting microbial flora that decarboxylate amino acids to amines. Little is known of the factors which induce biosynthesis of decarboxylating enzymes and/or which modulate their catalytic activity: the accumulation of amines is generally considered to be a mechanism that contrasts an acidic environment and/or that produces metabolic energy through coupling amino acid decarboxylation with electrogenic amino acid/amine antiporters. Two Lactobacillus strains, Lactobacillus sp. 30a (ATCC 33222), and a Lactobacillus sp. strain (w53) isolated from amine-contaminated wine, carrying genetic determinants for histidine decarboxylase (HDC) and ornithine decarboxylase (ODC), were studied and the influence of some environmental and nutritional parameters on amine production and protein biosynthesis was analyzed through a proteomic approach; this is the first report of a proteomic analysis of amine-producing bacteria. HDC and ODC biosynthesis were shown to be closely dependent on the presence of high concentrations of free amino acids in the growth medium and to be modulated by the growth phase. The stationary phase and high amounts of free amino acids also strongly induced the biosynthesis of an oligopeptide transport protein belonging to the proteolytic system of Lactic Acid Bacteria. At least two isoforms of glyceraldehyde-3-phosphate dehydrogenase, with different M(r), pI and expression profiles, were identified from Lactobacillus sp. w53: the biosynthesis of one isoform, in particular, is apparently repressed by high concentrations of free amino acids. Other proteins were identified from the Lactobacillus proteome, affording a global knowledge of protein biosynthesis modulation during biogenic amine production.

Histamine Production by Two Enterobacteriaceae Strains Isolated from Tuna (Thunnus thynnus) and Jack Mackerel (Trachurus murphyii)

Growth of Enterobacteriaceae in scombroids such as tuna (Thunnus thynnus) and jack mackerel (Trachurus murphyii) usually promotes aminoacid decarboxylation to biogenic amines. Among these compounds, histamine, produced by histidin-decarboxylase activity, is reported as toxic. The objective of this study was to compare histamine production by a strain, identified as Serratia liquefaciens, isolated from tuna (T. thynnus) caught in the Gulf of Mexico and Morganella morganii isolated from Chilean jack mackerel (T. murphyii). Morganella morganii had higher histamine producing ability in a synthetic medium than S. liquefaciens, presumably as a result of stressing conditions on M. morganii. However, in a medium formulated with muscle protein extracts obtained from tuna S. liquefaciens showed higher histamine production at similar microbial counts. In this medium, histidine was extracted together with proteins from tuna striated muscle, it was assumed that it acted as histamine production promoter.

Escherichia coli glutamate- and arginine-dependent acid resistance systems increase internal pH and reverse transmembrane potential.

Due to the acidic nature of the stomach, enteric organisms must withstand extreme acid stress for colonization and pathogenesis. Escherichia coli contains several acid resistance systems that protect cells to pH 2. One acid resistance system, acid resistance system 2 (AR2), requires extracellular glutamate, while another (AR3) requires extracellular arginine. Little is known about how these systems protect cells from acid stress. AR2 and AR3 are thought to consume intracellular protons through amino acid decarboxylation. Antiport mechanisms then exchange decarboxylation products for new amino acid substrates. This form of proton consumption could maintain an internal pH (pHi) conducive to cell survival. The model was tested by estimating the pHi and transmembrane potential (DeltaPsi) of cells acid stressed at pH 2.5. During acid challenge, glutamate- and arginine-dependent systems elevated pHi from 3.6 to 4.2 and 4.7, respectively. However, when pHi was manipulated to 4.0 in the presence or absence of glutamate, only cultures challenged in the presence of glutamate survived, indicating that a physiological parameter aside from pHi was also important. Measurements of DeltaPsi indicated that amino acid-dependent acid resistance systems help convert membrane potential from an inside negative to inside positive charge, an established acidophile strategy used to survive extreme acidic environments. Thus, reversing DeltaPsi may be a more important acid resistance strategy than maintaining a specific pHi value.

Cleavage of 14C-labeled glycine and its polycondensation with pyrogallol as catalyzed by birnessite

Deamination as well as decarboxylation of amino acids and their polycondensation with polyphenols catalyzed by Mn(IV) oxide have been reported. However, the mechanisms of cleavage of amino acids and their polycondensation with polyphenols by soil inorganic components are not well understood. The objective of this study was, thus, to investigate the enhanced abiotic decarboxylation of carboxyl group and dealkylation of alkyl group of glycine by birnessite (δ-MnO 2, 0.2–2 μm) both in the presence and absence of pyrogallol and the resultant formation of humic polycondensates in systems free of microbial activity. The degradation of 14C-labeled glycine through either carboxyl or alkyl group on the birnessite surface was demonstrated. Birnessite enhanced the dealkylation of alkyl C and especially decarboxylation of carboxyl C of glycine. Furthermore, it promoted the interaction of glycine and pyrogallol and the resulting simultaneous decarboxylation and dealkylation of glycine, ring cleavage of pyrogallol, and polycondensation. Carboxyl C and especially alkyl C of glycine were incorporated into the humic polycondensates. This study indicates that structural configuration and functionality of amino acids and polyphenols merits closer attention in understanding mechanisms of humification processes catalyzed by soil mineral colloids.

Transamination reactions with multiple turnovers catalyzed by hydrophobic pyridoxamine cofactors in the presence of polyethylenimine polymers.

Pyridoxamines carrying hydrophobic side chains reversibly bind into the hydrophobic core of polyethylenimines and transaminate ketoacids to amino acids with as much as a 725000-fold rate acceleration. Turnover catalysis was achieved by sacrificial oxidative decarboxylation of C-substituted amino acids, which reconverted the pyridoxals to pyridoxamines.

Effect of Putrefaction of Poultry Carcasses Prior to Rendering on Biogenic Amine Production

Biogenic amines, compounds produced by bacterial decarboxylation of amino acids, have been found at high levels in animal by-product meals and have been associated with negative effects on animal performance. The production of eight biogenic amines (tryptamine, phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine, and spermine) during putrefaction of poultry carcasses under common poultry house conditions was studied. Carcasses were allowed to putrefy for 0, 6, 12, 18, 24, 30, 36, 42, 48, or 72 h at 30°C and 70% relative humidity. Levels of biogenic amines were then determined using an HPLC method with fluorescence detection of dansylated derivatives. Levels of all amines increased with increasing putrefaction except spermidine and spermine, which decreased during putrefaction. Histamine, tyramine, and phenylethylamine had a common response. They remained low for approximately 24 h (less than 61, 59, and 58 μ g/g for histamine, tyramine, and phenylethylamine, respectively) after which they rose sharply to high levels, with histamine and phenylethylamine reaching 600 and 1,365 μ g/g, respectively, at 72 h. Putrescine, cadaverine, and tryptamine also exhibited a common response pattern. These amines remained low through the first 24 h and then rose to relatively high levels. However, they plateaued at 48 h and did not accumulate further. The data presented here suggest that putrefaction of poultry carcasses results in accumulation of high levels of biogenic amines, which might reduce the quality of the final rendered product.

Spectroscopy of Hydrothermal Reactions 25: Kinetics of the Decarboxylation of Protein Amino Acids and the Effect of Side Chains on Hydrothermal Stability

The stability toward decarboxylation of six protein amino acids with functionally substituted side chains [phenylalanine (Phe), serine (Ser), threonine (Thr), proline (Pro), histidine (His), and methionine (Met)] were studied with an FT-IR spectroscopy flow reactor at pH25 1.5−8.5, T = 270−340 °C, and P = 275 bar. The first-order (or pseudo-first-order) rate constants and Arrhenius parameters were obtained on the basis of the rate of CO2 formation. The decarboxylation rates of Phe, Ser, Thr, Pro, and Met are independent of pH in the range of 3−8.5. At pH 1.5−3, a maximum rate constant occurs at about 2.5. His has a different pH−rate profile. At its natural pH25 (7.44), the decarboxylation rate reaches a minimum, but increasing or decreasing the pH increases the rate. The activation energies and preexponential factors for the decarboxylation of the protein amino acids cover a wide range. However, a strong correlation between Ea and ln(A) (the kinetic compensation effect) exists, which implies that the amino acids share the same mechanism or at least the same rate-determining step regardless of the side chain. Combining the observations about decarboxylation, deamination, and dehydration, we discuss the effects of the side chains on the hydrothermal stability of protein amino acids in terms of their structures.

Biochemical Characteristics and Antimicrobials Susceptibility of Salmonella gallinarum Isolated in Korea

Fowl typhoid (FT) reported since 1992 in Korea is a septicemic disease of domestic birds caused by Salmonella gallinarum (S. gallinarum). The purpose of this study was to investigate the biochemical characteristics and antimicrobials susceptibility of field isolates of S. gallinarum isolated by year in Korea. A total of 258 isolates of S. gallinarum from 1995 to 2001 showed the same pattern in the majority of biochemical test such as IMViC (indole, methyl red, Voges-Proskauer and citrate utilization), carbohydrate fermentation and amino acid decarboxylation, and these results were almost in accordance with the traditional biochemical characteristics of S. gallinarum strain. When the antimicrobial susceptibility test against 258 isolates of S. gallinarum was performed by the disk diffusion method using 12 antimicrobial agents, all isolates from 1995 appeared to be susceptible to all of the antimicrobial agents tested except for tetracycline and oxytetracycline, whereas the vast majority of isolates from 2001 showed the reduced susceptibility to ampicillin (13.0%), gentamicin (43.4%), kanamycin (69.6%), enrofloxacin (6.5%), ciprofloxacin (10.9%), norfloxacin (52.5%) and ofloxacin (82. 6%). The prevalence of the prevalence of completely resistant isolates resistant isolates to one or more drugs rapidly increased from 0% in 1995 to 93.5% in 2001. The minimal concentrations range of the majority of antimicrobial agents to inhibit 50% (MIC50s) against S. gallinarum isolates increased from 0.06 approximately 8 microg/ml in 1997 to 2 approximately 256 microg/ml in 2001. Especially, MIC50s for gentamicin and fluoroquinolones of isolates from 2001 increased over 10-fold than those of isolates from 1997. Therefore, our results indicate that sorbitol fermentation and arginine decarboxylation showed the diversity by isolates and the vast majority of isolates from 2001 showed the reduced susceptibility to antimicrobials tested.

Spectroscopy of hydrothermal reactions, part 26: Kinetics of decarboxylation of aliphatic amino acids and comparison with the rates of racemization

AbstractThe kinetics of decarboxylation of six α‐amino acids (glycine, alanine, aminobutyric acid, valine, leucine, and isoleucine) and β‐aminobutyric acid were studied in aqueous solution at 310–330ˆC and 275 bar over the pH25 range 1.5–8.5 by using an in situ FT‐IR spectroscopy flow reactor. Based on the rate of formation of CO2, the first‐order or pseudo‐first‐order rate constants were obtained along with the Arrhenius parameters. The decarboxylation rates of amino acids follow the order Gly > Leu ≈ Ile ≈ Val > Ala > α‐Aib > β‐Aib. Differences in the concentration between 0.05 and 0.5 m had only a minor effect on the decarboxylation rate. The effect of the position of the amino group on the decarboxylation rate was investigated for α‐, β‐, and γ‐aminobutyric acid and the order was found to be α > β ≫ γ. Although the pH dependence is complex, the decarboxylation rates of α‐amino acids qualitatively have the inverse trend of the racemization rates. © 2003 Wiley Periodicals, Inc. Int J Chem Kinet 35: 602–610, 2003

Effect of White Versus Red Meat on Endogenous N-Nitrosation in the Human Colon and Further Evidence of a Dose Response

N-nitroso compounds are found in the colon and are formed endogenously because amines and amides are produced by bacterial decarboxylation of amino acids in the large gut. They can be N-nitrosated in the presence of a nitrosating agent. To test the hypothesis that increased nitrogenous residues from red meat would increase endogenous N-nitrosation, thus accounting for the epidemiologic association between red meat consumption and colorectal cancer, we fed increased levels of red meat and measured apparent total N-nitroso compounds (ATNCs) in fecal samples in a series of studies of volunteers maintained under controlled conditions. A result of these studies is that we have shown a consistent dose response to red meat consumption. Fiber, in the form of vegetables, bran or resistant starch, does not reduce the level of ATNCs formed, although transit time is reduced and fecal weight are increased. Here we show that the equivalent amount (420-600 g) of meat as white meat has no effect on fecal ATNCs in 12 volunteers (P = 0.338). At dosages of 0, 60, 120, 240 and 420 g of red meat/d, mean levels of ATNC output are highly correlated with dose of meat: for concentration ATNC versus dose of meat in g/d, r = 0.972, beta = 0.252 ng/g (SE 0.035); for total ATNC output versus dose of meat in g/d, r = 0.963, beta = 2.605 microg/d (SE 0.419). The effects of nonmeat protein and of heme on increased N-nitrosation and the genotoxic effects of the ATNCs produced are presently being investigated.

Stereoselectivity in reactions of amino acids catalyzed by pyridoxal derivatives carrying rigidly-Attached chirally-Mounted basic groups—transamination, racemization, decarboxylation, retro-Aldol reaction, and aldol condensation

A tetrahydroquinoline ring was used to mount the critical functional groups of pyridoxal, and also two examples of rigidly held chirally mounted basic groups. They were able to selectively catalyze decarboxylation, aldol reaction, and retro-aldol reaction of amino acids rather than transamination, and with stereoselectivity. In the aldol reaction of glycine with acetaldehyde to synthesize threonine and allo-threonine, one of the catalysts reversed its stereoselectivity when the basic group was protonated. The observed stereoselectivities were all consistent with prediction.

A biological role for prokaryotic ClC chloride channels.

An unexpected finding emerging from large-scale genome analyses is that prokaryotes express ion channels belonging to molecular families long studied in neurons. Bacteria and archaea are now known to carry genes for potassium channels of the voltage-gated, inward rectifier and calcium-activated classes, ClC-type chloride channels, an ionotropic glutamate receptor and a sodium channel. For two potassium channels and a chloride channel, these homologues have provided a means to direct structure determination. And yet the purposes of these ion channels in bacteria are unknown. Strong conservation of functionally important sequences from bacteria to vertebrates, and of structure itself, suggests that prokaryotes use ion channels in roles more adaptive than providing high-quality protein to structural biologists. Here we show that Escherichia coli uses chloride channels of the widespread ClC family in the extreme acid resistance response. We propose that the channels function as an electrical shunt for an outwardly directed virtual proton pump that is linked to amino acid decarboxylation.

Gold(III)‐Induced Oxidation of Amino Acids and Malonic Acid: Reaction Pathways Studied by NMR Spectroscopy with Isotope Labelling

AbstractReactions of Au(III) with biomolecules are of interest in relation to understanding the mechanism of action of therapeutic gold compounds. NMR investigations of 13C and 15N isotopically‐labelled glycine and alanine show that Au(III) induces deamination and subsequent decarboxylation of both amino acids with the same mechanism. For comparison, reactions of Au(III) with sarcosine and the dicarboxylic acid malonic acid were also investigated. The major intermediates and products have been identified.

High-performance liquid chromatographic determination of biogenic amines in poultry carcasses.

Biogenic amines, produced by bacterial decarboxylation of amino acids, have been associated with toxicological symptoms in broilers fed various poultry byproducts. A reversed-phase high-performance liquid chromatographic method is described for the quantitation of eight biogenic amines (tryptamine, phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine, and spermine) in chicken carcasses. Amines were extracted with perchloric acid, derivatized with dansyl chloride, separated using gradient elution (methanol and water), and detected by fluorescence. Benzylamine was used as the internal standard. Linearity, repeatability, and recovery of the method were evaluated. The method was linear for all of the amines studied at concentrations ranging from 0.05 to 25 microg/mL. Average recoveries ranged from 92.6% to 96.8% for all amines except for histamine, which was 74.6%.