View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Digital.CSIC 1 2 PCR methods for the detection of biogenic amine-producing bacteria on wine 3 4 5 6 7 José María Landete, Blanca de las Rivas, Angela Marcobal and Rosario Muñoz* 8 9 10 11 12 13 Departamento de Microbiología, Instituto de Fermentaciones Industriales, CSIC, Juan de la 14 Cierva 3, 28006 Madrid 15 16 17 18 19 *Corresponding author. Tel.: +34-91-5622900; fax: +34-91-5644853 20 E-mail address: [email protected] (R. Muñoz) 21 1 22 Abstract 23 24 Biogenic amines are low molecular weight organic bases frequently found in wine. 25 Several toxicological problems resulting from the ingestion of wine containing biogenic 26 amines have been described. Histamine, tyramine, phenylethylamine and putrescine are 27 mainly produced in wine by the decarboxylation of histidine, tyrosine, phenylalanine and 28 ornithine or arginine respectively by lactic acid bacteria action. Since the ability of 29 microorganisms to decarboxylate amino acid is highly variable, being in most cases strain- 30 specific, the detection of bacteria possessing amino acid decarboxylase activity is important 31 to estimate the risk of biogenic amine content and to prevent biogenic amine accumulation 32 in wine. Molecular methods for the early and rapid detection of these producer bacteria are 33 becoming an alternative to traditional culture methods. PCR methods offer the advantages 34 of speed, sensitivity, simplicity and specific detection of amino acid decarboxylase genes. 35 Moreover, these molecular methods detect potential biogenic amine risk formation in wine 36 before the amine is produced. Methods using quantitative PCR are efficient to enumerate 37 biogenic amines-producing lactic acid bacteria in wine. The aim of the present review is to 38 give a complete overview of the molecular methods proposed in the literature for the 39 detection of biogenic amine-producing bacteria in wine. The methods can help to better 40 control and to improve winemaking conditions in order to avoid biogenic amine 41 production. 42 43 Keywords: wine, histamine; tyramine; phenylethylamine, putrescine; PCR methods, Real 44 Time Quantitative PCR. 45 2 46 INTRODUCTION 47 BIOGENIC AMINE PRODUCING MICROORGANISMS IN WINE 48 Histamine-producing lactic acid bacteria in wine 49 Tyramine and phenylethylamine-producing lactic acid bacteria in wine 50 Putrescine producing lactic acid bacteria in wine 51 DETECTION OF BIOGENIC AMINE PRODUCING BACTERIA IN WINE 52 Detection of histamine-producing bacteria by PCR 53 Detection of phenylethylamine and tyramine-producing bacteria by PCR 54 3.3. Detection of putrescine-producing bacteria by PCR 55 Simultaneous detection of biogenic amine-producing bacteria by PCR 56 DETECTION OF LACTIC ACID BACTERIA PRODUCING BIOGENIC AMINES 57 IN WINE BY REAL TIME QUANTITAVE PCR 58 Detection of lactic acid bacteria carrying hdc gene by QPCR 59 Detection of lactic acid bacteria carrying tdc gene by QPCR 60 Detection of lactic acid bacteria carrying odc and/or agdi gene by QPCR 61 CONCLUSIONS 62 3 63 INTRODUCTION 64 65 Biogenic amines are organic bases endowed with biological activity that are 66 frequently found in wine. They are produced mainly as a consequence of the 67 decarboxylation of amino acids. Twenty-five different biogenic amines have been found in 68 wines, being the putrescine the most abundant (Soufleros et al., 1998). 69 High concentrations of biogenic amines can cause undesirable physiological effects 70 in sensitive humans, especially when alcohol and acetaldehyde are present (Bauza et al., 71 1995; Maynard and Schenker, 1996). More specifically, histamine is known to cause 72 headaches, low blood pressure, heart palpitations, edema, vomiting, and diarrhea (Bauza et 73 al., 1995; Lehtonen, 1996). Tyramine and phenylethylamine can produce hypertension 74 through the release of noradrenaline and norephedrine, respectively, which are 75 vasoconstrictor substances (Forsythe and Redmond, 1974). Putrescine and cadaverine, 76 although not toxic themselves, aggravate the adverse effects of histamine, tyramine, and 77 phenylethylamine, as they interfere with the enzymes that metabolize them (ten Brink et al., 78 1990; Straub et al., 1995). Some amines, such as putrescine, may already be present in 79 grapes (Broquedis et al., 1989), whereas others can be formed and accumulated during 80 winemaking. The main factors affecting its formation during vinification are free amino 81 acid concentrations and the presence of microorganisms able to decarboxylate these amino 82 acids. Amino acid concentration in grapes can be affected by fertilization treatments 83 (Broquedis et al., 1989) and in wines by winemaking treatments, such as time of 84 maceration with skins, addition of nutrients, and racking protocols (Rivas-Gonzalo et al., 85 1983; Zee et al., 1983; Vidal-Carou et al., 1990; Radler and Fäth, 1991 Lonvaud-Funel and 86 Joyeux, 1994). The concentration of biogenic amines in wines depends on the presence and 4 87 the concentration of microorganisms with decarboxylase activity (Rivas-Gonzalo, et al., 88 1983; Radler and Fäth, 1991; Vidal-Carou et al., 1990; Zee et al., 1993; Moreno-Arribas et 89 al., 2000) in addition to the precursors. The concentration of microorganisms is affected by 90 physicochemical factors of wine such as pH, temperature, or SO addition (Britz, et al., 2 91 1990; Baucom, et al., 1996). 92 Biogenic amine content in wines may be regulated in the future following the newly 93 implemented regulations by the U.S. Food and Drug Administration (FDA) for scombroid 94 fish (FDA). Upper limits for histamine in wine have been recommended in Germany (2 95 mg/L), Belgium (5-6 mg/L), and France (8 mg/ L) (Lehtonen, 1996). Switzerland has 96 established a limit of 10 mg/L as a tolerable value for histamine in wine (Les autorités 97 fédérales de la Confédération Suisse, 2002). 98 99 100 BIOGENIC AMINE PRODUCING MICROORGANISMS IN WINE 101 102 Many authors had implicated yeast and lactic acid bacteria as responsible for the 103 formation of amines in wine (Zee et al., 1983; Ough et al., 1987; Vidal-Carou et al., 1990; 104 Radler and Fäth, 1991; Baucom et al., 1996). However, data were complex and 105 contradictory, which suggested that more defined studies were necessary to elucidate which 106 kind of microorganism is the major contributor. Several researchers have demonstrated that 107 the amine content increases with microbial growth, specifically with that of bacteria, with 108 biogenic amine content suggested as an index of quality or of poor manufacturing practices 109 (Zee et al., 1983; Ough et al., 1987; Radler and Fäth, 1991; Baucom et al., 1996). 5 110 The biogenic amine production by 155 strains of lactic acid bacteria, 40 strains of acetic 111 bacteria and 36 strains of yeast isolated from wine were analysed by Landete et al., 112 (2007a). They did not observe biogenic amine production by acetic bacteria and yeast; 113 however, Landete et al. (2007a) found production of histamine, tyramine, phenylethylamine 114 and putrescine by lactic acid bacteria. Moreover, a correlation of 100% was observed 115 between biogenic amine production in synthetic medium and wine and between activity and 116 presence of gene. With the results expose by these authors and others (Lonvaud-Funel and 117 Joyeux, 1994; Le Jeune et al., 1995; Gerrini et al., 2002; Moreno-Arribas et al., 2003; 118 Landete et al., 2005), we can consider than the lactic acid bacteria are the microorganisms 119 responsible of histamine, tyramine, phenylethylamine and putrescine production in wine. 120 The authors previously cited have showed as several wine bacterial species are capable of 121 decarboxylating one or more amino acids, the bacterial ability to decarboxylate amino acids 122 is highly variable and this ability seems to be strain-dependent rather than being related to 123 species specificity. On the other hand, we can not consider that lactic acid bacteria, yeast or 124 acetic bacteria are responsible for tryptamine and cadaverine in wine (Landete et al., 125 2007a). Therefore, in this work, we show molecular methods to detect producing lactic acid 126 bacteria of histamine, tyramine, phenylethylamine and/or putrescine. 127 128 129 Histamine-producing lactic acid bacteria in wine 130 131 Histamine is the most important amine in food-borne intoxications, due to its strong 132 biological activity (Cabanis, 1985). The study of histamine in wine is of particular interest 133 as the presence of alcohol and other amines reportedly promotes its effects by inhibiting 6 134 human detoxification systems (Chu and Bjeldanes, 1981; Sessa et al., 1984). A high 135 concentration of histamine in wine is caused by the presence of histidine decarboxylase in 136 some lactic acid bacteria (Le Jeune et al., 1995; Lonvaud-Funel, 2001). There is great 137 interest in identifying and characterizing the bacteria that are able to produce histamine in 138 wine, in order to prevent its synthesis. In wines, high levels of histamine have been related 139 to spoilage by Pediococcus (Delfini, 1989). Pediococcus can be present in wine but usually 140 in a low proportion. It has been reported that some Oenococcus oeni strains are responsible 141 for histamine accumulation in wine (Castino, 1975; Le Jeune et al., 1995; Guerrini et al., 142 2002). The bacterial population in wine is a complex mixture of different species of lactic 143 acid bacteria (Lactobacillus, Leuconostoc, Pediococcus and Oenococcus), with O. oeni as 144 the predominant species in wine during and after malolactic fermentation. 145 Landete et al. (2005b) showed an increase in histamine during the malolactic fermentation; 146 As the histamine concentrations found in must are very low or non- existent (Landete et al., 147 2005b). So, it is normal that the concentrations of histamine must be attributed to strains of 148 lactic acid bacteria. Landete et al. (2005a) show that O. oeni, Lb. hilgardii, Lb. mali, L. 149 mesenteroides and P. parvulus can contribute to the histamine synthesis in wine, but the 150 main species responsible of high histamine production in wines seem to be Lb. hilgardii 151 and P. parvulus. Landete et al. (2005a) demonstrate in this work that histamine-producing 152 strains of O. oeni are very frequent in wine, in contrast with the paper of Moreno-Arribas et 153 al., (2003), where no Oenococcus histamine producer strains were detected. However, the 154 work of Landete et al. (2005a) agrees with Guerrini et al. (2002) who found a high number 155 of Oenococcus histamine producers in wine, but low levels of histamine production in 156 general. Histamine-producing strains of Lactobacillus, Pediococcus and Leuconostoc are 157 also detected, but with lower frequencies. The results showed by Landete et al. (2005a) do 7 158 not disagree with the most common idea that Pediococcus spp. (Delfini, 1989) is the main 159 organism responsible for histamine production, because although the percentage of 160 Pediococcus histamine producers is low, some strains can produce the highest 161 concentration of histamine. In addition, Lb. hilgardii is also capable of producing high 162 levels of histamine. 163 More recently, a histamine producing strain (Lactobacillus hilgardii IOEB 0006) proved to 164 retain or to lose the ability to produce histamine, depending on the culture conditions 165 (Lucas et al., 2005; 2008). Indeed, it was demonstrated that the hdcA gene in this strain was 166 located on an unstable 80-kb plasmid, suggesting an acceptable cause for the great 167 variability of histamine producing character among lactic acid bacteria. 168 169 170 Tyramine and phenylethylamine-producing lactic acid bacteria in wine 171 172 Tyrosine decarboxylase (TDC, EC 4.1.1.25) converts the amino acid tyrosine to the 173 biogenic amine tyramine. Bacterial tyrosine decarboxylase have been only thoroughly 174 studied and characterized in Gram-positive bacteria and, especially, in lactic acid bacteria 175 implicated in food fermentation as cheese or wine. 176 The study of phenylethylamine production has received less attention, it have been 177 demonstrated that enterococcal tyrosine decarboxylase is also able to decarboxylate 178 phenylalanine, an amino acid structurally related to tyrosine, originating the biogenic amine 179 phenylethylamine (Marcobal et al., 2006a). Some authors such as Moreno-Arribas et al. 180 (2000) and Landete et al. (2007) have demonstrated the simultaneous production of 181 tyramine and phenylethylamine in lactic acid bacteria isolated from wine. 8 182 Tyramine production is not a general trait among lactic acid bacteria. Several Lactobacillus 183 brevis tyramine-producing strains were isolated from wines (Moreno-Arribas et al., 2000) 184 and only 20 strains from 125 are showed to be tyramine producers (Landete et al., 2007). 185 This ability seems to be a general characteristic of L. brevis wine strains, however, for L. 186 hilgardii, this character is strain-dependent (Landete et al., 2007). 187 There are few reports concerning the ability of L. plantarum to produce tyramine in 188 fermented food. Arena et al. (2007) report the identification and characterization of a 189 tyramine-producing L. plantarum strain isolated from wine. These authors suggest that 190 some L. plantarum strains are able to decarboxylase tyrosine in wine. 191 192 193 Putrescine producing lactic acid bacteria in wine 194 195 Putrescine can be synthesized either directly from ornithine by ornithine 196 decarboxylase or indirectly from arginine via arginine decarboxylase. The arginine 197 decarboxylase converts arginine in agmatine, thus agmatine deiminase and N- 198 carbamoylputrescine amidohydrolase or putrescine carbamoyltransferase, biosynthetically 199 convert agmatine to putrescine in the ADI pathway. 200 O. oeni strains exhibited the capability to produce putrescine by decarboxylation of 201 ornithine (Guerrini et al., 2002). However, high concentrations of putrescine, as observed in 202 some wines after malolactic fermentation (Soufleros et al., 1998), cannot result only from 203 decarboxylation of free ornithine since its levels are usually low in wine. Indeed, ornithine 204 may also be produced by microorganisms from the degradation of arginine, as above 205 mentioned, the arginine is one of the major amino acids found in grape juice and wine. 9 206 Putrescine is the most abundant biogenic amine found in wine (Soufleros et al., 1998) and 207 agmatine is the most prevalent one in beer (Glória and Izquierdo Pulido, 1999). Arena and 208 Manca de Nadra (2001) reported that agmatine was formed as an intermediate in the 209 formation of putrescine from arginine in Lactobacillus hilgardii X1B, isolated from wine. 210 Putrescine is formed from agmatine through a pathway that does not involve amino acid 211 decarboxylase or formation of urea (Arena et al., 2001). 212 While performing malolactic fermentation, Guerrini et al. (2002) demonstrated as 213 Oenococcus oeni strains were very effective in forming putrescine from ornithine. The 214 formation of putrescine from arginine by some strains has been also demonstrated by these 215 authors. According to these authors, O. oeni can really and significantly contribute to the 216 overall biogenic amine content of wines. Marcobal et al. (2004) identified a putrescine- 217 producer O. oeni strains and sequenced its ornithine decarboxylase gene. Marcobal et al. 218 (2004) have also shown that the presence of an odc gene is a rare event in Spanish wine O. 219 oeni strains. Landete et al. (2008) did not find any microorganisms able to produce 220 putrescine; however, strains of Lb. hilgardii and the O. oeni coming on from others 221 laboratories were able to produce putrescine. Recently, Izquierdo-Cañas et al. (2009) found 222 only two strains able to produce putrescine, both on synthetic medium and wine. The 223 presence of the corresponding genes in these strains was also confirmed. According to these 224 authors, these results suggest that O. oeni does not significantly contribute to the overall 225 putrescine content of wines. 226 Broquedis et al. (1989) and Landete et al. (2005b) showed as the putrescine may be present 227 in grapes. Thus, we suggest that both, microorganisms and grapes, can be the responsible of 228 the presence of putrescine in wine. 229 10