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1.  Managing Your Wine Fermentation to Reduce the Risk of Biogenic Amine Formation 
Biogenic amines are nitrogenous organic compounds produced in wine from amino acid precursors mainly by microbial decarboxylation. The concentration of biogenic amines that can potentially be produced is dependent on the amount of amino acid precursors in the medium, the presence of decarboxylase positive microorganisms and conditions that enable microbial or biochemical activity such as the addition of nutrients to support the inoculated starter cultures for alcoholic and malolactic fermentation (MLF). MLF can be conducted using co-inoculation or an inoculation after the completion of alcoholic fermentation that may also affect the level of biogenic amines in wine. This study focused on the impact of the addition of complex commercial yeast and bacterial nutrients and the use of different MLF inoculation scenarios on the production of biogenic amines in wine. Results showed that the addition of complex nutrients to real grape must could potentially increase histamine concentrations in wine. The same experiment in synthetic grape must showed a similar trend for putrescine and cadaverine. The effect of different MLF inoculation scenarios was examined in two cultivars, Pinotage and Shiraz. Conflicting results was obtained. In the Shiraz, co-inoculation resulted in lower biogenic amine concentrations after MLF compared to before MLF, while the concentration was higher in the Pinotage. However, the production of biogenic amines was affected more by the presence of decarboxylase positive lactic acid bacteria than by the addition of complex nutrients or the inoculation scenario.
doi:10.3389/fmicb.2012.00076
PMCID: PMC3301445  PMID: 22419915
biogenic amines; nutrients; co-inoculation; malolactic fermentation; lactic acid bacteria; wine
2.  Tyramine biosynthesis in Enterococcus durans is transcriptionally regulated by the extracellular pH and tyrosine concentration 
Microbial biotechnology  2009;2(6):625-633.
Summary
The microbial decarboxylation of some amino acids leads to the undesirable presence of biogenic amines in foods. One of the most abundant and frequent biogenic amines found in fermented foods is tyramine, which is produced by the decarboxylation of tyrosine. In the present work, transcriptional analysis of tyramine biosynthesis in Enterococcus durans IPLA655, a strain isolated from cheese, was studied. The gene coding for the tyrosine decarboxylase (tdcA) and that coding for the tyrosine‐tyramine antiporter (tyrP) form an operon transcribed from the promoter PtdcA, the expression of which is regulated by the extracellular pH and tyrosine concentration. Quantification of gene expression during the log phase of growth showed high concentrations of tyrosine and acidic pH conditions to induce tdcA‐tyrP polycistronic messenger transcription.
doi:10.1111/j.1751-7915.2009.00117.x
PMCID: PMC3815318  PMID: 21255297
3.  Biogenic Amines Degradation by Lactobacillus plantarum: Toward a Potential Application in Wine 
Biogenic amines (BA) in wine represent a toxicological risk for the health of the consumer, with several trade implications. In this study 26 strains of Lactobacillus plantarum were analyzed for their ability to degrade BA commonly found during wine fermentation. Two strains of L. plantarum were selected in reason of their ability to degrade putrescine and tyramine. The degradation was assessed in vitro, both in presence of the BA and in presence of the specific chemical precursor and of producer bacteria. The two L. plantarum biotypes were found capable to work synergically. In addition, the survival in wine-like medium and the aptitude to degrade malic acid after alcoholic fermentation of the selected L. plantarum strains was analyzed. Our results suggest the potential application of wine L. plantarum strains to design malolactic starter cultures able to degrade BA in wine.
doi:10.3389/fmicb.2012.00122
PMCID: PMC3316997  PMID: 22485114
lactic acid bacteria; amine degradation; biogenic amines; malolactic fermentation; wine; Lactobacillus plantarum; putrescine; tyramine
4.  Three-Component Lysine/Ornithine Decarboxylation System in Lactobacillus saerimneri 30a 
Journal of Bacteriology  2013;195(6):1249-1254.
Lactic acid bacteria play a pivotal role in many food fermentations and sometimes represent a health threat due to the ability of some strains to produce biogenic amines that accumulate in foods and cause trouble following ingestion. These strains carry specific enzymatic systems catalyzing the uptake of amino acid precursors (e.g., ornithine and lysine), the decarboxylation inside the cell, and the release of the resulting biogenic amines (e.g., putrescine and cadaverine). This study aimed to identify the system involved in production of cadaverine from lysine, which has not been described to date for lactic acid bacteria. Strain Lactobacillus saerimneri 30a (formerly called Lactobacillus sp. 30a) produces both putrescine and cadaverine. The sequencing of its genome showed that the previously described ornithine decarboxylase gene was not associated with the gene encoding an ornithine/putrescine exchanger as in other bacteria. A new hypothetical decarboxylation system was detected in the proximity of the ornithine decarboxylase gene. It consisted of two genes encoding a putative decarboxylase sharing sequence similarities with ornithine decarboxylases and a putative amino acid transporter resembling the ornithine/putrescine exchangers. The two decarboxylases were produced in Escherichia coli, purified, and characterized in vitro, whereas the transporter was heterologously expressed in Lactococcus lactis and functionally characterized in vivo. The overall data led to the conclusion that the two decarboxylases and the transporter form a three-component decarboxylation system, with the new decarboxylase being a specific lysine decarboxylase and the transporter catalyzing both lysine/cadaverine and ornithine/putrescine exchange. To our knowledge, this is an unprecedented observation of a bacterial three-component decarboxylation system.
doi:10.1128/JB.02070-12
PMCID: PMC3592000  PMID: 23316036
5.  Control of Biogenic Amines in Food—Existing and Emerging Approaches 
Journal of Food Science  2010;75(7):R139-R150.
Biogenic amines have been reported in a variety of foods, such as fish, meat, cheese, vegetables, and wines. They are described as low molecular weight organic bases with aliphatic, aromatic, and heterocyclic structures. The most common biogenic amines found in foods are histamine, tyramine, cadaverine, 2-phenylethylamine, spermine, spermidine, putrescine, tryptamine, and agmatine. In addition octopamine and dopamine have been found in meat and meat products and fish. The formation of biogenic amines in food by the microbial decarboxylation of amino acids can result in consumers suffering allergic reactions, characterized by difficulty in breathing, itching, rash, vomiting, fever, and hypertension. Traditionally, biogenic amine formation in food has been prevented, primarily by limiting microbial growth through chilling and freezing. However, for many fishing based subsistence populations, such measures are not practical. Therefore, secondary control measures to prevent biogenic amine formation in foods or to reduce their levels once formed need to be considered as alternatives. Such approaches to limit microbial growth may include hydrostatic pressures, irradiation, controlled atmosphere packaging, or the use of food additives. Histamine may potentially be degraded by the use of bacterial amine oxidase or amine-negative bacteria. Only some will be cost-effective and practical for use in subsistence populations.
doi:10.1111/j.1750-3841.2010.01774.x
PMCID: PMC2995314  PMID: 21535566
biogenic amines; food additives; high hydrostatic pressure (HHP); irradiation; packaging; scombroid poisoning; starter cultures; temperature
6.  Reducing Biogenic-Amine-Producing Bacteria, Decarboxylase Activity, and Biogenic Amines in Raw Milk Cheese by High-Pressure Treatments 
Biogenic amines may reach concentrations of public health concern in some cheeses. To minimize biogenic amine buildup in raw milk cheese, high-pressure treatments of 400 or 600 MPa for 5 min were applied on days 21 and 35 of ripening. On day 60, counts of lactic acid bacteria, enterococci, and lactobacilli were 1 to 2 log units lower in cheeses treated at 400 MPa and 4 to 6 log units lower in cheeses treated at 600 MPa than in control cheese. At that time, aminopeptidase activity was 16 to 75% lower in cheeses treated at 400 MPa and 56 to 81% lower in cheeses treated at 600 MPa than in control cheese, while the total free amino acid concentration was 35 to 53% higher in cheeses treated at 400 MPa and 3 to 15% higher in cheeses treated at 600 MPa, and decarboxylase activity was 86 to 96% lower in cheeses treated at 400 MPa and 93 to 100% lower in cheeses treated at 600 MPa. Tyramine, putrescine, and cadaverine were the most abundant amines in control cheese. The total biogenic amine concentration on day 60, which reached a maximum of 1.089 mg/g dry matter in control cheese, was 27 to 33% lower in cheeses treated at 400 MPa and 40 to 65% lower in cheeses treated at 600 MPa. On day 240, total biogenic amines attained a concentration of 3.690 mg/g dry matter in control cheese and contents 11 to 45% lower in cheeses treated at 400 MPa and 73 to 76% lower in cheeses treated at 600 MPa. Over 80% of the histidine and 95% of the tyrosine had been converted into histamine and tyramine in control cheese by day 60. Substrate depletion played an important role in the rate of biogenic amine buildup, becoming a limiting factor in the case of some amino acids.
doi:10.1128/AEM.03368-12
PMCID: PMC3568586  PMID: 23241980
7.  Lactic acid bacteria in Hamei and Marcha of North East India 
Indian Journal of Microbiology  2007;47(2):119-125.
Hamei and Marcha are mixed dough inocula used as starters for preparation of various indigenous alcoholic beverages in Manipur and Sikkim in India, respectively. These starters are traditionally prepared from rice with wild herbs and spices. Samples of Hamei and Marcha, collected from Manipur and Sikkim, respectively, were analysed for lactic acid bacterial composition. The population of lactic acid bacteria (LAB) was 6.9 and 7.1 Log cfu/g in Hamei and Marcha, respectively. On the basis of phenotypic and genotypic characters, LAB strains isolated from Hamei and Marcha were identified as Pediococcus pentosaceus, Lactobacillus plantarum and Lactobacillus brevis. Technological properties of LAB such as antimicrobial properties, effect on acidification, ability to produce biogenic amines and ethanol, degree of hydrophobicity and enzymatic activities were also performed. Pediococcus pentosaceus HS: B1, isolated from Hamei, was found to produce bacteriocin. None of the strains produced biogenic amines. LAB strains showed a strong acidifying ability and they also produced a wide spectrum of enzymes.
doi:10.1007/s12088-007-0024-8
PMCID: PMC3450109  PMID: 23100653
LAB; Hamei; Marcha
8.  Evidence of Two Functionally Distinct Ornithine Decarboxylation Systems in Lactic Acid Bacteria 
Biogenic amines are low-molecular-weight organic bases whose presence in food can result in health problems. The biosynthesis of biogenic amines in fermented foods mostly proceeds through amino acid decarboxylation carried out by lactic acid bacteria (LAB), but not all systems leading to biogenic amine production by LAB have been thoroughly characterized. Here, putative ornithine decarboxylation pathways consisting of a putative ornithine decarboxylase and an amino acid transporter were identified in LAB by strain collection screening and database searches. The decarboxylases were produced in heterologous hosts and purified and characterized in vitro, whereas transporters were heterologously expressed in Lactococcus lactis and functionally characterized in vivo. Amino acid decarboxylation by whole cells of the original hosts was determined as well. We concluded that two distinct types of ornithine decarboxylation systems exist in LAB. One is composed of an ornithine decarboxylase coupled to an ornithine/putrescine transmembrane exchanger. Their combined activities results in the extracellular release of putrescine. This typical amino acid decarboxylation system is present in only a few LAB strains and may contribute to metabolic energy production and/or pH homeostasis. The second system is widespread among LAB. It is composed of a decarboxylase active on ornithine and l-2,4-diaminobutyric acid (DABA) and a transporter that mediates unidirectional transport of ornithine into the cytoplasm. Diamines that result from this second system are retained within the cytosol.
doi:10.1128/AEM.07161-11
PMCID: PMC3298143  PMID: 22247134
9.  Biogenic amine production by the wine Lactobacillus brevis IOEB 9809 in systems that partially mimic the gastrointestinal tract stress 
BMC Microbiology  2012;12:247.
Background
Ingestion of fermented foods containing high levels of biogenic amines (BA) can be deleterious to human health. Less obvious is the threat posed by BA producing organisms contained within the food which, in principle, could form BA after ingestion even if the food product itself does not initially contain high BA levels. In this work we have investigated the production of tyramine and putrescine by Lactobacillus brevis IOEB 9809, of wine origin, under simulated gastrointestinal tract (GIT) conditions.
Results
An in vitro model that simulates the normal physiological conditions in the human digestive tract, as well as Caco-2 epithelial human cell lines, was used to challenge L. brevis IOEB 9809, which produced both tyramine and putrescine under all conditions tested. In the presence of BA precursors and under mild gastric stress, a correlation between enhancement of bacterial survival and a synchronous transcriptional activation of the tyramine and putrescine biosynthetic pathways was detected. High levels of both BA were observed after exposure of the bacterium to Caco-2 cells.
Conclusions
L. brevis IOEB 9809 can produce tyramine and putrescine under simulated human digestive tract conditions. The results indicate that BA production may be a mechanism that increases bacterial survival under gastric stress.
doi:10.1186/1471-2180-12-247
PMCID: PMC3499163  PMID: 23113922
Biogenic amines; Lactic acid bacteria; Putrescine; Tyramine; Food safety; Food toxicity
10.  Lactobacillus oligofermentans sp. nov., Associated with Spoilage of Modified-Atmosphere-Packaged Poultry Products 
Unidentified lactic acid bacterium (LAB) isolates which had mainly been detected in spoiled, marinated, modified atmosphere packaged (MAP) broiler meat products during two previous studies, were identified and analyzed for their phenotypic properties and the capability to produce biogenic amines. To establish the taxonomic position of these isolates, 16S rRNA gene sequence analysis, numerical analysis of ribopatterns, and DNA-DNA hybridization experiments were done. Unexpectedly for a meat-spoilage-associated LAB, the strains utilized glucose very weakly. According to the API 50 CHL test, arabinose and xylose were the only carbohydrates strongly fermented. None of the six strains tested for production of histamine, tyramine, tryptamine, phenylethylamine, putrescine, and cadaverine were able to produce these main meat-associated biogenic amines in vitro. The polyphasic taxonomy approach showed that these strains represent a new Lactobacillus species. The six isolates sequenced for the 16S rRNA encoding genes shared the highest similarity (95.0 to 96.3%) with the sequence of the Lactobacillus durianis type strain. In the phylogenetic tree, these isolates formed a distinct cluster within the Lactobacillus reuteri group, which also includes L. durianis. Numerical analyses of HindIII-EcoRI ribotypes placed all isolates together in a cluster with seven subclusters well separated from the L. reuteri group reference strains. The DNA-DNA hybridization levels between Lactobacillus sp. nov. isolates varied from 67 to 96%, and low hybridization levels (3 to 15%) were obtained with the L. durianis type strain confirming that these isolates belong to the same species different from L. durianis. The name Lactobacillus oligofermentans sp. nov. is proposed, with strain LMG 22743T (also known as DSM 15707T or AMKR18T) as the type strain.
doi:10.1128/AEM.71.8.4400-4406.2005
PMCID: PMC1183308  PMID: 16085830
11.  The influences of fish infusion broth on the biogenic amines formation by lactic acid bacteria 
Brazilian Journal of Microbiology  2013;44(2):407-413.
The influences of fish infusion decarboxylase broth (IDB) on biogenic amines (BA) formation by lactic acid bacteria (LAB) were investigated. BA productions by single LAB strains were tested in five different fish (anchovy, mackerel, white shark, sardine and gilthead seabream) IDB. The result of the study showed that significant differences in ammonia (AMN) and BA production were observed among the LAB strains in fish IDB (p < 0.05). The highest AMN and TMA production by LAB strains were observed for white shark IDB. The all tested bacteria had decarboxylation activity in fish IDB. The uppermost accumulated amines by LAB strains were tyramine (TYM), dopamine, serotonin and spermidine. The maximum histamine production was observed in sardine (101.69 mg/L) and mackerel (100.84 mg/L) IDB by Leuconostoc mesenteroides subsp. cremoris and Pediococcus acidophilus, respectively. Lactobacillus delbrueckii subsp. lactis and Pediococcus acidophilus had a high TYM producing capability (2943 mg/L and 1157 mg/L) in sardine IDB.
doi:10.1590/S1517-83822013000200010
PMCID: PMC3833135  PMID: 24294229
biogenic amines; lactic acid bacteria; starter cultures; fish infusion broth
12.  Dual Role for the Tyrosine Decarboxylation Pathway in Enterococcus faecium E17: Response to an Acid Challenge and Generation of a Proton Motive Force ▿  
In this work we investigated the role of the tyrosine decarboxylation pathway in the response of Enterococcus faecium E17 cells to an acid challenge. It was found that 91% of the cells were able to remain viable in the presence of tyrosine when they were incubated for 3 h in a complex medium at pH 2.5. This effect was shown to be related to the tyrosine decarboxylation pathway. Therefore, the role of tyrosine decarboxylation in pH homeostasis was studied. The membrane potential and pH gradient, the parameters that compose the proton motive force (PMF), were measured at different pHs (pH 4.5 to 7). We obtained evidence showing that the tyrosine decarboxylation pathway generates a PMF composed of a pH gradient formed due to proton consumption in the decarboxylation reaction and by a membrane potential which results from electrogenic transport of tyrosine in exchange for the corresponding biogenic amine tyramine. The properties of the tyrosine transporter were also studied in this work by using whole cells and right-side-out vesicles. The results showed that the transporter catalyzes homologous tyrosine/tyrosine antiport, as well as electrogenic heterologous tyrosine-tyramine exchange. The tyrosine transporter had properties of a typical precursor-product exchanger operating in a proton motive decarboxylation pathway. Therefore, the tyrosine decarboxylation pathway contributes to an acid response mechanism in E. faecium E17. This decarboxylation pathway gives the strain a competitive advantage in nutrient-depleted conditions, as well as in harsh acidic environments, and a better chance of survival, which contributes to higher cell counts in food fermentation products.
doi:10.1128/AEM.01958-08
PMCID: PMC2620722  PMID: 19011061
13.  Effect of γ-Aminobutyric Acid (GABA) Producing Bacteria on In vitro Rumen Fermentation, Biogenic Amine Production and Anti-oxidation Using Corn Meal as Substrate 
The effects and significance of γ-amino butyric acid (GABA) producing bacteria (GPB) on in vitro rumen fermentation and reduction of biogenic amines (histamine, methylamine, ethylamine, and tyramine) using corn meal as a substrate were determined. Ruminal samples collected from ruminally fistulated Holstein cows served as inoculum and corn was used as substrate at 2% dry matter (DM). Different inclusion rates of GPB and GABA were evaluated. After incubation, addition of GPB had no significant effect on in vitro fermentation pH and total gas production, but significantly increased the ammonia nitrogen (NH3-N) concentration and reduced the total biogenic amines production (p<0.05). Furthermore, antioxidation activity was improved as indicated by the significantly higher concentration of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) among treated samples when compared to the control (p<0.05). Additionally, 0.2% GPB was established as the optimum inclusion level. Taken together, these results suggest the potential of utilizing GPB as feed additives to improve growth performance in ruminants by reducing biogenic amines and increasing anti-oxidation.
doi:10.5713/ajas.2012.12558
PMCID: PMC4093236  PMID: 25049853
GABA; Biogenic Amines; Feed Additives; In vitro; Antioxidant
14.  The Mechanism of the Tyrosine Transporter TyrP Supports a Proton Motive Tyrosine Decarboxylation Pathway in Lactobacillus brevis 
Journal of Bacteriology  2006;188(6):2198-2206.
The tyrosine decarboxylase operon of Lactobacillus brevis IOEB9809 contains, adjacent to the tyrosine decarboxylase gene, a gene for TyrP, a putative tyrosine transporter. The two genes potentially form a proton motive tyrosine decarboxylation pathway. The putative tyrosine transporter gene of L. brevis was expressed in Lactococcus lactis and functionally characterized using right-side-out membranes. The transporter very efficiently catalyzes homologous tyrosine-tyrosine exchange and heterologous exchange between tyrosine and its decarboxylation product tyramine. Tyrosine-tyramine exchange was shown to be electrogenic. In addition to the exchange mode, the transporter catalyzes tyrosine uniport but at a much lower rate. Analysis of the substrate specificity of the transporter by use of a set of 19 different tyrosine substrate analogues showed that the main interactions between the protein and the substrates involve the amino group and the phenyl ring with the para hydroxyl group. The carboxylate group that is removed in the decarboxylation reaction does not seem to contribute to the affinity of the protein for the substrates significantly. The properties of the TyrP protein are those typical for precursor-product exchangers that operate in proton motive decarboxylation pathways. It is proposed that tyrosine decarboxylation in L. brevis results in proton motive force generation by an indirect proton pumping mechanism.
doi:10.1128/JB.188.6.2198-2206.2006
PMCID: PMC1428153  PMID: 16513749
15.  Understanding the physiology of Lactobacillus plantarum at zero growth 
The physiology of Lactobacillus plantarum at extremely low growth rates, through cultivation in retentostats, is much closer to carbon-limited growth than to stationary phase, as evidenced from transcriptomics data, metabolic fluxes, and biomass composition and viability.Using a genome-scale metabolic model and constraint-based computational analyses, amino-acid fluxes—in particular, the rather paradoxical excretion of Asp, Arg, Met, and Ala—could be rationalized as a means to allow extensive metabolism of other amino acids, that is, that of branched-chain and aromatic amino acids.Catabolic products from aromatic amino acids are known to have putative plant-hormone action. The metabolism of amino acids, as well as transcription data, strongly suggested a plant environment-like response in slow-growing L. plantarum, which was confirmed by significant effects of fermented medium on plant root formation.
Natural ecosystems are usually characterized by extremely low and fluctuating nutrient availability. Hence, microorganisms in these environments live a ‘feast-and-famine' existence, with famine the most habitual state. As a result, extremely slow or no growth is the most common state of bacteria, and maintenance processes dominate their life.
In the present study, Lactobacillus plantarum was used as a model microorganism to investigate the physiology of slow growth. Besides fermented foods, this microorganism can be observed in a variety of environmental niches, including plants and lakes, in which nutrient supply is limited. To mimic these conditions, L. plantarum was grown in a glucose-limited chemostat with complete biomass retention (retentostat). During cultivation, biomass progressively accumulated, resulting in steadily decreasing specific substrate availability. Less energy was thus available for growth, and the specific growth rate decreased accordingly, with a final calculated doubling time greater than one year. Detailed measurements of metabolic fluxes were used as constraints in a genome-scale metabolic model to precisely calculate the amount of energy used for net biomass synthesis and for maintenance purposes: at the lowest growth rate investigated (μ=0.0002 h−1), maintenance accounted for 94% of all energy expenses.
Genome-scale metabolic analysis was used in combination with transcriptomics to study the adaptation of L. plantarum to extremely slow growth under limited carbon and energy supply. Importantly, slow growth as investigated here was fundamentally different from the widely studied carbon starvation-induced stationary phase: non-growing cells in retentostat conditions were glucose limited rather than starved, and the transition from a growing to a non-growing state under retentostat conditions was progressive, in contrast with the abrupt transition in batch cultures. These differences were reflected in various aspects of the cell physiology.
The metabolic behavior was remarkably stable during adaptation to slow growth. Although carbon catabolite repression was clearly relieved, as indicated by the upregulation of genes for the utilization of alternative carbohydrates, the metabolism remained largely based on the conversion of glucose to lactate.
Stress resistance mechanisms were also not massively induced. In particular, analysis of the biomass composition—which remained similar to fast-growing cells even under virtually non-growing conditions—and of the gene expression profile, failed to reveal clear stringent or general stress responses, which are generally triggered in glucose-starved cells. The observation that genes involved in growth-associated processes were not downregulated suggested that active synthesis of biomass components (RNA, proteins, and membranes) was required to account for the observed stable biomass and that turnover of macromolecules was high in slow-growing cells. Biomass viability or morphology was also not affected, compared with faster growth conditions. The only typical stress response was the induction of an SOS response—in particular, the upregulation of the two error-prone DNA polymerases—suggesting an increased potential for genetic diversity under adverse conditions. Although diversity was not apparent under the conditions studied here, such mechanisms of increased rates of mutagenesis are likely to have an important role in the adaptation of L. plantarum to slow growth.
A surprising response of L. plantarum during adaptation to slow growth was the production of several amino acids (Arg, Asp, Met, and Ala). A priori, this metabolic behavior seemed inefficient in a context of energy limitation. However, reduced cost analysis using the genome-scale metabolic model indicated that it had a positive effect on energy generation. In-depth analysis of metabolic flux distributions showed that biosynthesis of these amino acids was connected to the catabolism of branched-chain and aromatic amino acids (BCAAs and AAAs), under conditions of limited ammonium efflux. At a fixed ammonium efflux—fixed at the measured value—flux balance analysis indicated that BCAAs and AAAs were expensive to metabolize, because the regeneration of 2-ketoglutarate through glutamate dehydrogenase was limited by ammonium dissipation. Therefore, alternative pathways had to be active to supply the necessary pool of 2-ketoglutarate. At low growth rates, amino-acid production (Arg, Asp, Ala, and Met) accounted for most of the 2-ketoglutarate regeneration. Although it came at the expense of ATP, this metabolic alternative to glutamate dehydrogenase was less energy costly than other solutions such as purine biosynthesis. This is thus an excellent example in which precise, quantitative modeling results in new insights in physiology that intuition would never have achieved. It also shows that flux balance analysis can be used to accurately predict energetically inefficient metabolism, provided the appropriate fluxes are constrained (here, ammonium efflux).
The observation that BCAAs and AAAs were catabolized at the expense of energy was intriguing. However, several end products of these catabolic pathways can serve as signaling molecules for interactions with other organisms. In particular, precursors of plant hormones were predicted as possible end products in the model simulations. Accordingly, the production of compounds interfering with plant root development was demonstrated in slow-growing L. plantarum. The metabolic analysis thus suggested that slow-growing L. plantarum produced plant hormones—or precursors thereof—as a strategy to divert the plant metabolism towards its own interest. In support of this view, transcriptome analysis indicated the upregulation of genes involved in the catabolism of β-glucosides—typical sugars from plant cell wall—as well as a very high induction of six gene clusters encoding cell-surface protein complexes predicted to have a role in the utilization of plant polysaccharides (csc clusters). In such a plant context, limited ammonium production would also make sense, because of the well-documented toxicity of ammonium for plants: production of amino acids could represent an alternative to ammonium excretion while keeping both parties satisfied.
In conclusion, the physiology of L. plantarum at extremely low growth rates, as studied by genome-scale metabolic modeling and transcriptomics, is fundamentally different from that of starvation-induced stationary phase cells. Excitingly, these conditions seem to trigger responses that favor interactions with the environment, more specifically with plants. The reported observations were made in the absence of any plant-derived material, suggesting that this response might constitute a hardwired behavior.
Situations of extremely low substrate availability, resulting in slow growth, are common in natural environments. To mimic these conditions, Lactobacillus plantarum was grown in a carbon-limited retentostat with complete biomass retention. The physiology of extremely slow-growing L. plantarum—as studied by genome-scale modeling and transcriptomics—was fundamentally different from that of stationary-phase cells. Stress resistance mechanisms were not massively induced during transition to extremely slow growth. The energy-generating metabolism was remarkably stable and remained largely based on the conversion of glucose to lactate. The combination of metabolic and transcriptomic analyses revealed behaviors involved in interactions with the environment, more particularly with plants: production of plant hormones or precursors thereof, and preparedness for the utilization of plant-derived substrates. Accordingly, the production of compounds interfering with plant root development was demonstrated in slow-growing L. plantarum. Thus, conditions of slow growth and limited substrate availability seem to trigger a plant environment-like response, even in the absence of plant-derived material, suggesting that this might constitute an intrinsic behavior in L. plantarum.
doi:10.1038/msb.2010.67
PMCID: PMC2964122  PMID: 20865006
Lactobacillus plantarum; metabolic modeling; retentostat; slow growth; transcriptome analysis
16.  Genome Sequence of Lactobacillus saerimneri 30a (Formerly Lactobacillus sp. Strain 30a), a Reference Lactic Acid Bacterium Strain Producing Biogenic Amines 
Genome Announcements  2013;1(1):e00097-12.
Lactobacillus sp. strain 30a (Lactobacillus saerimneri) produces the biogenic amines histamine, putrescine, and cadaverine by decarboxylating their amino acid precursors. We report its draft genome sequence (1,634,278 bases, 42.6% G+C content) and the principal findings from its annotation, which might shed light onto the enzymatic machineries that are involved in its production of biogenic amines.
doi:10.1128/genomeA.00097-12
PMCID: PMC3569274  PMID: 23405290
17.  Biogenic amines determination in some traditional cheeses in West Azerbaijan province of Iran 
Veterinary Research Forum  2013;4(2):115-118.
Biogenic amines (BA) are nitrogenous compounds that possess biological activity. The source of production is the microbial decarboxylation of amino acids. This compounds are found in various types of cheese. The aim of this work was to evaluate the BA content of some traditional cheeses in West Azerbaijan province Iran. For this purpose, 70 samples of Koopeh, 10 samples of Lighvan and 5 samples of Red Salmas cheeses were obtained from local supermarkets of different cities of West Azerbaijan province. After preparation of samples, biogenic amines content was evaluated by modified HPLC method. The presence of histamine, cadaverine, putrescine and tyramine in tested cheeses were observed. Total amount of biogenic amines was highest in Red Salmas cheese with 1426.91 ppm. It followed by Lighvan cheese and Koopeh cheese with 1008.98 and 517.71 ppm, respectively. Putrescine, cadaverine, histamine and tyramine were detected in Koopeh cheese at levels up to 156.09, 282.34, 70.80, 8.48 ppm respectively. These amines were detected also in Lighvan cheese at levels up to 277.53, 342.74, 37.58, 351.12 ppm and in Red Salmas cheese samples at levels up to 438.03, 701.05, 105.21, 182.62 ppm, respectively. Large amounts of biogenic amines can indicate non hygienic conditions and contamination of used milk for cheese production.
PMCID: PMC4313012
Biogenic amines; Cheese; Histamine; HPLC
18.  Factors Influencing Biogenic Amines Accumulation in Dairy Products 
Fermented foods are among the food products more often complained of having caused episodes of biogenic amines (BA) poisoning. Concerning milk-based fermented foods, cheese is the main product likely to contain potentially harmful levels of BA, specially tyramine, histamine, and putrescine. Prompted by the increasing awareness of the risks related to dietary uptake of high biogenic amine loads, in this review we report all those elaboration and processing technological aspects affecting BA biosynthesis and accumulation in dairy foods. Improved knowledge of the factors involved in the synthesis and accumulation of BA should lead to a reduction in their incidence in milk products. Synthesis of BA is possible only when three conditions converge: (i) availability of the substrate amino acids; (ii) presence of microorganisms with the appropriate catabolic pathway activated; and (iii) environmental conditions favorable to the decarboxylation activity. These conditions depend on several factors such as milk treatment (pasteurization), use of starter cultures, NaCl concentration, time, and temperature of ripening and preservation, pH, temperature, or post-ripening technological processes, which will be discussed in this chapter.
doi:10.3389/fmicb.2012.00180
PMCID: PMC3390585  PMID: 22783233
biogenic amines; cheese; producing microorganisms; pasteurization; starters; ripening; chemico-physical factors
19.  Synthesis of γ-Aminobutyric Acid by Lactic Acid Bacteria Isolated from a Variety of Italian Cheeses▿  
Applied and Environmental Microbiology  2007;73(22):7283-7290.
The concentrations of γ-aminobutyric acid (GABA) in 22 Italian cheese varieties that differ in several technological traits markedly varied from 0.26 to 391 mg kg−1. Presumptive lactic acid bacteria were isolated from each cheese variety (total of 440 isolates) and screened for the capacity to synthesize GABA. Only 61 isolates showed this activity and were identified by partial sequencing of the 16S rRNA gene. Twelve species were found. Lactobacillus paracasei PF6, Lactobacillus delbrueckii subsp. bulgaricus PR1, Lactococcus lactis PU1, Lactobacillus plantarum C48, and Lactobacillus brevis PM17 were the best GABA-producing strains during fermentation of reconstituted skimmed milk. Except for L. plantarum C48, all these strains were isolated from cheeses with the highest concentrations of GABA. A core fragment of glutamate decarboxylase (GAD) DNA was isolated from L. paracasei PF6, L. delbrueckii subsp. bulgaricus PR1, L. lactis PU1, and L. plantarum C48 by using primers based on two highly conserved regions of GAD. A PCR product of ca. 540 bp was found for all the strains. The amino acid sequences deduced from nucleotide sequence analysis showed 98, 99, 90, and 85% identity to GadB of L. plantarum WCFS1 for L. paracasei PF6, L. delbrueckii subsp. bulgaricus PR1, L. lactis PU1, and L. plantarum C48, respectively. Except for L. lactis PU1, the three lactobacillus strains survived and synthesized GABA under simulated gastrointestinal conditions. The findings of this study provide a potential basis for exploiting selected cheese-related lactobacilli to develop health-promoting dairy products enriched in GABA.
doi:10.1128/AEM.01064-07
PMCID: PMC2168214  PMID: 17890341
20.  Biodiversity of Lactobacillus plantarum from traditional Italian wines 
In this study, 23 samples of traditional wines produced in Southern Italy were subjected to microbiological analyses with the aim to identify and biotype the predominant species of lactic acid bacilli. For this purpose, a multiple approach, consisting in the application of both phenotypic (API 50CHL test) and biomolecular methods (polymerase chain reaction-denaturing gradient gel electrophoresis and 16S rRNA gene sequencing) was used. The results showed that Lactobacillus plantarum was the predominant species, whereas Lb. brevis was detected in lower amount. In detail, out of 80 isolates 58 were ascribable to Lb. plantarum and 22 to Lb. brevis. Randomly amplified polymorphic DNA-polymerase chain reaction was used to highlight intraspecific variability among Lb. plantarum strains. Interestingly, the cluster analysis evidenced a relationship between different biotypes of Lb. plantarum and their origin, in terms of wine variety. Data acquired in this work show the possibility to obtain several malolactic fermentation starter cultures, composed by different Lb. plantarum biotypes, for their proper use in winemaking processes which are distinctive for each wine.
doi:10.1007/s11274-014-1654-8
PMCID: PMC4072923  PMID: 24817564
Lactobacillus plantarum; Wine; PCR-DGGE; RAPD-PCR; Malolactic fermentation
21.  Control of Biogenic Amines in Fermented Sausages: Role of Starter Cultures 
Biogenic amines show biological activity and exert undesirable physiological effects when absorbed at high concentrations. Biogenic amines are mainly formed by microbial decarboxylation of amino acids and thus are usually present in a wide range of foods, fermented sausages being one of the major biogenic amine sources. The use of selected starter cultures is one of the best technological measures to control aminogenesis during meat fermentation. Although with variable effectiveness, several works show the ability of some starters to render biogenic amine-free sausages. In this paper, the effect of different starter culture is reviewed and the factors determining their performance discussed.
doi:10.3389/fmicb.2012.00169
PMCID: PMC3345612  PMID: 22586423
starter cultures; biogenic amines; amino acid decarboxylase; fermented sausages; amino oxidase; autochthonous
22.  Arginine Catabolism by Sourdough Lactic Acid Bacteria: Purification and Characterization of the Arginine Deiminase Pathway Enzymes from Lactobacillus sanfranciscensis CB1 
Applied and Environmental Microbiology  2002;68(12):6193-6201.
The cytoplasmic extracts of 70 strains of the most frequently isolated sourdough lactic acid bacteria were screened initially for arginine deiminase (ADI), ornithine transcarbamoylase (OTC), and carbamate kinase (CK) activities, which comprise the ADI (or arginine dihydrolase) pathway. Only obligately heterofermentative strains such as Lactobacillus sanfranciscensis CB1; Lactobacillus brevis AM1, AM8, and 10A; Lactobacillus hilgardii 51B; and Lactobacillus fructivorans DD3 and DA106 showed all three enzyme activities. Lactobacillus plantarum B14 did not show CK activity. L. sanfranciscensis CB1 showed the highest activities, and the three enzymes were purified from this microorganism to homogeneity by several chromatographic steps. ADI, OTC, and CK had apparent molecular masses of ca. 46, 39, and 37 kDa, respectively, and the pIs were in the range of 5.07 to 5.2. The OTCs, CKs, and especially ADIs were well adapted to pH (acidic, pH 3.5 to 4.5) and temperature (30 to 37°C) conditions which are usually found during sourdough fermentation. Internal peptide sequences of the three enzymes had the highest level of homology with ADI, OTC, and CK of Lactobacillus sakei. L. sanfranciscensis CB1 expressed the ADI pathway either on MAM broth containing 17 mM arginine or during sourdough fermentation with 1 to 43 mM added arginine. Two-dimensional electrophoresis showed that ADI, OTC, and CK were induced by factors of ca. 10, 4, and 2 in the whole-cell extract of cells grown in MAM broth containing 17 mM arginine compared to cells cultivated without arginine. Arginine catabolism in L. sanfranciscensis CB1 depended on the presence of a carbon source and arginine; glucose at up to ca. 54 mM did not exert an inhibitory effect, and the pH was not relevant for induction. The pH of sourdoughs fermented by L. sanfranciscensis CB1 was dependent on the amount of arginine added to the dough. A low supply of arginine (6 mM) during sourdough fermentation by L. sanfranciscensis CB1 enhanced cell growth, cell survival during storage at 7°C, and tolerance to acid environmental stress and favored the production of ornithine, which is an important precursor of crust aroma compounds.
doi:10.1128/AEM.68.12.6193-6201.2002
PMCID: PMC134416  PMID: 12450844
23.  Pure Culture Fermentation of Brined Cucumbers1 
Applied Microbiology  1964;12(6):523-535.
The relative abilities of Pediococcus cerevisiae, Lactobacillus plantarum, L. brevis, and several other species of lactic acid bacteria to grow and produce acid in brined cucumbers were evaluated in pure culture fermentations. Such fermentations were made possibly by the use of two techniques, gamma radiation (0.83 to 1.00 Mrad) and hot-water blanching (66 to 80 C for 5 min), designed first to rid the cucumbers of naturally occurring, interfering, and competitive microbial groups prior to brining, followed by inoculation with the desired lactic acid bacteria. Of the nine species tested, strains of the three common to cucumber fermentations, P. cerevisiae, L. plantarum, and L. brevis, grew to the highest populations, and produced the highest levels of brine acidity and the lowest pH values in fermentations at 5.4 to 5.6% NaCl by weight; also, their sequence of active development in fermentations, with the use of a three-species mixture for inoculation, was in the species order just named. This sequence of occurrence was similar to that estimated by others for natural fermentations. The rates of growth and acid production in fermentations with a mixture of P. cerevisiae, L. plantarum, and L. brevis increased as the incubation temperature was increased from 21 to 27 to 32 C; however, the maximal populations and acidities attained were essentially the same for fermentations at each temperature. Further, these same three species were found to be the most salt tolerant of those tested; their upper limit for appreciable growth and measurable acid production was about 8% salt, whereas thermophilic species such as L. thermophilus, L. lactis, L. helveticus, L. fermenti, and L. delbrueckii exhibited a much lower salt tolerance, ranging from about 2.5 to 4.0%. However, certain strains of L. delbrueckii grew very rapidly in cucumbers brined at 2.5 to 3.0% salt, and produced sufficient acid in about 30 hr at 48 C to reduce the brine pH from above 7.0 to below 4.0. An inexpensive, pure culture fermentor which was suitable for gamma radiation, resistant to salt and acid, and which permitted repeated aseptic sampling of the fermenting brine, is illustrated and the specifications are given.
PMCID: PMC1058172  PMID: 16349651
24.  Taxonomic Structure and Monitoring of the Dominant Population of Lactic Acid Bacteria during Wheat Flour Sourdough Type I Propagation Using Lactobacillus sanfranciscensis Starters▿  
The structure and stability of the dominant lactic acid bacterium population were assessed during wheat flour sourdough type I propagation by using singly nine strains of Lactobacillus sanfranciscensis. Under back-slopping propagation with wheat flour type 0 F114, cell numbers of presumptive lactic acid bacteria varied slightly between and within starters. As determined by randomly amplified polymorphic DNA-PCR and restriction endonuclease analysis-pulsed-field gel electrophoresis analyses, only three (LS8, LS14, and LS44) starters dominated throughout 10 days of propagation. The others progressively decreased to less than 3 log CFU g−1. Partial sequence analysis of the 16S rRNA and recA genes and PCR-denaturating gradient gel electrophoresis analysis using the rpoB gene allowed identification of Weissella confusa, Lactobacillus sanfranciscensis, Lactobacillus plantarum, Lactobacillus rossiae, Lactobacillus brevis, Lactococcus lactis subsp. lactis, Pediococcus pentosaceus, and Lactobacillus spp. as the dominant species of the raw wheat flour. At the end of propagation, one autochthonous strain of L. sanfranciscensis was found in all the sourdoughs. Except for L. brevis, strains of the above species were variously found in the mature sourdoughs. Persistent starters were found in association with other biotypes of L. sanfranciscensis and with W. confusa or L. plantarum. Sourdoughs were characterized for acidification, quotient of fermentation, free amino acids, and community-level catabolic profiles by USING Biolog 96-well Eco microplates. In particular, catabolic profiles of sourdoughs containing persistent starters behaved similarly and were clearly differentiated from the others. The three persistent starters were further used for the production of sourdoughs and propagated by using another wheat flour whose lactic acid bacterium population in part differed from the previous one. Also, in this case all three starter strains persisted during propagation.
doi:10.1128/AEM.01524-08
PMCID: PMC2643576  PMID: 19088320
25.  Microcolumn Separation of Amine Metabolites in the Fruit Fly 
Analytical chemistry  2005;77(16):5349-5355.
Electrophoretic resolution of fourteen biogenic amines and metabolites with similar mobilities is addressed by employing micellar electrokinetic capillary chromatography coupled to amperometric electrochemical detection. The present study describes the optimization of separation conditions to achieve resolution of analytes of biological significance within 20 minutes in a single separation. They include dopamine, epinephrine, norepinephrine, octopamine (OA), l-3, 4-dihydroxyphenylalanine, tyramine (TA), and serotonin as well as metabolites 5-hydroxyindolacetic acid, 3,4-dihydroxyphenylacetic acid, homovanillic acid, and 3-methoxytyramine in addition to N-acetylated metabolites including N-acetyl dopamine, N-acetyl octopamine (naOA), and N-acetyl serotonin. The optimized conditions used result in excellent reproducibility and predictable peak shifting, thus enabling identification of several metabolites along with their biogenic amine precursors in biological samples, specifically from the fruit fly Drosophila melanogaster. The separation method is sensitive, selective and quantitative as demonstrated by its capacity to detect changes in TA, OA, and naOA present in the head homogenates of the Canton-S and mutant inactive1 Drosophila lines. Quantitative analysis of metabolites in conjunction with their biogenic amine precursors in a single separation offers tremendous potential to understand the physiological processes and underlying mechanisms mediated by various biogenic amines in Drosophila and other animals.
doi:10.1021/ac050474m
PMCID: PMC1351352  PMID: 16097779

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