Antiproliferative effect of indeno[1,2-d]thiazolo[3,2-a]pyrimidine analogues on IL-6 mediated STAT3 and role of the apoptotic pathway in albino Wistar rats of ethyl carbamate-induced lung carcinoma: In-silico, In-vitro, and In-vivo study.

Lung cancer (LC) ranks second most prevalent cancer in females after breast cancer and second in males after prostate cancer. Based on the GLOBOCAN 2020 report, India represented 5.9% of LC cases and 8.1% of deaths caused by the disease. Several clinical studies have shown that LC occurs because of biological and morphological abnormalities and the involvement of altered level of antioxidants, cytokines, and apoptotic markers. In the present study, we explored the antiproliferative activity of indeno[1,2-d]thiazolo[3,2-a]pyrimidine analogues against LC using in-vitro, in-silico, and in-vivo models. In-vitro screening against A549 cells revealed compounds 9B (8-methoxy-5-(3,4,5-trimethoxyphenyl)-5,6-dihydroindeno[1,2-d]thiazolo[3,2-a]pyrimidine) and 12B (5-(4-chlorophenyl)-5,6-dihydroindeno[1,2-d]thiazolo[3,2-a]pyrimidine) as potential pyrimidine analogues against LC. Compounds 9B and 12B were docked with different molecular targets IL-6, Cyt-C, Caspase9, and Caspase3 using AutoDock Vina 4.1 to evaluate the binding affinity. Subsequently, in-vivo studies were conducted in albino Wistar rats through ethyl-carbamate (EC)- induced LC. 9B and 12B imparted significant effects on physiological (weight variation), and biochemical (anti-oxidant [TBAR's, SOD, ProC, and GSH), lipid (TC, TG, LDL, VLDL, and HDL)], and cytokine (IL-2, IL-6, IL-10, and IL-1ß) markers in EC-induced LC in albino Wistar rats. Morphological examination (SEM and H&E) and western blotting (IL-6, STAT3, Cyt-C, BAX, Bcl-2, Caspase3, and caspase9) showed that compounds 9B and 12B had antiproliferative effects. Accordingly, from the in-vitro, in-silico, and in-vivo experimental findings, we concluded that 9B and 12B have significant antiproliferative potential and are potential candidates for further evaluation to meet the requirements of investigation of new drug application.

Structure-guided engineered urethanase from Candida parapsilosis with pH and ethanol tolerance to efficiently degrade ethyl carbamate in Chinese rice wine.

Urethane hydrolase can degrade the carcinogen ethyl carbamate (EC) in fermented food, but its stability and activity limit its application. In this study, a mutant G246A and a double mutant N194V/G246A with improved cpUH activity and stability of Candida parapsilosis were obtained by site-directed mutagenesis. The catalytic efficiency (Kcat/Km) of mutant G246A and double mutant N194V/G246A are 1.95 times and 1.88 times higher than that of WT, respectively. In addition, compared with WT, the thermal stability and pH stability of mutant G246A and double mutant N194V/G246A were enhanced. The ability of mutant G246A and double mutant N194V/G246A to degrade EC in rice wine was also stronger than that of WT. The mutation increased the stability of the enzyme, as evidenced by decreased root mean square deviation (RMSD) and increased hydrogen bonds between the enzyme and substrate by molecular dynamics simulation and molecular docking analysis. The molecule modification of new cpUH promotes the industrial process of EC degradation.

Formation and hazard of ethyl carbamate and construction of genetically engineered Saccharomyces cerevisiae strains in Huangjiu (Chinese grain wine).

Huangjiu, a well-known conventional fermented Chinese grain wine, is widely consumed in Asia for its distinct flavor. Trace amounts of ethyl carbamate (EC) may be generated during the fermentation or storage process. The International Agency for Research on Cancer elevated EC to a Class 2A carcinogen, so it is necessary to regulate EC content in Huangjiu. The risk of intake of dietary EC is mainly assessed through the margin of exposure (MOE) recommended by the European Food Safety Authority, with a smaller MOE indicating a higher risk. Interventions are necessary to reduce EC formation. As urea, one of the main precursors of EC formation in Huangjiu, is primarily produced by Saccharomyces cerevisiae through the catabolism of arginine, the construction of dominant engineered fermentation strains is a favorable trend for the future production and application of Huangjiu. This review summarized the formation and carcinogenic mechanism of EC from the perspectives of precursor substances, metabolic pathways after ingestion, and risk assessment. The methods of constructing dominant S. cerevisiae strains in Huangjiu by genetic engineering technology were reviewed, which provided an important theoretical basis for reducing EC content and strengthening practical control of Huangjiu safety, and the future research direction was prospected.

Research progress on the application of different controlling strategies to minimizing ethyl carbamate in grape wine.

Ethyl carbamate (EC) is a probable carcinogenic compound commonly found in fermented foods and alcoholic beverages and has been classified as a category 2A carcinogen by the International Agency for Research on Cancer (IARC). Alcoholic beverages are one of the main sources of EC intake by humans. Therefore, many countries have introduced a standard EC limit in alcoholic beverages. Wine is the second largest alcoholic beverage in the world after beer and is loved by consumers for its rich taste. However, different survey results showed that the detection rate of EC in wine was almost 100%, while the maximum content was as high as 100 µg/L, necessitating EC content regulation in wine. The existing methods for controlling the EC level in wine mainly include optimizing raw fermentation materials and processes, using genetically engineered strains, and enzymatic methods (urease or urethanase). This review focused on introducing and comparing the advantages, disadvantages, and applicability of methods for controlling EC, and proposes two possible new techniques, that is, changing the fermentation strain and exogenously adding phenolic compounds. In the future, it is hoped that the feasibility of this prospect will be verified by pilot-scale or large-scale application to provide new insight into the regulation of EC during wine production. The formation mechanism and influencing factors of EC in wine were also introduced and the analytical methods of EC were summarized.

Features and application potential of microbial urethanases.

Urethanase (EC 3.5.1.75) can reduce ethyl carbamate (EC), a group 2A carcinogen found in foods and liquor. However, it is not yet commercially available. Urethanase has been detected as an intracellular enzyme from yeast, filamentous fungi, and bacteria. Based on the most recent progress in the sequence analysis of this enzyme, it was observed that amidase-type enzyme can degrade EC. All five enzymes had highly conserved sequences of amidase signature family, and their molecular masses were in the range of 52-62 kDa. The enzymes of Candida parapsilosis and Aspergillus oryzae formed a homotetramer, and that of Rhodococcus equi strain TB-60 existed as a monomer. Most urethanases exhibited amidase activity, and those of C. parapsilosis and A. oryzae also demonstrated high activity against acrylamide, which is a group 2A carcinogen. It was recently reported that urease and esterase also exhibited urethanase activity. Although research on the enzymatic degradation of EC has been very limited, recently some sequences of EC-degrading enzyme have been elucidated, and it is anticipated that new enzymes would be developed and applied into practical use. KEY POINTS: • Recently, some urethanase sequences have been elucidated • The amino acid residues that formed the catalytic triad were conserved • Urethanase shows amidase activity and can also degrade acrylamide.

Molecularly imprinted bulk and solgel optosensing based on biomass carbon dots derived from watermelon peel for detection of ethyl carbamate in alcoholic beverages.

Biomass carbon dots synthesized by biological waste conform to the trend of ecological environmental protection and the requirements of green chemistry, which show great application potential in practice. In the study, we used watermelon peels as the raw materials to synthesize a novel blue biomass carbon dots (CDs) by a hydrothermal process with high fluorescence quantum yield of 22.8%. Through bulk polymerization and solgel method, two kinds of core-shell nanospheres were developed as fluorescent probes to recognize and detect ethyl carbamate (EC) rapidly without complex samples pretreatment. The obtained CDs@MIPs integrated the high-performance optical characteristics of CDs with excellent selectivity and adsorption of MIPs, which showed ideal linear relationships in the EC concentration range 1-120 µg L-1 and low LOD of 0.57 µg L-1 and 0.94 µg L-1, respectively. Both CDs@MIPs have a short equilibration time which was around 20 min, and the imprinting factors (IF) are 4.04 and 2.62. The recoveries of the six spiked samples were satisfying, and the RSD precisions were lower than 5.57%. Gas chromatography-mass spectrometry was seen as a parallel analysis to validate the correctness of the results, which indicated the practicability and reliability of the developed method. This proposal strategy of optical sensors provided an effective channel for trace EC recognition, with numerous advantages, involving eco-friendly, low cost, high sensitivity, separation effect, and good selectivity.

Identification and expression of bifunctional acid urea-degrading enzyme/urethanase from Enterobacter sp. R-SYB082 and its application in degradation of ethyl carbamate in Chinese rice wine (Huangjiu).

BACKGROUND: Ethyl carbamate (EC) is a potential carcinogen existing in fermented foods such as Chinese rice wine (Huangjiu). Since urea is an important precursor of EC, the degradation of urea could be an effective way to reduce EC in foods. RESULTS: In this study, an Enterobacter sp. R-SYB082 with acid urea degradation characteristics was obtained through microbial screening. Further research isolated a new acid urea-degrading enzyme from R-SYB082 strain - ureidoglycolate amidohydrolase (UAH) - which could degrade EC directly. The cloning and expression of UAH in Escherichia coli BL21 (DE3) suggested that the activity of urea-degrading enzyme reached 3560 U L-1 , while urethanase activity reached 2883 U L-1 in the optimal fermentation condition. The enzyme had the dual ability of degrading substrate urea and product EC. The removal rate of EC in Chinese rice wine could reach 90.7%. CONCLUSION: This study provided a new method for the integrated control of EC in Chinese rice wine and other fermented foods. © 2022 Society of Chemical Industry.

Ethyl carbamate in Chinese liquor (Baijiu): presence, analysis, formation, and control.

Ethyl carbamate (EC) is a genotoxic and carcinogenic compound that is also a by-product of fermented foods (bread, sour milk, soy cheese, etc.) and alcoholic beverages (wine, sake, distilled liquor, etc.). Studies have showed that ethyl carbamate is ingested by humans primarily through the consumption of alcoholic beverages. Many countries have thus established EC limits for alcoholic beverages. Chinese liquor (Baijiu) is a traditional and unique distilled liquor, which has a huge consumption in China due to its excellent color, flavor, and taste. Therefore, the control of EC in Chinese liquor is of great significance. This review summarized for the first time the progress in presence level, analysis method, formation mechanism, and elimination strategy of EC of Chinese liquor in recent decades. KEY POINTS: • GC-MS and HPLC are the main methods to quantify EC in Chinese liquor. • EC is formed in the fermentation, distillation, and storage stage. • EC content can be reduced from raw material, microorganism, and production process.

Fluorescent sensor based on quantum dots and nano-porphyrin for highly sensitive and specific determination of ethyl carbamate in fermented food.

BACKGROUND: Ethyl carbamate (EC) is a potentially toxic carcinogen produced during fermentation and storage of fermented foods, and many countries have set thresholds for its content in food. Therefore, sensitive, rapid and accurate detection of EC is meaningful to ensure the quality of fermented food. RESULTS: This study introduces a CdTe quantum dots/nano-5,10,15,20-tetrakis (4-methoxyphenyl)-porphyrin (nano TPP-OCH3 ) fluorescence sensor system detection of EC. The specificity of this sensing mainly relies on a photo-induced electron transfer and electrostatic force interaction between EC and nano TPP-OCH3 . This sensor presented a linear range of 10 to 1000 µg L-1 (R2  = 0.9903) with a low detection limit of 7.14 µg L-1 . Meanwhile, the recovery (91.19-101.09%) and precision [relative standard deviation (RSD) = 0.64-3.05%] of the sensor for the analysis of fermented food (yellow rice wine, soy sauce, Chinese spirits, Pu-erh tea) samples were good and could meet the requirements of practical detection. Moreover, the detection results of fermented food (yellow rice wine, soy sauce, Chinese spirits, Pu-erh tea) samples by this sensor are basically consistent with those of high-performance liquid chromatography with fluorescence detector (HPLC-FLD). CONCLUSION: This method was expected to provide a potential platform for sensitive and accurate detection of EC in food safety monitoring, which would provide knowledge of the flavor and quality related to fermented food. © 2021 Society of Chemical Industry.

New function of polysaccharide from Rubus chingii Hu: protective effect against ethyl carbamate induced cytotoxicity.

BACKGROUND: Rubus chingii Hu is a widely cultivated fruit in China and has declared multiple bioactivities including antioxidative activity. Ethyl carbamate (EC), mostly found in fermented food and alcoholic beverages, is a recognized human carcinogen, and researchers have proposed the correlation between oxidative stress and its toxicity. This study acquired the polysaccharide from R. chingii (RP) and explored its effect on EC-induced cytotoxicity using Caco-2 cells as the cell model. RESULTS: Results showed that RP exhibited protection against EC-induced toxicity by repairing redox imbalance as indicative of mitigated mitochondrial membrane potential collapse, attenuated reactive oxygen species overproduction, and impeded glutathione depletion. Moreover, the structural features of RP were characterized and revealed that it was mainly constituted by galacturonic acid and arabinose, with an average molecular weight of 7.039 × 105 g mol-1 . CONCLUSION: Overall, our results provided a new approach dealing with the toxicity caused by EC from the perspective of oxidative stress and described a new potential healthy value of R. chingii Hu, which could contribute to the development of a promising dietary supplement and functional food. © 2020 Society of Chemical Industry.

Effects of the pre-fermentative addition of chitosan on the nitrogenous fraction and the secondary fermentation products of SO2 -free red wines.

BACKGROUND: Different red winemaking were carried out to evaluate the effects of the prefermentative addition of chitosan, as an alternative to the use of SO2 , on the secondary products of alcoholic fermentation, yeast available nitrogen (YAN), biogenic amines and ethyl carbamate. RESULTS: The wines made with chitosan presented higher total acidity and higher content of tartaric and succinic acids than those made only with SO2 . The use of chitosan in winemaking resulted in wines with higher glycerol and diacetyl content without increasing the concentration of ethanol, acetic acid, acetaldehyde or butanediol. YAN was lower in wines made with chitosan, which may mean an advantage for the microbial stability of the wines. Furthermore, the use of chitosan at the beginning of alcoholic fermentation did not increase the concentration of biogenic amines or the formation of ethyl carbamate in SO2 -free red wines. CONCLUSION: The total or partial substitution of SO2 for chitosan at the beginning of the alcoholic fermentation gives rise to quality red wines without negatively affecting their nitrogen fraction or their very important secondary fermentation products such as acetic acid or acetaldehyde. © 2020 Society of Chemical Industry.

UHPLC-QTOFMS-based metabolomic analysis of serum and urine in rats treated with musalais containing varying ethyl carbamate content.

The aim of this work is to investigate the effect of the ethyl carbamate (EC) content in musalais on the metabolism of rats. Electron beam irradiation was performed to decrease the content of EC in musalais, and Sprague Dawley rats were subjected to intragastric administration of musalais with varying EC content (high, medium, and low groups). Control rats were fed normally without any treatment. Serum and urine samples were analyzed using ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry. Principal component analysis and orthogonal projections to latent structures discriminant analysis (OPLS-DA) were performed to detect changes in the metabolite profile in the serum and urine in order to identify the differential metabolites and metabolic pathways. The results demonstrated clear differences in the serum and urine metabolic patterns between control and treatment groups. Ions in treatment groups with variable importance in the projection of >1 (selected from the OPLS-DA loading plots) and Ps < 0.05 (Student t test) compared to control group were identified as candidate metabolites. Analysis of the metabolic pathways relevant to the identified differential metabolites revealed that high EC content in musalais (10 mg/kg) mainly affected rats through valine, leucine, and isoleucine biosynthesis and nicotinate and nicotinamide metabolism, which were associated with energy metabolism. In addition, this work suggests that EC can induce oxidative stress via inhibition of glycine content.

Metabolic engineering of Saccharomyces cerevisiae using the CRISPR/Cas9 system to minimize ethyl carbamate accumulation during Chinese rice wine fermentation.

Ethyl carbamate (EC) is a potential carcinogen to humans that is mainly produced through the spontaneous reaction between urea and ethanol during Chinese rice wine brewing. We metabolically engineered a strain by over-expressing the DUR3 gene in a previously modified strain using an improved CRISPR/Cas9 system to further decrease the EC level. Homologous recombination of the DUR3 over-expression cassette was performed at the HO locus by individual transformation of the constructed plasmid CRISPR-DUR3-gBlock-HO, generating the engineered strain N85DUR1,2/DUR3-c. Consequently, the DUR3 expression level was significantly enhanced in the modified strain, resulting in increased utilization of urea. The brewing test showed that N85DUR1,2/DUR3-c reduced urea and EC concentrations by 92.0% and 58.5%, respectively, compared with those of the original N85 strain. Moreover, the engineered strain showed good genetic stability in reducing urea content during the repeated brewing experiments. Importantly, the genetic manipulation had a negligible effect on the growth and fermentation characteristics of the yeast strain. Therefore, the constructed strain is potentially suitable for application to reduce urea and EC contents during production of Chinese rice wine. KEY POINTS: • An efficient CRISPR vector was constructed and applied for DUR3 over-expression. • Multi-modification of urea cycle had synergistic effect on reducing EC level. • Fermentation performance of engineered strain was similar with the parental strain. • No residual heterologous genes were left in the genome after genetic manipulation. • An efficient CRISPR vector was constructed and applied for DUR3 over-expression. • Multi-modification of urea cycle had synergistic effect on reducing EC level. • Fermentation performance of engineered strain was similar with the parental strain. • No residual heterologous genes were left in the genome after genetic manipulation.

Development of Cu(II)/Cu(I)-induced quantum dot-mediated fluorescence immunoassay for the sensitive determination of ethyl carbamate.

A series of haptens were rationally designed for producing monoclonal antibodies specific for EC and a simple fluorescence immunoassay platform was developed for the sensitive determination of EC based on alkaline phosphatase (ALP)-triggered Cu+ quenching of CdSe quantum dots (QDs). It was noted that Cd as a fluorescence substrate in CdSe QDs can be selectively substituted by Cu+ that resulted in a more significant fluorescence quenching in comparison with Cu2+. Meanwhile, because ALP catalyzed ascorbic acid phosphate and then assisted the transformation of Cu2+ to Cu+, the change in fluorescence intensity was found to be proportional to ALP concentration. After simple magnetic separation, the sensitivity and linear range of the established assay were improved approximately 53-fold and an order of magnitude, respectively, when compared with the conventional ELISA. The proposed platform was able to both amplify the signal and eliminate matrix interferences, making it a promising to determine EC as well as other contaminants in complex food matrix in a highly sensitive and simple manner. Graphical abstract.

Chinese Yellow Rice Wine Processing with Reduced Ethyl Carbamate Formation by Deleting Transcriptional Regulator Dal80p in Saccharomyces cerevisiae.

Ethyl carbamate (EC) is a potential carcinogen that forms spontaneously during Chinese rice wine fermentation. The primary precursor for EC formation is urea, which originates from both external sources and arginine degradation. Urea degradation is suppressed by nitrogen catabolite repression (NCR) in Saccharomyces cerevisiae. The regulation of NCR is mediated by two positive regulators (Gln3p, Gat1p/Nil1p) and two negative regulators (Dal80p/Uga43p, Deh1p/Nil2p/GZF3p). DAL80 revealed higher transcriptional level when yeast cells were cultivated under nitrogen-limited conditions. In this study, when DAL80-deleted yeast cells were compared to wild-type BY4741 cells, less urea was accumulated, and genes involved in urea utilization were up-regulated. Furthermore, Chinese rice wine fermentation was conducted using dal80Δ cells; the concentrations of urea and EC were both reduced when compared to the BY4741 and traditional fermentation starter. The findings of this work indicated Dal80p is involved in EC formation possibly through regulating urea metabolism and may be used as the potential target for EC reduction.

Transposition of IS4 Family Insertion Sequences ISTeha3, ISTeha4, and ISTeha5 into the arc Operon Disrupts Arginine Deiminase System in Tetragenococcus halophilus.

Tetragenococcus halophilus, a halophilic lactic acid bacterium, is often used as a starter culture in the manufacturing of soy sauce. T. halophilus possesses an arginine deiminase system, which is responsible for the accumulation of citrulline, the main precursor of the potential carcinogen ethyl carbamate. In this study, we generated five derivatives lacking arginine deiminase activity from T. halophilus NBRC 12172 by UV irradiation. Using these derivatives as a fermentation starter prevented arginine deimination in soy sauce. DNA sequence analysis of the derivatives revealed that novel IS4 family insertion sequences, designated ISTeha3, ISTeha4, and ISTeha5, were transposed into the region around the arginine deiminase (arc) operon in the mutants. These insertion sequences contain a single open reading frame encoding a putative transposase and 13- to 15-bp inverted repeats at both termini, which are adjacent to 7- to 9-bp duplications of the target sequence. Investigation of wild strains isolated from soy sauce mash incapable of arginine deimination also indicated that insertion sequences are involved in the disruption of the arginine deiminase system in T. halophilusIMPORTANCE Insertion sequences play important roles in bacterial evolution and are frequently utilized in mutagenesis systems. However, the intrinsic insertion sequences of tetragenococci are not well characterized. Here, we identified three active insertion sequences of T. halophilus by transposition into the region around the arc operon. This report provides an example of insertion sequence-mediated generation and evolution of T. halophilus and primary information about their characteristics.

The impacts of Schizosaccharomyces on winemaking.

In the past century, yeasts from the genus Saccharomyces represented the only option in fermentation industries, such as winemaking, to produce wine, beer, and other fermented products. However, other genera are currently emerging to solve challenges in modern enology. Schizosaccharomyces pombe is showing promising results in solving specific challenges in northern, cool viticulture regions with highly acidic wines by deacidifying these wines through its malic acid metabolism. In addition, this microorganism is considered beneficial in warm growing regions with challenges such as the control of wine food safety problems such as the presence of biogenic amines, ochratoxin A, or ethyl carbamate. Indeed, the genus Schizosaccharomyces positively influences other important wine quality parameters, such as color and polysaccharide content. However, the main challenge of using this genus remains the selection of proper strains that alleviate problems such as the production of high acetate concentrations. Industries other than wine production such as ginger fermentation, apple wine, Kei-apple fermentation, plum wine, sparkling wine, and bilberry fermentation industries have also started to study Schizosaccharomyces species as an alternative tool for solving specific related problems. The review discusses the influence of Schizosaccharomyces on different fermentation quality parameters and its main applications in different industries.

Environment-friendly synthesis of diethyl carbonate via ethyl carbamate alcoholysis over cerium oxide catalyst.

A series of cerium oxide catalysts were prepared by simply calcination of nitrate cerium, and used for clean synthesis of diethyl carbonate (DEC) by ethyl carbamate (EC) alcoholysis. 51% conversion and 82% selectivity were obtained with catalyst CeO2-600. Almost the same activity as the fresh sample could be regained if the used catalyst was recalcined at 600 °C. XPS, XRD, CO2-TPD and BET study suggested that high surface area and the presence of abundant basic sites are important to achieve a higher catalytic performance.

Formation of Ethyl Carbamate during the Production Process of Cantonese Soy Sauce.

The aim of this work was to clarify the formation of ethyl carbamate (EC) and its influence factors throughout the production process of Cantonese soy sauce. The results showed that EC was not detected in the koji-making and early moromi fermentation stages, but started to be generated when pH of the moromi decreased to about 4.9-at the same time, the levels of ethanol, urea and citrulline increased significantly. Most EC was formed during raw soy sauce hot extraction (40.6%) and sterilization (42.9%) stages. The EC content exhibited the highest correlation with ethanol throughout the whole production process (R = 0.97). The simulation soy sauce produced in laboratory led the same conclusion-moreover, the contents of EC, ethanol and citrulline were higher in soy sauce fermented at 30 °C than in soy sauce fermented at 15 °C. Extraction of raw soy sauce by squeezing contributed little to EC formation. Further research showed that citrulline and ethanol led to significant increases in EC levels in raw soy sauce upon heating. These results indicate that ethanol and citrulline are two critical precursors of EC and that EC is mainly formed during the heat treatment stage of soy sauce.

Arginine and Citrulline Catabolic Pathways Encoded by the arc Gene Cluster of Lactobacillus brevis ATCC 367.

High concentrations of l-arginine or l-citrulline in the growth medium provided the wine bacterium Lactobacillus brevis with a significant growth advantage. The arginine deiminase pathway (ADI) arc gene cluster of Lactobacillus brevis contains three genes-arcD, arcE1, and arcE2-encoding putative l-arginine/l-ornithine exchangers. Uptake experiments with Lactococcus lactis cells expressing the genes showed that all three transported l-ornithine with affinities in the micromolar range. Similarly, ArcD and ArcE2 transported l-arginine, while ArcE1 transported l-citrulline, an intermediate of the ADI pathway. Chase experiments showed very efficient exchange of l-arginine and l-ornithine by ArcD and ArcE2 and of l-citrulline and l-ornithine by ArcE1. Low affinities (millimolar range) combined with low translocation rates were found for ArcD and ArcE2 with l-citrulline and for ArcE1 with l-arginine. Resting cells of Lactobacillus brevis grown in the presence of l-arginine and l-citrulline rapidly consumed l-arginine and l-citrulline, respectively, while producing ammonia and l-ornithine. About 10% of l-arginine degraded was excreted by the cells as l-citrulline. Degradation of l-arginine and l-citrulline was not subject to carbon catabolite repression by glucose in the medium. At a high medium pH, l-citrulline in the medium was required for induction of the l-citrulline degradation pathway. Pathways are proposed for the catabolic breakdown of l-arginine and l-citrulline that merge at the level of ornithine transcarbamylase in the ADI pathway. l-Arginine uptake is catalyzed by ArcD and/or ArcE2, l-citrulline by ArcE1. l-Citrulline excretion during l-arginine breakdown is proposed to be catalyzed by ArcD and/or ArcE2 through l-arginine/l-citrulline exchange.IMPORTANCELactobacillus brevis, a bacterium isolated from wine, as well as other food environments, expresses a catabolic pathway for the breakdown of l-citrulline in the medium that consists of the l-citrulline/l-ornithine exchanger ArcE1 and part of the catabolic arginine deiminase (ADI) pathway enzymes. The proposed pathways for l-arginine and l-citrulline breakdown provide a mechanism for l-citrulline accumulation in fermented food products that is the precursor of the carcinogen ethyl carbamate.