© INRA, EDP Sciences, 2009

1. INTRODUCTION

Recent trends in food safety are directed toward an increasing search for trace compounds that adversely affect human health. The presence of biogenic amines (BAs) in foods is of public concern for the food industry and the regulatory agencies [13]. BAs represent a group of low molecular weight basic nitrogenous compounds that are mainly produced by the microbial decarboxylation of certain amino acids [14]. BAs, however, may be of endogenous origin at very low concentration in non-fermented foods such as fruits, vegetables, meat, milk and fish. Unfortunately, all fermented foods (dairy products, beer, wine, sausages, etc.) carry the risk of high concentration of BAs as a result of growth of contaminating or indigenous microflora exhibiting amino acid decarboxylase activity [28].

Histamine, tyramine, putrescine, cadaverine and phenylethylamine are the most common BAs that are found in fermented foods [14] and are mainly produced by lactic acid bacteria (LAB) [19]. Their occurrence and accumulation is influenced by the environmental factors (temperature, pH, availability of free amino acid, etc.) that affect the growth and decarboxylase activity of these organisms [9, 27]. High amounts of these BAs may negatively alter the organoleptic properties of the contaminated products, and consumption of these foods could have several toxicological effects such as respiratory distress, headache, hyper- or hypotension and allergies [14, 27]. These problems are particularly more severe in sensitive consumers having low levels of monoamine and diamino-oxidase enzymes (belonging to the BA detoxification system) [3]. Moreover, alcohol ingestion increases the undesirable effects that are produced due to the presence of BAs [22]. cis-Urocanic acid has been recognized as a mast cell degranulator and showed synergistic effect with endogenous histamine in spoiled fish [17]. More specifically, tyramine is the most studied BA and is known to cause headaches, low blood pressure, hypertension, edema, vomiting, diarrhea, etc. [14, 27]. Although there are no regulations governing the BA content in most foodstuffs, the Nutritional codex of the Slovak Republic had determined the maximal tolerable limit for the tyramine in cheese (200 mg·kg⁻¹) [13]. The presence of BA in the milk is quite low, about 1 mg·mL⁻¹, but in the cheese their content reaches about 1052 mg·kg⁻¹ [10].

The genus Enterococcus like other LAB forms an important part of food and is known to have an ambiguous relationship to human nutrition. They play a significant role in the development of typical taste, flavor and aroma of several fermented foods. Furthermore, the production of bacteriocins by enterococci (enterocins) is well documented [11]. These technological applications have led us to propose enterococci as adjunct starters or protective cultures in fermented foods. Moreover, enterococci are nowadays promoted as probiotics that have several beneficial health claims [1, 11]. However, unlike most of LAB, Enterococcus species are not having a “generally recognized as safe” (GRAS, USA) or “qualified presumption of safety” (QPS, Europe) status. This is because some of the enterococcal strains are typical opportunistic pathogens that cause disease especially in the nosocomial settings, which may in part be linked to the presence of antibiotic resistance and virulence determinants [23]. Furthermore, it has also been observed that the prolific growth of enterococci in food products leads to the formation of significant levels of tyramine [10, 18, 19].

Methods that can rapidly detect BAs producing strains in foodstuffs are required if food quality and safety is to be assured. This can help the food industry to inspect raw materials destined for use in food production [9]. Specific differential culture media for the presumptive identification of BA-producer bacteria have been developed [6, 26]. Analytical chromatographic methods used for routine BA analysis of food substrates have also been applied to bacterial cultures [20, 24]. Recently, several polymerase chain reaction (PCR)-based methods have been developed targeting specifically the amino acid decarboxylase gene. These molecular methods, in addition to their rapidity and specificity, offer the advantage of the identification of producer bacteria before the amine is synthesized [15].

As there are few reports on the relative comparison among these methods of analysis of BAs produced by enterococci, the present study aims at the evaluation of the 28 strains of Enterococcus sp. that were isolated in a previous study [12] for tyramine production by qualitative (decarboxylating medium), quantitative (high performance liquid chromatography, HPLC) and molecular methods (detection of tdc gene by PCR).

2. MATERIALS AND METHODS

2.1. Bacterial strains and growth conditions

The 28 enterococcal strains used in this study are listed in Table I. Enterococus faecalis NCDC 114 and Lactococcus lactis subsp. cremoris NCDC 61 used as positive and negative control, respectively, were procured from the National Collection of Dairy Cultures (NCDC) and National Dairy Research Institute, Karnal, India. All strains were grown in deMan, Rogosa and Sharpe (MRS) medium that was purchased from HiMedia Laboratories, Mumbai, India and incubated under aerobic conditions in a shaker incubator (Innova42, New Brunswick Scientific, New Jersey, USA) at 70 rpm at 37 °C until mid-log phase.

2.2. Molecular characterization of Enterococcus sp.

All 28 biochemically characterized enterococcal isolates [12] were subjected to molecular characterization by the PCR method for definite confirmation of species. The genomic DNA from all the cultures was extracted by the method described by Pospiech and Newmann [25]. Primer pairs EM1A (5′-TTGAGGCAGACCAGATTGACG-3′)/EM1B (5′-TATGACAGCGACTCCGATTCC-3′) [4] targeted ATP-dependent DNA helicase RecG gene and E1 (5′-ATCAAGTACAGTTAGTTCT-3′)/E2 (5′-ACGATTCAAAGCTAACTG-3′) [8] for D-ala:D-ala ligase (ddl) gene was used for the characterization of E. faecium and E. faecalis, respectively. The amplification program used was as follows: 95 °C for 5 min, then by 35 cycles at 95 °C for 45 s, 51 °C for 45 s and 45 °C for 1 min for E. faecium and E. faecalis, respectively, and 72 °C for 1 min and a final extension at 72 °C for 10 min in a thermocycler, EP Gradient (Eppendorf Mastercycler, Hamburg, Germany). The integrity of PCR products was assayed by the development of single bands following electrophoresis for 1 h at 100 V in 1.5% (w/v) agarose gels in Tris-EDTA buffer. E. faecium DSM 900 that was kindly provided by Ulrich Schillinger (Institute of Microbiology and Toxicology, Federal Research Centre for Nutrition, Karlsruhe, Germany) and E. faecalis ATCC 29212 that was kindly provided by Annalisa Serio (Dipartimento di Scienze degli Alimenti, Universita degli Studi di Teramo, Via C.R. Lerici, Mosciano Stazione TE, Italy) were used as reference strains in the PCR-based identification.

2.3. Qualitative estimation of tyramine-forming ability

The primary screening and qualitative estimation of tyrosine decarboxylation ability of the 28 enterococcal strains were done by the two different decarboxylating media, the improved medium [2], based on the color change of indicator and the modified tyramine production medium (TPM) [16]. All the enterococcal strains along with both positive and negative cultures were subcultured 5–10 times in an MRS broth containing 0.1% tyrosine and 0.005% pyridoxal-5-phosphate to promote enzyme induction before the actual screening test. After that, the strains were inoculated in the improved broth medium as well as streaked on TPM plates simultaneously and incubated for four days at 37 °C under both aerobic and anaerobic conditions in a gas jar (GasPak™ 100 system, complete, BBL Systems, USA). Strains were considered tyrosine decarboxylase positive if the color of the indicator turned yellow to violet in improved medium and a clear zone due to solubilization of tyrosine around the colonies in the case of TPM.

2.4. Detection of the tyrosine decarboxylase gene

The tdc gene detection was performed using a duplex PCR method, with two sets of primers, TD2/TD5 [7], targeted tdc gene and BSF8/BSR1541, 16S rRNA universal primers, as PCR internal control [30] (both sets of primers synthesized by Imperial Life Science, Gurgaon, India). The PCR mixture consisted of 1 ng of total DNA, primer concentrations 20 pmol for TD2 and TD5 and 5 pmol for BSF8 and BSR1541, 1 U of Taq DNA polymerase, 5 μL of 10 X Taq buffer and 1 μL dNTPs (50 pmol) in a final volume of 50 μL. The amplification program was as follows: 95 °C for 5 min, then by 35 cycles at 95 °C for 45 s, 52 °C for 45 s and 72 °C for 1 min and a final extension at 72 °C for 5 min in thermocycler, EP Gradient (Eppendorf Mastercycler, Hamburg, Germany). The presence of PCR products was assayed by electrophoresis as described in Section 2.2.

2.5. Quantitative estimation of tyramine by HPLC

Tyramine concentration produced by enterococcal strains was quantified by HPLC. All tested strains were inoculated in a TPM broth that was supplemented with 10 g·L⁻¹ of tyrosine and incubated simultaneously under both aerobic and anaerobic (in GasPak™ 100 system, complete, BBL systems, USA) conditions at 37 °C for three days in a shaker incubator to see the effect of the presence and the absence of oxygen. Sample preparation involved centrifugation of the broth, filtration through a 0.22 µm filter and derivatized with orthophthalaldehyde. Finally, the sample was injected into the port of the HPLC system (Shimadzu Corporation, Kyoto, Japan), equipped with a UV detector and an intelligent pump. HPLC separation was performed on a C₁₈ (2) column (250 × 4.60 mm, 100 Å, particle diameter 5 μm) that was purchased from Phenomenex (Macclesfield, Cheshire, UK). A gradient elution was used comprising two buffer systems, viz: (i) 0.1 mol·L⁻¹ ammonium acetate buffer, pH 7.3 (buffer A) and acetonitrile (buffer B). The eluant gradient began with 45% 0.1 mol·L⁻¹ ammonium acetate and 55% acetonitrile and ended with 10% 0.1 mol·L⁻¹ ammonium acetate and 90% acetonitrile for 11 min at 40 °C oven temperature with the flow rate of 1 mL·min⁻¹. The detection was done at the wavelength of 254 nm. Standard tyramine was purchased from HiMedia Laboratories, Mumbai, India. Regression coefficient (r) of peak area against tyramine concentration was calculated.

3. RESULTS

3.1. Screening for tyrosine decarboxylase activity

Preliminary screening of the tyrosine decarboxylase activity among the 28 strains of Enterococcus was done using the two different media, improved medium and TPM. Of the 28 strains, 22 (78%) showed positive results in improved medium indicated by a color change of medium [2]; while only 19 strains (68%) produced a clear zone (Fig. 1) on TPM indicating tyrosine decarboxylase activity [16] (Tab. I). However, with improved medium three strains (DH 28, DH 56 and RH 106) showed negative, while six strains (KH 24, KH 98, FH 99, KH 111, DH 115 and FH 133) showed positive result, which was contradictory to the results of TPM.

Figure 1. Tyrosine production medium (TPM) to detect tyrosine decarboxylating enterococcal strains. a, E. faecalis KH 12; b, E. faecium DH 28; c, E. faecium RH 31; d, E. faecium RH 33; e, E. faecalis NCDC 114 (positive control); f, L. lactis subsp. cremoris NCDC 61 (negative control); g, E. faecium RH 38; h, E. faecalis KH 62 and i, E. faecalis KH 67.

3.2. Molecular characterization of Enterococcus sp.

The results for the molecular identification of bacteriocinogenic isolates are presented in Figures 2A and 2B. Of the 28 all the 19 biochemically identified E. faecium strains were confirmed as E. faecium and similarly, all nine E. faecalis strains that were identified through biochemical tests were confirmed on molecular basis by the gene-specific PCR.

Figure 2. Molecular identification of Enterococcus sp. (A) E. faecium 1, E. faecium BFE 900 (positive control); 2, E. faecium KH 24; 3, E. faecium DH 28; 4, E. faecium RH 31; 5, E. faecium RH 33; 6, E. faecium RH 38; 7, E. faecium DH 56; 8, E. faecium KH 58; 9, E. faecium DH 59; 10, E. faecium CH 60; 11, E. faecium RH 78; 12, E. faecium KH 79; 13, E. faecium KH 81; 14, E. faecium FH 99; 15, E. faecium FH 102; 16, E. faecium RH 106; 17, E. faecium KH 110; 18, E. faecium DH 115; 19, E. faecium KH 126; 20, E. faecium FH 133 and 21, L. lactis subsp. cremoris NCDC 61 (negative control). (B) E. faecalis 1, E. faecalis ATCC 29212 (positive control); 2, E. faecalis KH 12; 3, E. faecalis KH 62; 4, E. faecalis KH 67; 5, E. faecalis KH 70; 6, E. faecalis KH 72; 7, E. faecalis KH 91; 8, E. faecalis KH 93; 9, E. faecalis KH 98; 10, E. faecalis KH 111 and 11, L. lactis subsp. cremoris NCDC 61 (negative control).

3.3. Molecular identification of the tyrosine decarboxylase gene (tdc)

Primers TD2/TD5 developed by Coton et al. [7] targeting the tdc gene allowing for the amplification of a 1100 bp fragment were used in combination with the primers BSF8/BSF1541 (PCR internal control), for the detection of the tdc gene. On PCR amplification of all the 28 enterococcal strains, 19 strains (those found positive on TPM) gave two amplicons of 1100 and 1560 bp (for tdc gene and internal control, respectively) similar to the positive control, while as expected the negative control only showed amplification of the internal control (Fig. 3). No silent tdc gene (i.e. negative in TPM and found to be positive in PCR) was observed in any of the isolates.

Figure 3. PCR-based detection of tyrosine decarboxylase (tdc) gene among different enterococcal strains tested. 1, E. faecalis KH 12; 2, E. faecium KH 24; 3, E. faecium DH 28; 4, E. faecium RH 31; 5, E. faecium RH 33; 6, E. faecium RH 38; 7, E. faecium DH 56; 8, E. faecium KH 58; 9, E. faecium DH 59; 10, E. faecium CH 60; 11, E. faecalis KH 62; 12, E. faecalis KH 67; 13, E. faecalis KH 70; 14, E. faecalis KH 72; 15, E. faecium RH 78; 16, E. faecium KH 79; 17, L. lactis subsp. cremoris NCDC 61 (negative control); 18, E. faecalis NCDC 114 (positive control); 19, E. faecalis NCDC 114 (positive control); 20, L. lactis subsp. cremoris NCDC 61 (negative control); 21, E. faecium KH 81; 22, E. faecalis KH 91; 23, E. faecalis KH 93; 24, E. faecalis KH 98; 25, E. faecium FH 99; 26, E. faecium FH 102; 27, E. faecium RH 106; 28, E. faecium KH 110; 29, E. faecalis KH 111; 30, E. faecium DH 115; 31, E. faecium KH 126 and 32, E. faecium FH 133.

3.4. Quantitative analysis of tyramine by HPLC

Quantitative estimation of the tyramine concentration in the fermenting broth under both aerobic and anaerobic incubation was done by the HPLC method. Standard tyramine was used for the standard curve preparation. Tyramine standard gave a distinct peak of good resolution in < 3 min of run time. A standard curve was plotted between the peak area and the amount of tyramine. An exact linear curve was obtained with a regression coefficient (r) of 0.991, which indicated a good linear relationship between tyramine concentration and area, hence justifying the acceptability of the gradient elution program used. Nineteen of the 28 enterococcal strains were found to be positive for the production of tyramine. These 19 strains were the same as those found positive on TPM as well as PCR amplification (giving an amplicon of 1100 bp). The levels of tyramine produced by enterococcal isolates ranged from 717 to 9063 mg·mL⁻¹ and from 512 to 8234 mg·mL⁻¹ under aerobic and anaerobic conditions, respectively (Tab. I). About 20–40% higher tyramine concentration was observed under aerobic condition as compared to anaerobic condition.

4. DISCUSSION

Tyramine is the most extensively studied BA. This is of bacterial origin in fermented foods and has severe toxicological effects on human health. In the present study, the tyramine production ability was screened among several strains of Enterococcus sp. by different methods: decarboxylating media, PCR and HPLC. Of the 28 bacteriocinogenic enterococcal strains, six strains (KH 24, KH 98, FH 99, KH 111, DH 115 and FH 133) showed positive results for tyrosine decarboxylation in the improved medium, i.e. produced a color change, but were found to be negative in TPM, PCR as well as HPLC estimation. Thus, it can be concluded that these strains showed a false positive result on improved medium. Similar, false positive results have also been reported by several workers for this medium [2, 16]. However, three enterococcal strains (DH 28, DH 56 and RH 106) showed negative results in improved medium i.e. no color change, while, they showed positive results in the case of other three methods. Similarly, a false negative result was also observed by Bover-Cid and Holzapfel [2]. This may be due to the fact that the pH change of the improved medium was too low to change the color of bromocresol purple. Significantly, in the case of TPM no false positives or false negatives were observed because there was no interference with the acidification produced by the fermentation of sugars. As a result, some amount of additional glucose or fructose may be added in TPM for the better growth of microorganisms. All the tested strains grew well in TPM because TPM can provide all the nutrients required for the growth of even fastidious LAB. Moreover, the low glucose concentration, low pH (below 5.5) [21] and the presence of pyridoxal-5-phosphate have a strong enhancing effect on the amino decarboxylase activity [29] and, therefore, an improved tyramine production on TPM plates. Thus, TPM is an easy, conventional and suitable method to screen LAB for tyrosine decarboxylase activity in laboratories lacking sophisticated equipment.

We also evaluated the usefulness of the gene-specific PCR method to detect enterococcal strains possessing tdc gene responsible for the production of tyramine. Several tdc gene-specific primers are reported for the PCR identification of tdc gene [15, 20]. However, we used a set of primers TD2/TD5 [7] for this purpose because they have been shown to give the right response in several species of LAB including Enterococcus [5]. Using this set of primers all these 19 test strains and a reference strain that showed positive results on the TPM also gave 1100 and 1560 bp amplification products in the PCR. The presence of amplicon 1560 bp for PCR internal control in all negative strains justifies that the tdc-targeted PCR negative results are not due to an inhibition of PCR. We did not observe any silent tdc gene (i.e. giving a band of 1100 bp in the PCR, but found to be negative for decarboxylase activity) as observed by Serio et al. [26]. Therefore, PCR results were also in 100% correlation with the results obtained on TPM. Similarly, 100% correlation between the results of TPM and PCR has recently been reported by Landete et al. [16]. Thus, tdc gene-specific PCR is an easy and quick genetic tool and provides a rapid means of detecting tyramine-producing microorganism in fermented foodstuffs.

The concentration of tyramine produced by enterococci in the fermenting broth under both aerobic and anaerobic incubation was quantified by the HPLC method. Tyramine production in the broth after three days showed a wide variation, from 717 to 9063 mg·L⁻¹ (incubated under aerobic conditions), being > 4000 mg·mL⁻¹ in most of the cases. Relatively a higher tyramine concentration was observed in the case of incubation under aerobic conditions as compared to anaerobic incubation. This is supported by the observations of Landete et al. [16]. Low levels of tyramine under anaerobic incubation may probably be due to the less growth of enterococci as compared to under aerobic incubation. However, levels of tyramine formed by strains of Enterococcus sp. in this study were relatively high compared to those previously reported (379–4986 mg·L⁻¹) in broth [2] and 242–476 mg·mL⁻¹ in cheese [18]. This variability can be justified by the difference in the composition and the concentration of the precursor in fermenting broth or cheese. Several other factors such as incubation time and temperature may also influence the production of tyramine by these strains.

The present study thus establishes a positive correlation between the results of decarboxylating medium, PCR and HPLC with respect to the production of tyramine by the enterococcal strains. However, the results of the two decarboxylating media are not in agreement with each other. Hence, between the two decarboxylating media used for the screening of the decarboxylase activity, TPM was found to be better as the results of this medium showed complete correlation with the other two methods. Since the results of HPLC and PCR were found to be showing 100% correlation, these two methods may serve as a complementary approach to attain the same goal. On one hand where HPLC analysis is essential for quantification i.e. determination of the exact concentration of tyramine in the samples, PCR on the other hand is an easy and rapid method for analyzing large numbers of samples for the presence of putative tyramine-producing microorganisms. Moreover, it can also facilitate in the detection of silent tdc gene if present in any strain. Thus, all the three methods used in this study are efficient by themselves in one or the other way in the detection of BA.

In the present study as some strains of both E. faecalis and E. faecium tested were found to be negative for the tyramine production and also, variability was observed in the concentration of tyramine that was produced by the positive strains of both species. Hence, it seems to indicate that the tyramine-producing ability is not related to a particular species of the genus Enterococcus or to the genus itself but that it is rather a strain-specific attribute. Recently, Coton and Coton have also shown that the tyrosine decarboxylating ability is not a species-specific trait of Lactobacillus brevis but that it is a strain-dependent attribute [6].

Thus, it can be concluded that the tyramine production trait seems to be more common among most strains of genus Enterococcus species. As several strains of Enterococcus sp. are being used in the food industry as a starter, protective and probiotic cultures, it is, therefore, important that strains used in the food industry must be screened for the production of BAs in order to select the safe strain.

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All Tables

All Figures

	Figure 1. Tyrosine production medium (TPM) to detect tyrosine decarboxylating enterococcal strains. a, E. faecalis KH 12; b, E. faecium DH 28; c, E. faecium RH 31; d, E. faecium RH 33; e, E. faecalis NCDC 114 (positive control); f, L. lactis subsp. cremoris NCDC 61 (negative control); g, E. faecium RH 38; h, E. faecalis KH 62 and i, E. faecalis KH 67.
In the text

Figure 2. Molecular identification of Enterococcus sp. (A) E. faecium 1, E. faecium BFE 900 (positive control); 2, E. faecium KH 24; 3, E. faecium DH 28; 4, E. faecium RH 31; 5, E. faecium RH 33; 6, E. faecium RH 38; 7, E. faecium DH 56; 8, E. faecium KH 58; 9, E. faecium DH 59; 10, E. faecium CH 60; 11, E. faecium RH 78; 12, E. faecium KH 79; 13, E. faecium KH 81; 14, E. faecium FH 99; 15, E. faecium FH 102; 16, E. faecium RH 106; 17, E. faecium KH 110; 18, E. faecium DH 115; 19, E. faecium KH 126; 20, E. faecium FH 133 and 21, L. lactis subsp. cremoris NCDC 61 (negative control). (B) E. faecalis 1, E. faecalis ATCC 29212 (positive control); 2, E. faecalis KH 12; 3, E. faecalis KH 62; 4, E. faecalis KH 67; 5, E. faecalis KH 70; 6, E. faecalis KH 72; 7, E. faecalis KH 91; 8, E. faecalis KH 93; 9, E. faecalis KH 98; 10, E. faecalis KH 111 and 11, L. lactis subsp. cremoris NCDC 61 (negative control).

In the text

Figure 3. PCR-based detection of tyrosine decarboxylase (tdc) gene among different enterococcal strains tested. 1, E. faecalis KH 12; 2, E. faecium KH 24; 3, E. faecium DH 28; 4, E. faecium RH 31; 5, E. faecium RH 33; 6, E. faecium RH 38; 7, E. faecium DH 56; 8, E. faecium KH 58; 9, E. faecium DH 59; 10, E. faecium CH 60; 11, E. faecalis KH 62; 12, E. faecalis KH 67; 13, E. faecalis KH 70; 14, E. faecalis KH 72; 15, E. faecium RH 78; 16, E. faecium KH 79; 17, L. lactis subsp. cremoris NCDC 61 (negative control); 18, E. faecalis NCDC 114 (positive control); 19, E. faecalis NCDC 114 (positive control); 20, L. lactis subsp. cremoris NCDC 61 (negative control); 21, E. faecium KH 81; 22, E. faecalis KH 91; 23, E. faecalis KH 93; 24, E. faecalis KH 98; 25, E. faecium FH 99; 26, E. faecium FH 102; 27, E. faecium RH 106; 28, E. faecium KH 110; 29, E. faecalis KH 111; 30, E. faecium DH 115; 31, E. faecium KH 126 and 32, E. faecium FH 133.

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