Biogenic Amines in Dairy Products

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1 This article was downloaded by: [Miguel A. Alvarez] On: 28 July 2011, At: 00:39 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: Registered office: Mortimer House, Mortimer Street, London W1T 3JH, UK Critical Reviews in Food Science and Nutrition Publication details, including instructions for authors and subscription information: Biogenic Amines in Dairy Products Daniel M. Linares a, MaCruz Martín a, Victor Ladero a, Miguel A. Alvarez a & María Fernández a a Instituto de Productos Lácteos de Asturias (IPLA CSIC), 33300, Villaviciosa, Asturias, Spain Available online: 27 Jul 2011 To cite this article: Daniel M. Linares, MaCruz Martín, Victor Ladero, Miguel A. Alvarez & María Fernández (2011): Biogenic Amines in Dairy Products, Critical Reviews in Food Science and Nutrition, 51:7, To link to this article: PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: This article may be used for research, teaching and private study purposes. Any substantial or systematic reproduction, re-distribution, re-selling, loan, sub-licensing, systematic supply or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material.

2 Critical Reviews in Food Science and Nutrition, 51: (2011) Copyright C Taylor and Francis Group, LLC ISSN: print / online DOI: / Biogenic Amines in Dairy Products DANIEL M. LINARES, M a CRUZ MARTÍN, VICTOR LADERO, MIGUEL A. ALVAREZ, and MARÍA FERNÁNDEZ Instituto de Productos Lácteos de Asturias (IPLA CSIC), Villaviciosa, Asturias, Spain Biogenic amines (BA) are organic, basic, nitrogenous compounds with biological activity, mainly formed by the decarboxylation of amino acids. BA are present in a wide range of foods, including dairy products, and can accumulate in high concentrations. In some cheeses more than 1000 mg of BA have been detected per kilogram of cheese. The consumption of food containing large amounts of these amines can have toxicological consequences. Although there is no specific legislation regarding the BA content in dairy products, it is generally assumed that they should not be allowed to accumulate. Greater knowledge of the factors involved in the synthesis and accumulation of BA should lead to a reduction in their incidence in foods. This article focuses on the factors that affect BA production, in particular environmental conditions, the microorganisms that produce them, the genetic organization and regulation of the biosynthetic pathways involved, and the available methods for detecting the presence of BA or BA-producing microorganisms in dairy products. Keywords INTRODUCTION biogenic amines, dairy products, toxicology, microbiology, biochemistry, genetics, regulation, detection Biogenic amines (BA) are organic, basic, nitrogenous compounds with biological activity, mainly formed by the decarboxylation of amino acids. Although they are produced naturally by plants, animals, and microorganisms, the consumption of foods containing large amounts of these amines can have toxicological consequences (Shalaby, 1996). These problems are more severe in consumers in whom detoxification is less efficient because of their genetic constitution or their undergoing pharmacological treatment (Bodmer et al., 1999). Foods likely to contain high levels of biogenic amines include fish, fish products, and fermented foodstuffs (meat, dairy, vegetables, beers, and wines). The most important BA, both qualitatively and quantitatively, in foods and beverages are histamine, tyramine, putrescine, cadaverine, and β-phenyl ethylamine, products of the decarboxylation of histidine, tyrosine, ornithine, lysine, and β-phenylalanine respectively. The presence of BA in foods has traditionally been used as an indicator of undesired microbial activity. Relatively high levels of certain BA have also been reported to indicate the deterioration of food products and/or their defective manufacture. Address correspondence to Miguel A. Alvarez, Instituto de Productos Lácteos de Asturias (IPLA CSIC), Apdo. de Correos 85, Villaviciosa, Asturias, Spain. Tel.: , Fax: maag@ipla.csic.es Dairy products, especially cheese, can accumulate BA, with concentrations varying from just traces to more than 1000 mg Kg 1. Their toxicity had led to the general agreement that they should not be allowed to accumulate in food. This article describes the physiological role and toxic effects of BA, their presence in dairy products, the environmental conditions that favor their synthesis and accumulation, their production by microorganisms (including an in-depth description of the genetic organization and regulation of their biosynthetic pathways), and finally the methods available for detecting the presence of BA or BA-producing microorganisms. The factors affecting BA accumulation are analyzed in order to suggest possible means of reducing their presence, particularly in cheese. Knowledge in this area might help us reduce the formation of these toxic compounds and establish safety limits for their concentration in foods. CLASSIFICATION There are several ways to classify BA. According to their chemical structure they can be classified as aliphatic (putrescine, cadaverine, spermine, and spermidine), aromatic (tyramine, phenylethylamine) or heterocyclic (histamine, tryptamine). According to their number of amine groups they can be divided as monoamines (tyramine, phenylethylamine), diamines (putrescine, cadaverine), or polyamines (spermine 691

3 692 D. M. LINARES ET AL. and spermidine). Some authors indicate that since they are produced by a condensation process following decarboxylation, polyamines such as spermine and spermidine should not be classified as BA at all (Bardócz, 1999). PHYSIOLOGICAL ROLE In eukaryotic cells, BA biosynthesis is essential since these compounds act as precursors for the synthesis of hormones, alkaloids, nucleic acids, and proteins (Premont et al., 2001). Some BA play an important role as neurotransmitters while others, such as putrescine and spermidine, are needed for critical biological functions such as modulating DNA, RNA, and protein synthesis (Tabor and Tabor, 1985; Igarashi et al., 2001). In prokaryotic cells the physiological role of BA synthesis remains unresolved, although it appears mainly related to the performance of decarboxylation reactions one of the defence mechanisms used by bacteria to withstand an acidic environment. BA production in response to acidic stress has been extensively studied in the cadaverine producers Eschericha coli, Salmonella enterica serovar Thyphimurium, and Vibrio vulnificus (Park et al., 1996; Rhee et al., 2002; Lee et al., 2007). Decarboxylation increases survival under acidic stress condition (Rhee et al., 2002) via the consumption of protons and the excretion of amines and CO 2, helping to restore the external ph (Silla Santos, 1996; Schelp et al., 2001; van de Guchte et al., 2002). BA production may also offer a way of obtaining energy the electrogenic amino acid/amine antiport can lead to the generation of a proton motive force (Molenaar et al., 1993). This function would be particularly important to microorganisms such as lactic acid bacteria (LAB) lacking a respiratory chain for generating high yields of ATP (Vido et al., 2004). BA production may also mediate other physiological functions in bacteria, such as osmotic and oxidative stress responses (Schiller et al., 2000; Tkachenko et al., 2001). TOXICOLOGICAL EFFECTS Although BA are required for many critical biological functions (Tabor and Tabor, 1985; Igarashi et al., 2001) the consumption of foods containing large amounts of BA can have toxicological consequences. After food consumption, small quantities of BA are commonly metabolized in the human gut to physiologically less active forms via the action of amine oxidases (monoamine oxidases, MAO, and diamine oxidase, DAO). Histamine can also be detoxified by methylation (via the action of methyl transferases) (Taylor and Sumner, 1986) or acetylated (Lehane and Olley, 2000). However, the intake of foods with high BA loads, or inadequate detoxification (for genetic reasons or due to the inhibitory effects of some medicines or alcohol) (Bodmer et al., 1999) can lead to BA entering the systemic circulation and causing the release of adrenaline and noradrenaline, provoking gastric acid secretion, increased cardiac output, migraine, tachycardia, increased blood sugar levels, and higher blood pressure (Shalaby, 1996). Premont et al. (2001) also report BA levels to be higher in patients with Parkinson s disease, schizophrenia, and depression. Although the establishment of toxic levels of BA is difficult since this depends on the characteristics of different individuals, Wöhrl et al. (2004) report that 75 mg of pure liquid oral histamine a dose found in normal meals can provoke immediate as well as delayed symptoms in 50% of healthy females with no history of food intolerance. An additional problem in determining toxic levels of BA is their synergistic effects. For example, experiments in rats and guinea pigs indicate that cadaverine and other amines act as potentiators of histamine toxicity. These amines play a role as diamine oxidase inhibitors (Lehane and Olley, 2000) and their presence may explain why the intake of aged cheese is more toxic than an equivalent amount of histamine in aqueous solution (Taylor and Sumner, 1986). Secondary amines such as putrescine and cadaverine can also react with nitrite to form carcinogenic nitrosamines (Ten Brink et al., 1990), and the adherence to intestinal mucosa of some enteropathogens, such as E. coli O157:H7 is increased in the presence of tyramine (Lyte, 2004). It has been suggested that BA have been the causative agents behind a number of food poisoning episodes, the most notorious being those caused by histamine. Histamine poisoning is also known as scombroid poisoning owing to the association of this illness with the consumption of scombroid fish (Taylor, 1983). With respect to cheese, BA food poisoning is caused by high levels of tyramine; indeed, this is known as cheese reaction (Silla Santos, 1996). There is little specific legislation concerning BA content in foods. While for fish products there are clear limits for histamine (European Union Commission (EC) n o 2073/2005 and the Food and Drug Administration USA (FDA, 2001)), upper limits for BA in other foods have only been recommended or suggested (e.g., 100 mg of histamine per kg of food, or 2 mg of histamine per liter of alcoholic beverage). In the case of tyramine, a limit of between 100 and 800 mg Kg 1 has been recommended, and a limit of 30 mg Kg 1 of β-phenylethylamine has been proposed (Ten Brink et al., 1990; Halász et al., 1994). Although more research is necessary, there is a general consensus that food BA contents should be kept at minimum levels. BIOGENIC AMINES IN DAIRY PRODUCTS Dairy products are an important component of the diet in developed countries, a fact highlighted by the 2010 production of cheese in the EU at some 7.7 million tonnes (Eurostat, EU). Together with fish and wine, dairy products, especially cheese, can accumulate high levels of BA. Table 1 shows the BA content of different dairy products. In the raw material (milk),

4 BIOGENIC AMINES IN DAIRY PRODUCTS 693 Table 1 Biogenic amine content in different dairy products. Ud: undetect Biogenic amines (mg Kg 1 ) Product Histamine Tyramine Cadaverine Putrescine Tryptamine β-phenyethylamine Spermidine Spermine References Milk 0.13 ud Novella-Rodríguez et al., 2004 Curd Novella-Rodríguez et al., 2004 Whey ud 0.24 ud Novella-Rodríguez et al., 2002 Yogur 13 Bodmer et al., 1999 Unripenned cheeses ud ud ud ud Fernández et al., 2007b raw milk Unripenned cheeses pasteurized milk Fernández et al., 2007b Hard cheeses raw milk Fernández et al., 2007b Hard cheeses Fernández et al., 2007b pasteurized milk Blue cheeses raw milk Fernández et al., 2007b Blue cheeses Fernández et al., 2007b pasteurized milk Idiazabal ud 238,0 77, ud ud ud ud Ordoñez et al., 1997 Feta Valsamaki et al., 2000 Terrincho cheese Pinho et al., 2004 Semicotto Caprino 1.8 3, Galgano et al., 2001 Semihard Italian Innocente and D Agostin, 2002 polyamines are the most abundant. However, in the final product, tyramine, histamine, putrescine, cadaverine, and, at lower concentrations, β-phenylethylamine and tryptamine, are all detected. Since cheese is the dairy product that accumulates the highest BA concentrations, this review focuses mainly on this type of food (Table 1). The BA content of different types of cheese varies. Indeed, it can also vary within the same type of cheese and even between different sections of the same cheese (Novella-Rodríguez et al., 2003a). The synthesis and accumulation of BA in foods requires the presence of bacteria with decarboxylase activity, the environmental conditions that allow for their growth and the activity of the latter enzyme, and the presence of amino acid substrates. Microorganisms with Amino Acid Decarboxylase Activity Many microorganisms have the capacity to produce BA, including Gram positive and Gram negative bacteria of different genera and species, although such capacity is generally considered a strain-level characteristic. Although yeast seems to have great potential for the production of aliphatic amines (putrescine and cadaverine), only a few Debaryomyces hansenii strains isolated from cheese actually seem able to produce histamine (Gardini et al., 2006). Histamine, pustrescine, and tryptamine production in cheese has also been attributed to the activities of Yarrowia lipolytica, Pichia jadinii (Wyder et al., 1999) and Geotrichum candidum (Roig-Sagués et al., 2002). Most of the Gram negative bacteria described as the usual contaminants of milk are able to produce histamine, for example, Hafnia alvei, E. coli, Klebsiella pneumoniae, and Serratia spp. Putrescine and cadaverine production has mainly been related to Gram negative bacteria, especially Enterobacteriaceae (Ten Brink et al., 1990; Pircher et al., 2007). This has led to the suggestion that the presence of BA is an indicator of poor quality or bad manufacturing practices. However, the main BA producers in cheese are Gram positive bacteria, with LAB being the main histamine and tyramine producers. The genera Enterococcus, Lactobacillus, Leuconostoc, and Streptococcus include some strains that have been described as BA producers. These can be present in the milk microbiota or introduced by contamination before, during, or after the processing of dairy products. BA + -LAB may even form part of starter or adjunct cultures. Several authors have reported the presence of tyrosine and histamine decarboxylase activity in strains from various starter cultures (Botazzi, 1993; Burdychova and Komprda 2007; Komprda et al., 2008). It is therefore important to include the failure to produce BA as an indispensable condition of strains intended to be used as starters. Certainly, the use of good-quality milk is also important for reducing the risk of amine formation. Some authors relate the presence of high counts of Enterococci in milk with the later presence of large quantities of tyramine in cheese (Joosten and Northolt, 1987). Availability of Substrate Amino Acids The availability of substrate amino acids is a limiting factor in BA synthesis. Amine formation has been studied during the

5 694 D. M. LINARES ET AL. ripening of different type of cheeses and the influence of proteolysis on BA formation is now well known (Novella-Rodríguez et al., 2004; Pinho et al., 2004; Komprda et al., 2008). The proteolysis of casein during cheese ripening is critical for BA accumulation since it releases the amino acid substrates for the reaction. The influence of proteolysis is observed when the BA content of cheeses ripened over long and short periods is compared; the former can contain anything from 10 to 2000 times more BA (Innocente and D Agostin, 2002). This is because the content of free amino acids increases over ripening (from g tyrosine Kg 1 cheese at 3 days of ripening to 2191 g tyrosine Kg 1 cheese at 90 days of ripening). Thus, in Cabrales (a Spanish, blue, raw milk cheese ripened for 90 days), the concentration of the precursor amino acids is very low over the first few days of ripening and BA are not detected; however, after 15 days an increase in the amino acid concentration is observed and BA become detectable (Fernández et al., 2006a). Similar results have been reported for other types of cheese such as Feta, Terrhinco, and Semicotto Caprino (Valsamaki et al., 2000; Galgano et al., 2001; Pinho et al., 2004). High BA concentrations ( mg Kg 1 tyramine and 920 mg Kg 1 histamine) are observed in blue cheeses, the proteolytic activity of their molds helping these compounds to accumulate (Novella-Rodríguez et al., 2003a; Fernández et al., 2007a). Fernández-García et al. (2000) showed that adding proteinase significantly increased tyramine and histamine concentrations in Manchego cheese. A similar effect has been reported by Leuschner et al. (1998) for Gouda cheese. Environmental Conditions Related to BA Synthesis One of the key factors involved in BA production is the ph of the medium. Although Gardini et al. (2001) indicate that rapid acidification can lead to reduced BA production via a reduction in the growth of contaminant Gram negative decarboxylating microorganisms, other authors report a relationship between an acidic ph and an increase in BA synthesis (Marcobal et al., 2006c; Fernández et al., 2007b). It has been shown that acidic environments favor tyramine production. Earlier synthesis and higher final concentrations of tyramine were obtained when an Enterococcus durans BA-producing strain isolated from cheese was grown at ph 5.0 compared to neutral ph conditions (Fernández et al., 2007b). Decarboxylase enzymes have an optimum ph around 5.0 (Moreno-Arribas et al., 2001) and in the case of histidine decarboxylase, acidic ph s induce structural changes necessary for its activity (Schelp et al., 2001). Previous results of our group indicate that an increase in production is related to the induced expression of decarboxylases and transporter genes (Linares et al., 2009). Temperature control has been suggested to be the most efficient way to prevent BA formation (Stratton et al., 1991). Santos et al. (2003) observed a reduction in BA formation in cheeses made at lower incubation temperatures (20 C instead of 32 C). The addition of NaCl to the milk can also prevent BA formation. However, it is important to note that it is not always possible to make such modifications to the cheese manufacturing process without causing changes in texture, flavor, or quality. BIOCHEMISTRY The presence of BA depends on the activity of at least two proteins, a decarboxylase, the enzyme that catalyzes the decarboxylation of the substrate amino acid, and a transporter responsible for amino acid-ba interchange (Fig. 1). Decarboxylases Decarboxylases (EC ) belong to the pyridoxal-pdependent enzyme group, whose members use pyridoxal-5 - phosphate (PLP) as a coenzyme. Histidine decarboxylases (HDC) (EC ) fall into two different classes those from eukaryotic cells and Gram-negative bacteria, which require pyridoxal phosphate as a cofactor, and those from Gram-positive bacteria, which use a covalently bound pyruvoyl moiety as the prosthetic group (van Poelje and Snell, 1990). HDC from Lactobacillus 30A has been well characterized. This enzyme is synthesized in an inactive form called the π chain, and these π chains activate themselves via cleavage at a Ser residue. The N- terminal fragment of the π chain is known as the β-chain, and the larger C-terminal fragment as the α-chain. In the active enzyme, the N-terminal Ser of the α-chain is converted to a pyruvoyl moiety, which serves as the prosthetic group for the decarboxylation reaction (Recsei et al., 1983; Recsei and Snell, 1985). The X-ray diffraction structure of the HDC complex of Lactobacillus 30A shows that HDC forms a cup-shaped trimmer with a deep, central cavity containing three active sites (Hackert et al., 1981; Parks et al., 1985). HDC is active at low ph and inactive at neutral to alkaline and neutral ph induces structural changes at the substrate binding site that greatly reduce activity (Schelp et al., 2001). Tyrosine decarboxylase (TDC) enzymes (EC ) were initially characterized in eukaryotic organims where the same enzyme catalyzes tyrosine and L-dopa decarboxylation. In prokaryotic microorganims, this enzyme has only been characterized in two strains of the LAB group, Enterococcus faecalis (Børrensen et al., 1989) and Lactobacillus brevis IOEB 9809 (Moreno-Arribas et al., 2001). The E. faecalis enzyme can decarboxylate tyrosine and L-dopa whereas Lb. brevis TDC is specific for tyrosine. The structure of these characterized enzymes is different; that of a E. faecalis is a heterodimer with 74.5 and 76 kd subunits, while that of Lb. brevis isa70kd homodimer. TDC enzymes are also able to use phenylalanine as a substrate to produce β-phenylethylamine; this enzyme is therefore also responsible for the β-phenylethylamine content detected in some dairy products. This dual activity has recently been confirmed with the expression of TDC enzyme from Enterococcus faecium RM58 in E. coli, which showed phenylalanine and tyrosine decarboxylase activities (Marcobal et al., 2006a).

6 BIOGENIC AMINES IN DAIRY PRODUCTS 695 Figure 1 Biogenic amine biosynthesis pathways in lactic acid bacteria. Arginine decarboxylase (ADC), agmatine deiminase (AGD), arginine deiminase (AD), histidine decarboxylase (HDC), lysine decarboxylase (LDC), tyrosine decarboxylase (TDC), ornithine decarboxylase (ODC), carbamate kinase (CK), and putrescine carbamoyl transferase (PTC). Tryptophan decarboxylase activity (EC ) has been determined in some strains of Lactobacillus (Gao et al., 1997; Gonzalez de Llano et al., 1998) and tryptamine detected in some cheese samples (Stratton et al., 1991). However, no data are available on this enzyme. The arginine, lysine, and ornithine decarboxylase enzymes have been the most extensively studied decarboxylases. These can be divided into two groups: Degradative. These are induced by different conditions such as low oxygen tension, high acidity, and high concentrations of the respective substrate amino acids. Their activity seems to play a role in ph homeostasis (Meng and Bennett, 1992). Biosynthetic. These are constitutively produced at low levels in normal culture conditions and their role appears to be the catalysis of the first steps of polyamine synthesis needed for optimal ribosome function and growth (Tabor and Tabor, 1985). In LAB, only the degradative group has been described in strains of the Lactobacillus and Oenococcus genera. Putrescine can be formed from ornithine by ornithine decarboxylase (ODC) (EC ). This activity has been described in Lactobacillus 30A (Hackert et al., 1994) and Oenococcus oeni BIFI-83 (Marcobal et al., 2004). The three-dimensional structure of ODC from Lactobacillus 30A is a dodecamer assembled from six homodimers with 731 amino acid residues per subunit (Momany et al., 1995). The homodimers have an ellipsoid core and two protruding wing domains, one from each monomer, pointing into the central cavity region. Since casein does not contain ornithine, in the case of dairy products putrescine has to be formed from arginine. In a

7 696 D. M. LINARES ET AL. first step, arginine is decarboxylated by arginine decarboxylase (ADC) (EC ) (Fig. 1) to produce agmatine. Arginine decarboxylase activity (Fig. 1) has been reported in Lactobacillus hilgardii X1B (Arena et al., 2001) but no more data are available regarding these enzymes in LAB. Agmatine can be converted to putrescine by the agmatine deiminase system which is formed by three enzymes: agmatine deiminase (AgDI) (EC ), putrescine carbamoyltransferase (PTC) (EC ), and carbamate kinase (CK) (E.C ). This pathway was initially described in E. faecalis (Simon and Stalon, 1982). The genes encoding these enzymes have been identified in different LAB and in some have been linked to the tyrosine decarboxylase cluster (Lucas et al., 2007). The lysine decarboxylase (LDC) (EC ) activity involved in lysine conversion to cadaverine has not been characterized in LAB, and it has been mostly associated with Gram negative bacteria. However, the completely sequenced genomes of Streptococcus pneumoniae strains R6 and TIGR4 harbor genes homologous to those encoding the LDC enzyme (cad and SP0916 respectively) (Hoskins et al., 2001; Tettelin et al., 2001). The Transporter Proteins The second component in the decarboxylation process is an inner membrane antiporter used to deliver the amino acid substrate into the cell and to remove (excrete) the decarboxylated product from the cytoplasm. The proteins involved in the amino acid/amine exchange belong to the amino acid/polyamine/organocation (APC) superfamily [TC (transporter classification) no. 2.A.3-] (Jack et al., 2000). These proteins show a uniform topology with α-helical transmembrane spanners (TMSs) as a single polypeptide, the size of which varies from 400 to 800 aminoacyl residues (Sophianopoulou and Diallinas, 1995). The histidine/histamine antiporter, HdcP, has been studied in the LAB Lb. hilgardii 006 and Streptococcus thermophilus (Molenaar et al., 1993; Lucas et al., 2005; Trip et al., 2011). Lucas et al. (2005) demonstrated that the uptake of substrate and excretion of products are coupled events. The tyrosine/tyramine transport mechanism has been characterized in Lb. brevis IOEB 9809 and Sporolactobacillus sp. P3J (Wolken et al., 2006; Coton et al., 2011). The transporter protein, TyrP, catalyzes homologous tyrosine-tyrosine exchange and heterologous exchange between tyrosine and tyramine. In addition to this electrogenic exchange, the transporter catalyzes tyrosine uniport but at a much lower rate (Wolken et al., 2006). The putrescine and cadaverine transport proteins (PotE and CadB respectively) have been well characterized in E. coli. PotE and CadB can function not only as a putrescine/ornithine and lysine/cadaverine antiporter respectively to excrete putrescine and cadaverine, but also as putrescine and cadaverine uptake proteins (Kashiwagi et al., 1997; Soksawatmaekhin et al., 2004). While the polyamine uptake activity depends on the proton motive force, the polyamine excretion activity acts as a polyamine/amino acid antiporter. At neutral ph, CadB is involved in cadaverine uptake; at acidic ph, CadB functions as the cadaverine/lysine antiporter. Lb. brevis agmatine/putrescine transport has recently been characterized (Lucas et al., 2007). Functionally, this system resembles the arginine/ornithine antiporter induced in cells grown in the presence of agmatine (Driessen et al., 1988). GENETICS Genetic Organization BA formation requires the expression of at least two genes, one encoding the decarboxylase and the other the transporter involved in amino acid/amine interchange. These genes are always linked and in many cases organized into a cluster with a third gene involved in regulation. Figure 2 shows the genetic organization of the clusters from different microorganisms involved in BA formation in dairy products. The hdc gene has been described in different LAB of diverse origin: O. oeni, Lactobacillus hilgardii, Lactobacillus sakei, Lactobacillus reuteri, Tetragenococcus muriaticus, Tetragenococcus halophilus, Lactobacillus saerimneri 30A, Leuconostc oeni, Lactobacillus buchneri and Streptococcus thermophilus (access numbers: AJ831547, AY800122, AB040487, AB076394, J02613, U09485, AJ749838, AY651779, ABQ and FN ). The comparison of the deduced amino acid sequences, including those for the hdc genes of Gram negative bacteria, revealed two different groups that reflected different modes of action: PLP-dependent enzymes from Gram negative bacteria, and pyruvoyl-dependent enzymes from Gram positive bacteria (Fig. 3). The flanking regions of hdc have been characterized in Lb. buchneri, Lb saerimneri 30A, O. oeni, and Lb. hilgardii. In these LAB the hdca gene is co-transcribed with hdcb (Fig. 2), involved in hdca cleavage and maturation (Trip et al., 2011). The gene encoding the transporter and a fourth gene encoding a protein similar to the histidyl t-rna synthetases are also part of these clusters. In most of the histamine producer strains, the histamine cluster is located on the bacterial chromosome, while in Lb. hilgardii and O. oeni strains, isolated from wine (Lucas et al., 2005; 2008), the cluster is located on an unstable plasmid. The latter authors suggest that the histamine-producers T. muriaticus and O. oeni 9204 harbor the same plasmid. The tdc gene has been characterized in E. faecalis (Connil et al., 2002), Lb. brevis (Lucas et al., 2003), E. durans (Fernández et al., 2004), St. thermophilus (La Gioia et al., 2011) and Sporolactobacillus sp. P3J (Coton et al., 2011). It has also been annotated in the genome sequence of E. faecalis V583 (Paulsen et al., 2003), E. faecium DO (httpp/genome.jgipsf.org/mic home.html), and Lb. brevis ATCC 367 (Makarova et al., 2006), and a partial or total tdc gene has been identified in Lactobacillus fermentum, Lb. brevis IOEB 9809, E. faecalis

8 BIOGENIC AMINES IN DAIRY PRODUCTS 697 Figure 2 Cluster organization of decarboxylase pathways. Tyrosine decarboxylase (tdc), histidine decarboxylase (hdc), lysine decarboxylase (cdc), and ornithine decarboxylase (odc). Figure 3 Phylogenetic three for amino acid decarboxylases. Tyrosine decarboxylase (Tdc), histidine decarboxylase (Hdc), lysine decarboxylase (Cdc), and ornithine decarboxylase (Odc). Protein sequences were aligned using the Clustal W programme, and the phylogenetic tree constructed using the TreeTrop routine of GeneBee. Boostrap confidence intervals are shown.

9 698 D. M. LINARES ET AL. JH2 2, E. faecium RM58, Lactobacillus plantarum, Enterococcus hirae, Carnobacterium divergens IO8, T. halophilus, and Lb. curvatus HSCC 1736, CTC 6677, CTC 6513, HSCC 1737 (access numbers: CAH04395, ABQ53170, ABM55261, BAD93616, CAI39170, CAI39169, BAE02560, BAE02559, ABC68277, and AAQ73505). All the latter have different food origins (wine, dairy, and meat) except for E. faecalis V583, which has a clinical origin. The same genetic organization has been found in all the available sequences: a gene encoding a protein that shows similarity with tyrosil t-rna synthetases (tyrs), tdc, the gene encoding the transporter (tyrp), and a gene encoding a Na + /H + antiporter (Fig. 2). The deduced amino acid sequences of tdc from the different LAB strains show great similarity. Indeed, the phylogenetic tree shows the degree of these similarities to be little different to species-related similarity (Fig. 3). The ornithine decarboxylase (odc) cluster is the most simple decarboxylase cluster since it involves only the decarboxylase (spef) and the transporter gene (pote). In LAB this gene cluster has been identified in Lactobacillus 30A (AA64830) and O. oeni (Marcobal et al., 2004). Genome analysis projects have also identified this cluster in Lactobacillus gasseri, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus jhonsonii, Lactobacillus herlveticus, and Lactobacillus delbrueckii ATCC and BAA 365 (Access numbers: NP965822, YP535038, YP , ABJ61155, YP618646, and ABJ59110). The lysine decarboxylase (cdc) cluster in E. coli has been much studied, and is formed by the decarboxylase cada and transporter gene cadb, together with a third gene encoding for a regulatory protein (cadc) (Fig. 2) (Meng and Bennett, 1992). Although BA are mainly produced by decarboxylation reactions, pustrescine can be synthesized from agmatine by the agmatine deiminase pathway. Agmatine deiminase and arginine deiminase are analogous pathways with similar genetic organizations, while the arginine deiminase pathway is widely distributed in LAB. A number of LAB that produce putrescine by the agmatine deiminase pathway have been detected (Lucas et al., 2007). Beside this, Enterococcus faecalis putrescine producer strains have been described (Llácer et al., 2006); and our group has recently identified several putrescine producer Lactococcus lactic strains (unpublished results). Four genes encoding an agmatine deiminase (AgDI), a putrescine transcarbamylase, a carbamate kinase, and an amino acid transporter take part in this pathway. Transcription of this operon is under the control of the AguR regulator encoded by agur. Given the similarity between the arginine and agmatine pathways it might be hypothesized that the AgDI pathway arose from a duplication of the ADI pathway, followed by changes in the specificities of the encoded enzymes. However, recent results suggest that the enzymes of the AgDI pathway evolved independently (Llácer et al., 2006), as shown by the specificity of the exchanger proteins. BA production has been assumed as a strain characteristic and the same genetic organization of the gene clusters in the different strains suggests horizontal gene transfer has occurred. Thus, BA production may be acquired as a mechanism to improve survival under stress conditions (Marcobal et al., 2006b; Lucas et al., 2007). Lucas et al. (2007) also demonstrated that the tdc and agdi clusters in Lactobacillus strains are linked, further suggesting that this region of the chromosome represents a genomic island involved in acid stress resistance and/or energy generation that is transferred horizontally between different organisms. Regulation of Decarboxylation Gene Clusters Since aminoacyl decarboxylation has been described as an adaptation to acidic conditions, the ph and amino acid substrate concentrations have been the main factors analyzed in studies on decarboxylase gene expression. The requirement of an amino acid substrate for the reaction is evident, and the induction of hdca by histidine (the amino acid substrate) has been demonstrated (Copeland et al., 1989; Martín et al., 2005; Calles Enríquer et al., 2011). As described for other decarboxylase genes, the decarboxylase gene of the tdc cluster of E. faecalis, E. durans, and Lb. brevis is co-transcribed with the gene encoding the transporter protein. Transcriptional studies of the tdc cluster in E. durans 655 have shown that tyramine biosynthesis is regulated by extracellular ph. The tyrosine concentration and the quantification of expression of these genes during the log phase of growth are induced by high concentrations of tyrosine, but only under acidic conditions (Linares et al., 2009). The expression of the ornithine decarboxylase gene (spef), which catalyzes the conversion of ornithine into putrescine, is induced by ph and ornithine in E. coli (Kashiwagi et al., 1994). In this case no gene encoding a regulator protein has been found as a part of the cluster. RNase III increases the translational efficiency of this gene by cutting the 5 un-translated region of the mrna, although it remains to be seen whether RNase III is activated at acidic ph or not. The regulation of the cad operon has been much studied in E. coli. The decarboxylase and transporter genes (cadb and cada) are transcribed as a single transcriptional unit induced by ph and lysine. This requires the expression of an activator protein (CadC), which directly binds to the cadba promoter (Neely and Olson, 1996; Rhee et al., 2005). In E. coli, CadC also represses the expression of lysp (originally named cadr), which negatively regulates cadba expression (Neely and Olson, 1996). At present, no data are available regarding the AgDI regulation pathway, although Arena et al. (2008) have reported the influence of ph, amino acids, and sugar concentration on pustrescine formation. DETECTION METHODS An important area of applied research on BA is the development of methods for the detection of these compounds in foods.

10 BIOGENIC AMINES IN DAIRY PRODUCTS 699 These methods can be divided into two groups: those based on the detection of BA themselves, and those based on the detection of the producer microorganisms. The detection of BA has improved alongside chromatography. Early techniques for the determination of BA in foods were based on thin-layer chromatography. However, more modern analytical techniques that allow the acquisition of reliable quantitative data and the better separation/resolution of various amines now exist. The quantitative determination of BA in foods is generally accomplished by overpressure-layer chromatography, high-performance liquid chromatography (HPLC), and gas chromatography (Rogers and Staruszkiewicz, 1997). UV, electrochemical, and fluorescence techniques BA-detection techniques, in combination with pre- and post- column derivatization with o-phthalaldehyde (OPT), fluorescamine, or dansyl chloride, are also available (for a review, see Önal, 2007). Other methods based on capillary electrophoresis have also been developed and their sensitivity, reproducibility, linear range, accuracy, and efficiency all optimized (for a review, see Oguri, 2000). Several biochemical assays for certain BA, for example, histamine, have also been described (López- Sabater et al., 1994). However, cheese contains high proportions of fat and protein which hinder the analytical process; there is, therefore, still room for improvements in analytical efficiency. With respect to the detection of producer microorganisms, screening methods were initially based on the use of differential media containing a ph indicator to identify the BA-producer strains (Maijala, 1993; Bover-Cid and Holzapfel, 1999). However, these methods require the isolation and growth of the producer microorganism, and are therefore laborious and time-consuming. The characterization of the genes encoding the decarboxylating enzymes led to the development of new methods based on PCR. A relationship between the presence of the gene encoding the decarboxylase and the capacity to synthesize BA has been shown by several authors (Lucas et al., 2005; Landete et al., 2005; Fernández et al., 2004). A strain carrying aminoacyl-decarboxylase genes is a potential BA producer and its presence should be avoided in food. Therefore, these genes are good candidates for the design of specific primers for detecting BA-producer strains by PCR; in fact, numerous studies have been undertaken in this area (for a review, see Landete et al., 2007). Several sets of primers have been developed for the detection of tyramine-producing LAB (Lucas and Lonvaud-Funel, 2002; Fernández et al., 2004; Coton and Coton, 2005). PCR has successfully been used with milk curd and cheese samples in this respect (Fernández et al., 2004), and has been also used for the detection of tyramine-producing bacteria during cheese manufacture (Fernández et al., 2006a). An important benefit of this technique is the detection of producer microorganisms before BA themselves are detected in the sample; such detection can help predict the likely accumulation of BA in the final product. This method can be used at any stage of cheese manufacture and could be a useful tool for dairy product companies. The method was also used on marketed cheese samples; a good correlation was seen between PCR and HPLC results (Fernández et al., 2007a). Three sets of primers have been developed for the detection of Gram positive histamine-producing bacteria (Le Jeune et al.,1995; Landete et al., 2005; Coton and Coton, 2005), and two sets of primers have been proposed for the detection of Gram negative histamine producing bacteria (Takahashi et al., 2003; de las Rivas et al., 2006). In addition, several sets of primers have been proposed for the detection of cadaverineand pustrescine-producing strains (Marcobal et al., 2005; de las Rivas et al., 2006). A multiplex PCR method for the simultaneous detection of histamine-, tyramine-, and pustrescine-producing LAB has also been proposed (Marcobal et al., 2005; Coton and Coton, 2005) although this method has not yet been used with dairy samples. Although sensitive and specific under optimized conditions, conventional PCR has two drawbacks: the need to analyze the data by traditional endpoint analysis, and the impossibility of template quantification. Real time quantitative PCR (q-pcr) offers a potential alternative. This allows continuous monitoring of the PCR amplification process and, under appropriate conditions, the quantification of the target DNA present in the sample. A new set of primers has been designed for the detection of histamine-producing LAB by q-pcr (Fernández et al., 2006b; Lucas et al., 2008). In two hours this method can determine the histamine-producing capacity of a strain, and it has been successfully used in the different steps of cheese manufacture and on studies of the final product (Ladero et al., 2008). Low C T values (which correspond to large numbers of histamineproducing LAB) in the early stages of manufacture correlated well with high concentrations of histamine in mature cheese samples. These PCR methods are useful not only for the early detection of BA producers, but also for the characterization of LAB selected as starters. REDUCING THE BA CONTENT OF DAIRY PRODUCTS Current knowledge on BA-producing microorganisms and the conditions required for BA production now allow the conditions required to prevent BA accumulation in dairy products to be better defined. Since the presence of BA depends on the presence of decarboxylating microorganisms, and given that these microorganisms can be part of the milk microbiota, the relationship between the treatment of milk and BA content has been studied by several authors. The analysis of the BA content of different types of cheese showed these compounds to be more common in those made from raw milk (87.5% of the samples analyzed compared to 68.9% of samples from cheeses made from pasteurized milk) (Fernández et al., 2007a). The BA concentration is also generally higher in cheeses made from raw milk than pasteurized milk (Novella-Rodríguez et al., 2003a; Fernández et al., 2007a). Pasteurization is the most common

11 700 D. M. LINARES ET AL. milk treatment used during cheesemaking to reduce the numbers of pathogenic and spoilage microorganisms. Many authors conclude that pasteurization reduces the presence of BA-producing microorganisms, and therefore the BA content. Certainly, Enterobacteriaceae, and enterococci counts are 2 to 3 logarithmic units lower in cheeses made with pasteurized milk (Novella- Rodríguez et al., 2004). Since Enterobacteriaceae are the main producers of cadaverine in dairy products, Marino et al. (2000) suggest this BA in cheese might be limited by controlling the Enterobacteriaceae population. Enterococcus strains are the main producers of tyramine (the most commonly found and most abundant BA in cheese), the concentration of which is around 30 times higher in cheeses made from raw milk rather than from pasteurized milk (Novella-Rodríguez et al., 2004); this is probably explained by the marked differences in enterococci counts between such cheeses (Marino et al., 2000). Other authors relate the lower BA content of pasteurized milk cheeses with a slower rate of proteolysis and a consequently slower release of the substrate amino acids (Lau et al., 1991). The heat sensitivity of the pyridoxal phosphate cofactor necessary for decarboxylation activity has also been suggested (Ordoñez et al., 1997). Other milk treatments, such as the use of pressure, have also been investigated as a means of reducing BA contents. No differences in BA profiles were observed between cheeses made with pressurized or pasteurized milk, even though pressure can provoke greater proteolysis rendering substrates more available. This indicates that the control of BA-producing microorganisms by adequate treatment of milk is one of the most important factors for reducing BA accumulation in dairy products (Novella- Rodríguez et al., 2003b). Amino acid availability, an important factor related to BA production, is difficult to reduce in dairy products since proteolysis is essential in cheese ripening and amino acids are required for flavor development (Fernández and Zuñiga, 2006). The selection of starters without BA synthesis capability is an important means of reducing BA contents in dairy products. As mentioned above, PCR based on decarboxylase gene detection is an excellent tool in this respect. The in-depth study of the factors required for the expression of the decarboxylase- and transporter-coding genes, and the activity of their products may lead to new strategies for preventing BA accumulation. CONCLUSIONS The demand for safer foods has promoted much research into BA in recent years, but questions remain. Dairy products, especially cheese, are fermented products that can accumulate large quantities of BA. These toxic compounds have been related to different pathologies, although more research is needed to determine how these compounds affect consumers. The influence of milk quality and the treatment of milk have been analyzed and there is general agreement on the importance of these factors in reducing the presence of BA in dairy products. Further, there is no doubt regarding the importance of selecting starter strains without the genes required for BA synthesis. Other active research areas include the development of more sensitive and faster techniques to detect the presence of these compounds in food, and the development of PCR methods for the early detection and quantification of producer microorganisms. Knowledge of the metabolic pathways involved in BA production, of the mechanisms that regulate BA production, and of the physiological role of these compounds may lead to the development of strategies that prevent the synthesis and accumulation of these toxic compounds. ACKNOWLEDGMENTS The authors thank Adrian Burton for linguistic assistance. D. M. Linares was the recipient of a fellowship from the Spanish Ministry of Education and Science. V. Ladero is the beneficiary of a I3P CSIC contract financed by the European Social Fund. This research was performed with the financial support of the Ministry of Science and Innovation, Spain (AGL ), and the European Community s Seventh Framework Programme (KBBE BIAMFOOD). REFERENCES Arena, M.E., Landete, J.M., Manca de Nadra, M.C., Pardo, I., and Ferrer, S. (2008). Factors affecting the production of putrescine from agmatine by Lactobacillus hilgardii X 1 B isolated from wine. J. Appl. Microbiol. 105: Arena, M.E. and Manca de Nadra, M. C. (2001). Biogenic amine production by Lactobacillus. J. Appl. Microbiol. 90: Bardócz, S. (1999). Role of biogenic amines-summing up or what is it we do not know? In: Biogenically Active Amines in Food, Vol. III. pp 1 4. European Community, Ed. Bodmer, S., Imark, C., and Kneubühl, M. (1999). Biogenic amines in foods: Histamine and food processing. Inflam. Research. 48: Børrensen, T., Klausen, N.K., Larsen, L.M., and Sørensen, H. (1989). Purification and characterization of tyrosine decarboxylase and aromatic- L-amino acid decarboxylase. Biochim. Biophys. Acta. 993: Botazzi, V. (1993). Microbiologia lattiero-casearia. Editorial Edagricole, Bologna. Bover-Cid, S. and Holzapfel, W.H. (1999). Improved screening procedure for biogenic amine production by lactic acid bacteria. Int. J Food Microbiol. 53: Burdychova, R. and Komprda, T. (2007). Biogenic amine-forming microbial communities in cheese. FEMS Microbiol. Lett. 276: Connil, N., Breton, Y.L., Dousset, X., Auffray, Y., Rincé, A., and Prévost, H. (2002). Identification of the Enterococcus faecalis tyrosine decarboxylase operon involved in tyramine production. Appl. Environ. Microbiol. 68: Copeland, W.C., Domena, J.D., and Robertus, J.D. (1989). The molecular cloning, sequence and expression of the hdcb gene from Lactobacillus 30A. Gene. 85:

12 BIOGENIC AMINES IN DAIRY PRODUCTS 701 Coton, E. and Coton, M. (2005). Multiplex PCR for colony direct detection of Gram-positive histamine- and tyramine-producing bacteria. J. Microbiol. Methods. 63: de las Rivas, B., Marcobal, A., Carrascosa, A.V., and Muñoz, R. (2006). PCR detection of foodborne bacteria producing the biogenic amines histamine, tyramine, putrescine, and cadaverine. J. Food Prot. 69: Driessen, A.J., Smid, E.J., and Konings, W.N. (1988). Transport of diamines by Enterococcus faecalis is mediated by an agmatine-putrescine antiporter. J. Bacteriol. 170: Fernández, M., Linares, D.M., del Río, B., Ladero, V., and Alvarez, M.A. (2007a). HPLC quantification of biogenic amines in cheeses: correlation with PCR-detection of tyramine-producing microorganisms. J. Dairy Res. 74: Fernández, M., Linares, D.M., Rodríguez, A., and Alvarez, M.A. (2007b). Factors affecting tyramine production in Enterococcus durans IPLA 655. Appl. Microbiol. Biotechnol. 73: Fernández, M., Flórez, A.B., Linares, D.M., Mayo, B., and Alvarez, M.A. (2006a). Early PCR detection of tyramine-producing bacteria during cheese production J. Dairy Res. 73: Fernández, M., del Río, B., Linares, D.M., Martín, M.C., and Alvarez, M.A. (2006b). Real-time polymerase chain reaction for quantitative detection of histamine-producing bacteria: use in cheese production. J. Dairy Sci. 89: Fernández, M., Linares, D.M., and Alvarez, M.A. (2004). Sequencing of the tyrosine decarboxylase cluster of Lactococcus lactis IPLA 655 and the development of a PCR method for detecting tyrosine decarboxylating lactic acid bacteria J. Food Prot. 67: Fernández, M. and Zuñiga, M. (2006). Amino acid catabolic pathways in lactic acid bacteria. Crit. Rev. Microbiol. 32: Fernández-García, E., Tomillo, J., and Nuñez, M. (2000) Formation of biogenic amines in raw milk Hispanico cheese manufactured with proteinases and different levels of starter culture. J. Food Prot. 63: Galgano, F., Suzzi, G., Favati, F., Caruso, M., Martuscelli, M., Gardini, F., and Salzano, G. (2001). Biogenic amines during ripening in Semicotto Caprino cheese: Role of enterococci. Int. J. Food Sci. Technol. 36: Gao, S., Oh, D.H, Broadbent, J.R., Johnson, M.E., Weimer, B.C., and Steele, J.L. (1997). Aromatic amino acid catabolism by lactococci. Lait. 77: Gardini, F., Tofalo, R., Belletti, N., Iucci, L., Suzzi, G., Torriani, S., Guerzoni, M.E., and Lanciotti, R. (2006). Characterization of yeasts involved in the ripening of Pecorino Crotonese cheese. Food Microbiol. 23: Gardini, F., Martuscelli, M., Caruso, M.C., Galgano, F., Crudele, M.A., Favati, F., Guerzoni, M.E., and. Suzzi, G. (2001). Effects of ph, temperature and NaCl concentration on the growth kinetics, proteolytic activity and biogenic amine production of Enterococcus faecalsi. Int. J. Food. Microbiol. 64: González de Llano, D., Cuesta, P., and Rodríguez, A. (1998). Biogenic amine production by wild lactococcal and leuconostoc strains. Lett. Appl. Microbiol. 26: Hackert, M.L., Carroll, D.W., Davidson, L., Kim, S.O., Momany, C., Vaaler, G.L., and Zhang, L. (1994). Sequence of ornithine decarboxylase from Lactobacillus sp. strain 30a. J. Bacteriol. 176: Hackert, M.L., Meador, W.E., Oliver, R.M., Salmon, J.B., Recsei, P.A., and Snell, E.E. (1981). Crystallization and subunit structure of histidinedecarboxylase from Lactobacillus-30A. J. Biol. Chem. 256: Hoskins, J., Alborn, W.E., Arnold, J., et al. (2001). Genome of the bacterium Streptococcus pneumoniae strain R6. J. Bacteriol. 183: Halász, A., Baráth, A., Simon-Sarkadi, L., and Holzapfel, W.H. (1994). Biogenic amines and their production by microorganisms in food. Trends Food Sci. Technol. 5: Igarashi, K., Ito, K., and Kashiwagi, K. (2001). Polyamine uptake systems in Escherichia coli. Res. Microbiol. 152: Innocente, N. and D Agostin, P. (2002). Formation of biogenic amines in a typical semihard Italian cheese. J. Food Prot. 65: Jack, D.L., Paulsen, I.T., and Saier, M.H. (2000). The amino acid/polyamine/ organocation (APC) superfamily of transporters specific for amino acids, polyamines and organocations. Microbiol. 146: Joosten, H.M.L.J. and Northolt, M.D. (1987). Conditions allowing the formation of biogenic amines in cheese 2. Decarboxylative properties of some non starter bacteria. Neth. Milk Dairy J. 41: Kashiwagi, K., Shibuya, S., Tomitori, H., Kuraishi, A., and Igarashi, K. (1997). Excretion and uptake of putrescine by the PotE protein in Escherichia coli. J. Biol. Chem. 272: Kashiwagi, K., Watanabe, R., and Igarashi, K. (1994). Involvement of ribonuclease III in the enhancement of expression of the spef-pote operon encoding inducible ornithine decarboxylase and polyamine transport protein. Biochem. Biophys. Res. Commun. 15: Komprda, T., Burdychová, R., Dohnal, V., Cwiková, O., Sládková, P., and Dvorácková, H. (2008). Tyramine production in Dutch-type semi-hard cheese from two different producers. Food Microbiol. 25: Ladero, V., Linares, D.M., Fernández, M., and Alvarez, M.A. (2008). Real Time Quantitative PCR detection of histamine-producing lactic acid bacteria in cheese: relation with histamine content. Food Res Int. 41: Landete, J.M., de Las Rivas, B., Marcobal, A., and Muñoz, R. (2007). Molecular methods for the detection of biogenic amine-producing bacteria on foods. Int. J. Food Microbiol. 117: Landete, J.M., Ferrer, S., and Pardo, I. (2005). Which lactic acid bacteria are responsible for histamine production in wine? J. Appl. Microbiol. 99: Lau, K.L., Barbano, M., and Rasmussen, R.R. (1991). Influence of pasteurization of milk on protein breakdown in Cheddar cheese during aging. J. Dairy Sci. 74: Lee Y.H., Kim, B.H., Kim, J.H., Yoon, W.S. Bang, S.H., and Park, Y.K. (2007). CadC has a global translational effect during acid adaptation in Salmonella enterica serovar Typhimurium. J. Bacteriol. 189: Le Jeune, C., Lonvaud-Funel, A., Ten Brink, B., Holstra, H., and. van der Vossen, J.M.B.M. (1995). Development of a detection system for histidine decarboxylating lactic acid bacteria based on DNA probes, PCR and activity test. J. Appl. Bacteriol. 78: Lehane, L. and Olley, J. (2000). Histamine fish poisoning revisited. Int. J. Food Microbiol. 58: Leuschner, R.G.K., Kurihara, R., and Hammes, W.P. (1998). Effect of enhanced proteolysis on formation of biogenic amines by lactobacilli during Gouda cheese ripening. Int. J. Food Microbiol. 44: Lácer, J.L., Polo, L.M., Tavarez, S., Alarcón, B., Hilario, R., and Rubio, V. (2006). The gene cluster for agmatine catabolism of Enterococcus faecalis. Studies of recombinant putrescine transcarbamylase and agmatine deiminase catalyzing this reaction. J. Bacteriol. 189: López-Sabater, E.I., Rodríguez-Jerez, J.J., Hernández-Herrero, M., and Mora- Ventura, M.T. (1994). Evaluation of histidine decarboxylase activity of bacteria isolated from sardine (Sardina pilchardus) by an enzymatic method. Lett. Appl. Microbiol. 19: Lucas, P.M., Claisse, O., and Lonvaud-Funel, A. (2008). High frequency of histamine-producing bacteria in the enological environment and instability of the histidine decarboxylase production phenotype. Appl. Environ. Microbiol. 74: Lucas, P.M., Blancato, V.S., Claisse, O., Magni, C., Lolkema, J.S., and Lonvaud- Funel, A. (2007). Agmatine deiminase pathway genes in Lactobacillus brevis are linked to the tyrosine decarboxylation operon in a putative acid resistance locus. Microbiology 153: Lucas, P.M., Wolken, W.A., Claisse, O., Lolkema, J.S., and Lonvaud-Funel, A. (2005). Histamine-producing pathway encoded on an unstable plasmid in Lactobacillus hilgardii 0006 Appl. Environ. Microbiol. 71: Lucas, P.M., Landete, J., Coton, M., Coton, E., and Lonvaud-Funel, A. (2003). The tyrosine decarboxylase operon of Lactobacillus brevis IOEB 9809: Characterization and conservation in tyramine producing bacteria. FEMS Microbiol. Lett. 229: Lucas, P.M. and Lonvaud-Funel, A. (2002). Purification and partial gene sequence of the tyrosine decarboxylase of Lactobacillus brevis IOEB FEMS Microbiol. Lett. 211: Lyte, M. (2004). The biogenic amine tyramine modulates the adherence of Escherichia coli O157:H7 to intestinal mucosa. J. Food Prot. 67: