Lactobacillus casei strains isolated from cheese reduce biogenic amine accumulation in an experimental model

International Journal of Food Microbiology 157 (2012) 297–304

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Lactobacillus casei strains isolated from cheese reduce biogenic amine accumulation in an experimental model Ana Herrero-Fresno 1, Noelia Martínez, Esther Sánchez-Llana, María Díaz, María Fernández, Maria Cruz Martin, Victor Ladero, Miguel A. Alvarez ⁎ Instituto de Productos Lácteos de Asturias (IPLA-CSIC), Crta. Infiesto s/n, 33300 Villaviciosa, Asturias, Spain

a r t i c l e

i n f o

Article history: Received 22 March 2012 Received in revised form 17 May 2012 Accepted 1 June 2012 Available online 7 June 2012 Keywords: Biogenic amines Tyramine Histamine Cheese Degradation Lactobacillus casei

a b s t r a c t Tyramine and histamine are the biogenic amines (BAs) most commonly found in cheese, in which they appear as a result of the microbial enzymatic decarboxylation of tyrosine and histidine respectively. Given their toxic effects, their presence in high concentrations in foods should be avoided. In this work, samples of three cheeses (Zamorano, Cabrales and Emmental) with long ripening periods, and that often have high BA concentrations, were screened for the presence of BA-degrading lactic acid bacteria (LAB). Seventeen isolates were found that were able to degrade tyramine and histamine in broth culture. All 17 isolates were identified by 16S rRNA sequencing as belonging to Lactobacillus casei. They were typed by plasmid S1-PFGE and genomic macrorestriction-PFGE analysis. Two strains (L. casei 4a and 5b) associated with high degradation rates for both BAs were selected to test how this ability might affect histamine and tyramine accumulation in a Cabrales-like mini-cheese manufacturing model. The quantification of BAs and the monitoring of the strains' growth over ripening were undertaken by RP-HPLC and qPCR respectively. Both strains were found to reduce histamine and tyramine accumulation. These two strains might be suitable for use as adjunct cultures for reducing the presence of BAs in cheese. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Biogenic amines (BAs) are low molecular weight organic bases that are synthesized and degraded during normal metabolism in animals, plants and microorganisms. BAs are mainly produced by the decarboxylation of certain amino acids and play important roles in many human physiological functions such as brain activity, gastric acid secretion and the immune response (Shalaby, 1996). However, an excessive oral intake of BAs can cause nausea, headache, rashes and alterations of the blood pressure (Ladero et al., 2010c). This is especially the case in sensitive individuals whose detoxifying systems work less well because of genetic reasons or as consequence of pharmacological treatment (Bodmer et al., 1999). Given their adverse health effects, the accumulation of BAs in foods needs to be prevented (EFSA, 2011). Foods likely to contain high levels of BAs are fish, fish derivatives and fermented products (Halász et al., 1994; Linares et al., 2011; ten Brink et al., 1990). Cheese is one of the fermented foods most commonly associated with BA poisoning; indeed, the term “cheese

⁎ Corresponding author. Tel.: + 34 985 89 21 31; fax: + 34 985 89 22 33. E-mail address: [email protected] (M.A. Alvarez). 1 Present address: National Food Institute (DTU), Kemitorvet, Building 204, Ground floor, Room 039, 2800 Lyngby, Copenhagen, Denmark. 0168-1605/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2012.06.002

reaction” is even used to refer to tyramine intoxication (ten Brink et al., 1990). Concentrations of over 1 g/kg have been reported in cheese, with tyramine and histamine the most commonly present and most abundant of all BAs (Fernández et al., 2007a; Stratton et al., 1991). Different strategies have been proposed for reducing BA accumulation in foods, such as the inhibition of BA-producing bacteria (e.g., by adding sulphite to wine), reducing the number of BA producers via the pasteurisation of milk to be used in cheese manufacture, reducing the amount of proteolytic activity (thus reducing the availability of the amino acid precursors of BAs), and reducing ripening times. However, in some cases the characteristics of fermented foods render these strategies difficult to follow. Another way to reduce the BA content of foods would be to eliminate them from the food matrix. This is the strategy used in the gastrointestinal tract where mono- and di-amino-oxidases catalyse the detoxifying oxidation of BAs (Ladero et al., 2010c). Leuschner et al. (1998), demonstrated the ability of some bacteria isolated from foods to degrade BAs in vitro, in particular strains belonging to the genera Brevibacterium, Lactobacillus, Pediococcus and Micrococcus. García-Ruiz et al. (2011) reported some strains of Lactobacillus casei and Pediococcus isolated from wine as capable of reducing histamine, tyramine and putrescine concentrations in this medium, while Leuschner and Hammes (1998) underscored the potential role of some Brevibacterium linens strains in reducing the BA content in Munster cheese. In studies performed on fish, silage and

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dry sausages too, the presence of certain bacterial strains has been found to reduce the BA content, although the exact mechanism via which this occurs remains unknown (Enes-Dapkevicius et al., 2000; Fadda et al., 2001; Gardini et al., 2002). Together, these findings suggest that the addition of bacterial strains able to reduce BA contents could be of use in cheese manufacture. The aim of the present work was to screen for BA-degrading lactic acid bacteria (LAB) in different cheeses usually rich in BAs. The isolates detected were identified, characterized, and their ability to degrade histamine and tyramine in broth culture quantified. Finally, the capacity of two selected strains with high BA-degradation rates to prevent BA accumulation in a Cabrales-like mini-cheese model was evaluated. Cabrales is the most famous traditional Spanish blue cheese and has had Protected Designation of Origin status since 1981. However, due to its characteristics – it is made from raw milk, is blue-veined, its proteolytic activity is high, and it has a long ripening period – it can develop high BA concentrations (Ladero et al., 2010a). The results suggest that the selected strains could be used as adjunct cultures to avoid the accumulation of high concentrations of histamine and tyramine in cheese.

England) at 37 °C or M17 (Oxoid) supplemented with 1% w/v glucose (Sigma, Madrid, Spain) (GM17) at 30 °C, respectively.

2. Materials and methods

2.3. Detection of the tdcA and hdcA genes in the BA-degrading isolates

2.1. Bacterial and fungal strains and plasmids used in the present work

The presence of the histidine and tyrosine decarboxylase genes (hdcA and tdcA respectively) in the isolates was evaluated by PCR using the previously described primers hdc1 and hdc2 (Fernández et al., 2006) and QGtdcAF and QGtdcAR (Ladero et al., 2010b) respectively. PCR was performed directly using plate colonies resuspended in 50 μl of MilliQ water; 1 μl was added as template. The PCR conditions where those described by Fernández et al. (2006) and Ladero et al. (2010b). Lactobacillus parabuchneri DSM 5987 and Enterococcus durans 655 were used as positive controls for hdcA and tdcA respectively.

Table 1 provides a general list of all the bacterial and fungal strains and plasmids used in the present work. Unless otherwise stated, the Lactobacillus and Enterococcus strains used in all experiments were grown without aeration in MRS (Oxoid, Basingstoke, Hampshire,

Table 1 Microorganisms used in the present work.

2.2. Isolation of BA-degrading bacteria from cheese samples Cheese samples (5 g) were homogenized in 45 ml of 2% w/v sodium citrate for 2 min using a Lab-Blender 400 stomacher (Seward Ltd., London, UK). The cheese homogenates were transferred to 1% w/v chemically defined medium (CDM), as described by Miladinov et al. (2001) but with minor modifications. This medium was supplemented with cycloheximide (50 μg/ml) (Sigma) to inhibit yeast and mould growth, and with 0.1% w/v Tween 20 (Bio-Rad, Hercules, CA) to improve the growth of lactobacilli. Tyramine (2.5 mM) (Sigma) or histamine (1 mM) (Sigma) was added as the single nitrogen source. Enrichment cultures were grown for 48 h without aeration at two different temperatures: 30 °C and 37 °C. After two rounds of enrichment, culture dilutions were spread on CDM agar plates supplemented with one of the BAs and grown for 48 h at 30 °C and 37 °C. Isolated colonies with different morphologies were individually transferred to GM17 or MRS agar plates and incubated at 30 °C or 37 °C.

Strain or plasmid

Relevant characteristics

Reference or source

2.4. Molecular identification of BA-degrading strains

Strain Penicillium roqueforti 1AM8

Protelolytic mould from Cabrales cheese

IPLA collection

BA-Producing E. durans 655

Tyramine producer

L. parabuchneri DSM 5987

Histamine producer

Fernández et al., 2007b DSMZ collection

BA-Degrading L. casei 4a L. casei 5b L. casei 13b L. casei 16b L. casei 18b L. casei 54b L. casei 61b L. casei 63b L. casei E2 L. casei 36b L. casei 37c L. casei 39b L. casei 41b L. casei 43c L. casei 49b L. casei 50b L. casei 68b L. casei 4aEM76

Potential BA-degrading strain Potential BA-degrading strain Potential BA-degrading strain Potential BA-degrading strain Potential BA-degrading strain Potential BA-degrading strain Potential BA-degrading strain Potential BA-degrading strain Potential BA-degrading strain Potential BA-degrading strain Potential BA-degrading strain Potential BA-degrading strain Potential BA-degrading strain Potential BA-degrading strain Potential BA-degrading strain Potential BA-degrading strain Potential BA-degrading strain L. casei 4a carrying A2 int-attP

This This This This This This This This This This This This This This This This This This

Selected BA-degrading isolates were identified by partial amplification of the 16S rRNA gene. Total DNA was isolated from bacterial cultures using 2× Kirby lytic mix as previously described (Hopwood et al., 1985). The DNA was used as a template for PCR amplification involving primers pA and pH* (Edwards et al., 1989). Amplifications were performed in an iCyclerTM thermocycler (Bio-Rad) using conditions described by the latter authors. The PCR products were purified using the GenElute PCR Clean-Up Kit (Sigma) and sequenced by Macrogen (Seoul, Korea). The resulting sequences were aligned and compared with all eubacterial 16S rRNA gene sequences available in the GenBank and EMBL databases using the BLAST programme (Altschul et al., 1997). Sequences with a percentage similarity of 98% or higher were considered the same species (Stackebrandt and Goebel, 1994). All the sequences (n = 17) obtained were 100% identical to that of L. casei. Therefore, the nucleotide sequence of one representative strain (L. casei 5b) was deposited in the EMBL database under the accession number HE647132.

Non-degrading L. casei EM116

L casei 393 carrying A2 int-attP

Martín et al., 2004

Plasmids pEM76 pEM94

pUC19-borne A2 int-attP, six, Eryr pG + host9-borne β-gene, Cmr

Martín et al., 2000 Martín et al., 2004

work work work work work work work work work work work work work work work work work work

DSMZ: German collection of micro-organisms and cell cultures. IPLA: Instituto de Productos Lácteos de Asturias collection. Cmr: chloramphenicol resistance gene; Eryr: erythromycin resistance gene

2.5. Macrorestriction of genomic DNA and analysis by pulsed-field gel electrophoresis (PFGE), plus S1-PFGE analysis of plasmids The 17 L. casei isolates identified were grown in MRS (Oxoid) supplemented with 40 mM threonine (Merck, Darmstadt, Germany) at 37 °C. Cells (1.5 ml of overnight cultures) were harvested by centrifugation (8000×g, 10 min), washed twice with 0.5 ml of STE-100 buffer (100 mM Tris–HCl, 100 mM EDTA, 0.3 M saccharose) and resuspended in 500 μl of STE-100 buffer containing 1 μl of mutanolysin, (150 U/μl) (Sigma). The bacterial suspension was incubated for 30 min at 37 °C. A 200 μl volume of this suspension was then mixed with 240 μl of liquid 1.8% w/v low melting point agarose (USB Corporation, Cleveland, OH, USA) at 40 °C. Portions of 200 μl of this mixture were pipetted into plug moulds and left to solidify at 4 °C. The agarose plugs were removed and

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incubated in 2 ml of a lysis solution containing 6 mM Tris HCl pH8, 1 M NaCl, 100 mM EDTA, 0.5% w/v N-lauroylsarcosine (Merck), 0.2% w/v deoxycholic acid (Alfa Aesar, Karlsruhe, Germany), 0.5% w/v Brij 58 (Merck) and 2 mg/ml lysozyme (Sigma), for 2 h at 37 °C. After this period, the agarose plugs were transferred to ESP buffer (0.5 M EDTA, 0.5% w/v N-lauroylsarcosine, 1 mg/ml proteinase K) (Roche, Basel, Switzerland) and incubated overnight at 55 °C. After removing the ESP buffer, the plugs were first equilibrated for 15 min at 50 °C with TE (10 mM Tris HCl, 1 mM EDTA) and 1 mM phenylmethylsulphonyl fluoride (PMSF) (Roche) and then equilibrated five times with TE for 15 min at 50 °C. The agarose blocks were treated with SfiI endonuclease (Fermentas, Vilnius, Lithuania) and PFGE was performed using a CHEFDR III System at 14 °C (Bio-Rad) in a 1% w/v agarose gel containing 0.5× Tris, borate and EDTA buffer at 4.5 V/cm, employing pulse ramps from 0.5 to 25 s for 12 h and 25 to 50 s for 6 h. The DNA was visualized by staining with ethidium bromide. Plasmids were extracted and analysed by the S1-PFGE method described by Barton et al. (1995). 2.6. Construction of L. casei 4aEM76 In order to monitor the growth of a BA-degrading strain during cheesemaking, a food grade L. casei 4aEM76 strain carrying the sitespecific integration apparatus of temperate phage A2 was constructed using the strategy described by Martín et al. (2004). Briefly, the integrative plasmid pEM76 containing the integration region of phage A2 (int-attP) (Martín et al., 2000) was introduced by electroporation into L. casei 4a using conditions previously described (Wei et al., 1995). The resulting strain was subsequently electrotransformed with pEM94, a replicative plasmid that carries the β-resolvase gene. The latter allows the removal, by site-specific recombination, of the ‘non-food grade’ DNA (antibiotic resistance genes and Escherichia coli DNA), which is located between two six (β-resolvase recognition sequence) sites (Martín et al., 2004). After this purification step the strains were cultured at 37 °C to eliminate pEM94 (which carries a temperature-sensitive origin of replication point). The obtained strain was designated L. casei 4aEM76. Each step (integration, purification, and plasmid curing) was confirmed by PCR analysis and Southern blotting, using the primers and conditions described by Martín et al. (2000).

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330 g/l at pH 5.4. After 5 min, the saline solution was removed and the mini-cheeses placed in a ripening chamber at 15 °C. Samples were collected after inoculation (T0) and at 1, 2, 3 and 4 at months (T1, T2, T3 and T4 respectively). Each sample was analysed by RPHPLC to quantify the BAs, and by qPCR to assess bacterial growth. 2.8. Monitoring of the BA-degrading and BA-producing strains during mini-cheese manufacture qPCR was used to monitor the growth of the BA-degrading strain L. casei 4aEM76 and the non-degrading control L. casei EM116 in the mini-cheese model. For this, specific primers were designed to target the attP region of the integrated plasmid (qattP 5′GCAAGAATGCCGGTTTAAAGC-3′) and the attB target in the bacterial chromosome (qattB 5′-GCGGAATTGGCAGACGC-3′). The fluorescent probe attP-NED (5′-TGAGCACTAAAAAAGACCCT-3′MGB) was also designed. The amplification conditions were: 20 s denaturation at 95 °C, followed by 40 cycles of 5 s at 95 °C and 30 s at 60 °C. To determine the sensitivity of the method, total DNA was obtained from 1 ml of 10-fold-diluted overnight cultures of L. casei 4aEM76 and L. casei EM116 (10 9 cfu/ml). A standard regression curve plotting log cfu against the cycle threshold (CT) showed a linear correlation between these variables (R 2 0.996, slope − 3.44, detection limit 10 4 cfu/ml). To determine bacterial growth in the mini-cheeses, DNA was extracted from 1 g of cheese in 10 ml of 2% w/v sodium citrate and homogenized in a Lab-Blender 400 stomacher (Seward Medical, London, UK) for 2 min. This DNA was then purified using the QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. 2.5 μl of the purified DNA solution was added to 12.5 μl of qPCR TaqMan Universal PCR master mix (Applied Biosystems, Warrington, UK), 0.9 μM of each primer and 0.25 μM of the TaqMan probe (when necessary). All reaction volumes were made up to 25 μl with distilled water. The growth of the BA-producing strains was determined by quantifying the tdcA and hdcA genes present by specific qPCR using the primers and conditions described by Ladero et al. (2010b) and Fernández et al. (2006) respectively. 2.9. Reversed-phase high performance liquid chromatography quantification of BAs

2.7. Cabrales-like mini-cheese model Table 1 shows the strains used with the mini-cheese model. Cabrales-like mini-cheeses were made based on the model of Hynes et al. (2000). The starter culture Lactococcus lactis subsp. lactis CABI (BIOGES, León, Spain) (10 5 cfu/ml), the mould P. roqueforti 1AM8 (10 3 cfu/ml) and CaCl2 (2% w/v) were placed in sterilized bottles containing 200 ml of pasteurised milk. A BA-producing strain (E. durans 655 or L. parabuchneri DSM5987) (10 5 cfu/ml) was then added either alone or in combination with a BA-degrading strain (L. casei 4aEM76 or L. casei 5b) (10 6 cfu/ml) or a non-degrading strain as a negative control (L. casei EM116) (10 6 cfu/ml). Bottles containing only the non-degrading strain (10 6 cfu/ml) were also made up. Finally, bottles with none of the previous strains, i.e., containing only the mould and the starter culture, were prepared. The mixtures were left at 30 °C for 45 min to initiate the growth of the cultures. After this time, liquid rennet extract of bovine origin (NIEVI, Barcelona, Spain) was added (0.33 ml/l). The bottles were inverted three times and left to coagulate under supervision in a water bath at 30 °C for approximately 1 h. After coagulation, the bottles were left for 30 min more at 30 °C to achieve the appropriate curd consistency. The curd was then cut and inverted for 20 min to promote draining. The bottles were then centrifuged at 220 ×g for 10 min at room temperature and the whey discarded. All steps were performed under sterile conditions. The cheeses were then transferred to new sterilized recipients and salted with 35 ml of NaCl

To test the individual capacity of each initial isolate to degrade tyramine or histamine, colonies were inoculated into MRS or GM17 broth supplemented with tyramine (2.5 mM) or histamine (1 mM) and incubated at 37 °C or 30 °C for 24 h. Samples were taken just after inoculation and after 24 h of incubation and centrifuged at 8000 ×g for 10 min. The supernatants were then filtered through 0.45 μm PTFE filters (VWR International, Radnor, PA, USA). 5 μl of these filtrates were derivatised as described by Krause et al. (1995). BAs were separated and quantified by reversed-phase high performance liquid chromatography (RP-HPLC) using a Waters Nova-pack C18 column (150 × 3.9 mm) in a Waters liquid chromatography apparatus controlled by Millenium 32 software (Waters Milford, MA, USA) following the protocol described by Krause et al. (1995). The RP-HPLC analysis experiments were performed in triplicate. To test BAs accumulated in the mini-cheese model, one gram of mini-cheese was placed in 10 ml of 0.1 M HCl containing 0.2% w/v 3,3′thiodipropionic acid (TDPA), and mixed using a UltraTurrax T50 homogeniser (OMNI International, CT, USA) for 2 min at 2000 ×g. The sample was then disrupted for 30 min in an ultrasonic bath and centrifuged at 5000 ×g for 20 min. After removing the top fat layer, the supernatant was filtered through 0.45 μm PTFE filters. The filtrates were deproteinised by passing through ultrafiltration inserts (Amicon Biomax5K; Millipore, MA, USA) by centrifugation at 3500 g for 1 h. 5 μl was then derivatised and the BAs separated and quantified as previously described.

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reduce the content of either BA tested (Table 2) and was used as negative control in this and further experiments.

3. Results 3.1. Isolation of BA-degrading bacteria from cheese samples Samples of three different cheeses that commonly show high BA contents (Fernández et al., 2007a) were used as starting material for screening for bacteria capable of degrading BAs: Cabrales (a raw milk blue cheese rich in tyramine and histamine), Zamorano (a long duration ripening raw milk cheese rich in tyramine and histamine), and grated Emmental (a processed cheese rich in histamine). As described in Materials and methods, cheese samples were prepared and inoculated into liquid CDM with tyramine or histamine as the single nitrogen source. When Emmental cheese was screened, only histaminedegrading bacteria were sought. After two rounds of enrichment, colonies were isolated on solid CDM supplemented with the corresponding BA. A total of 300 colonies were then picked off and placed on new GM17 and MRS plates and subjected to functional and genetic selection. 3.2. Identification of tyramine and histamine producers The first step in the selection of appropriate BA-degraders was to discard any isolates that synthesized tyramine or histamine. Each of the 300 colonies selected in the step above was examined for the presence of hdcA and tdcA by PCR. None was positive for the hdcA gene, while 143 were discarded because they were positive for tdcA, indicating their potential to produce tyramine. The remaining 157 isolates – 88 isolated from CDM with histamine and 69 from CDM with tyramine – were selected for further analysis. 3.3. BA-degrading ability of the selected non-BA-producing isolates The 157 isolates were tested for their capacity to degrade histamine (88 isolates) or tyramine (69 isolates). Single colonies were inoculated into 10 ml of MRS supplemented with 2.5 mM tyramine or 1 mM histamine. After 24 h (Time 24) at 37 °C, the supernatants were analysed for their BA content by RP-HPLC. Seventeen isolates (12 from Zamorano, 4 from Cabrales and 1 from Emmental) showed a degradation rate of over 25% (compared to Time 0) for at least one of the tested BAs. These were selected for further characterization. The 17 isolates were tested (in triplicate) for both tyramine and histamine degradation; all degraded both BAs but with different efficiency (Table 2). L. casei EM116 (Table 1) did not significantly

3.4. Identification and characterization of the selected BA-degrading strains 16S rRNA gene sequence comparisons showed the selected isolates to all belong to L. casei (100% similarity). To characterize the 17 BA-degrading strains, SfiI restriction of the genomes and fragment separation by PFGE were initially performed. To define the SfiI profiles, only bands between ca. 10 and 700 kb were taken into account. Between 16 and 23 bands were resolvable under these conditions. Nine SfiI profiles were distinguishable (SfiI-1 to SfiI-9; Fig. 1A), confirming the wide diversity of the isolates. Table 2 shows the SfiI profile for each isolate. Four isolates showed the SfiI-1 and SfiI-3 profiles and three showed the SfiI-7 profile; 6 profiles (SfiI-2, SfiI-4, SfiI-5, SfiI-6, SfiI-8 and SfiI-9) were shown by only one isolate each. The similarity between the SfiI-8 and SfiI-0 profiles of L. casei E2 and L. casei EM116 respectively should be noted; note also the similarity between the SfiI-3 and SfiI-4 profiles of L. casei 13b and L. casei 18b respectively (Fig. 1A). To determine the number and molecular size of any plasmids, total DNA was digested with S1 nuclease and subjected to PFGE (S1-PFGE). Nine plasmid profiles of varying size were distinguished (S1-I to S1-IX; Fig. 1B, Table 2). All the isolates harboured between one and six plasmids. As expected, the control strain L. casei EM116 (S1-0) carried no plasmid. It is noteworthy that the S1-I profile was the most common (five isolates) followed by S1-III (4 isolates), S1-VI (2 isolates), and S1-II, S1-IV, S1-V, S1-VII, S1-VIII and S1-IX (one isolate each). Interestingly, the S1-VI (L. casei 63b and L. casei 68b) and S1-IX (L. casei 61b) profiles differed by only one plasmid. Finally, the only difference between L. casei EM116 (S1-0) and L. casei E2 (S1-VIII) was the presence of a plasmid (ca. 50 kb) in the latter. 3.5. Ability of the selected strains to prevent BA accumulation in the Cabrales-like mini-cheese model Two of the 17 strains (L. casei 4a and L. casei 5b) that showed high degradation rates for BAs (Table 2) were selected for the minicheesemaking experiment. In order to follow the growth of the selected strains in the cheese model, a derivative strain carrying the integration region from the A2 phage was constructed (L. casei

Table 2 Features of the selected strains. The BA degradation rate is given as a percentage and was measured after 24 h post-inoculation. The S1 and SfiI PFGE profiles are those described in Fig. 1. Strain

L. casei 4a L. casei 5b L. casei 13b L. casei 16b L. casei 18b L. casei 54b L. casei 61b L. casei 63b L. casei E2 L. casei 36b L. casei 37c L. casei 39b L. casei 41b L. casei 43c L. casei 49b L. casei 50b L. casei 68b Control L. casei EM116 n.d.: not detected.

Cheese origin

Amine for enrichment

Biogenic amine degradation rate Tyramine

Histamine

Zamorano Zamorano Zamorano Zamorano Zamorano Cabrales Cabrales Cabrales Emmental Zamorano Zamorano Zamorano Zamorano Zamorano Zamorano Zamorano Cabrales

Histamine Histamine Histamine Histamine Histamine Histamine Histamine Histamine Histamine Tyramine Tyramine Tyramine Tyramine Tyramine Tyramine Tyramine Tyramine

36.22 ± 0.90 40.55 ± 13.60 21,92 ± 2.60 37.50 ± 14.21 34.30 ± 14.65 24.35 ± 9.60 49.17 ± 12.29 27.15 ± 11.12 14.92 ± 2.60 22.49 ± 7.70 50.37 ± 15.80 49.63 ± 8.30 56.52 ± 13.22 55.66 ± 18.10 50.71 ± 19.38 52.45 ± 13.80 54.03 ± 15.40

47.90 ± 1.43 43.64 ± 0.12 17.13 ± 0.49 37.77 ± 0.58 45.43 ± 0.27 34.20 ± 0.78 32.36 ± 0.89 36.56 ± 0.76 42.86 ± 0.32 27,13 ± 0.29 32.07 ± 1.50 39.63 ± 0.96 19.17 ± 0.41 23.56 ± 1.10 12.752 ± 0.20 18.75 ± 0.86 22.10 ± 0.45

n.d.

3.24 ± 2.70

Plasmid Profile (S1-PFGE)

SfiI PFGE profile

S1-I S1-II S1-III S1-V S1-IV S1-I S1-IX SI-VI S1-VIII S1-I S1-III S1-III S1-VII S1-III S1-I S1-I S1-VI

SfiI-1 SfiI-2 SfiI-3 SfiI-5 SfiI-4 SfiI-1 SfiI-7 SfiI-7 SfiI-8 SfiI-1 SfiI-3 SfiI-3 SfiI-7 SfiI-3 SfiI-9 SfiI-1 SfiI-6

S1-0

SfiI-0

5b 13b 18b 16b 41b 49b 61b E2 EM116 M

M

S1-0

S1-VI

S1-VIII

S1-VII

S1-IX

S1-V

S1-IV

S1-III

S1-I

SfiI-0

SfiI-8

SfiI-6

SfiI-9

SfiI-7

SfiI-5

SfiI-4

SfiI-3

B

301

S1-II

M 4a

SfiI-2

A

SfiI-1

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4a

5b 13b 18b 16b 41b 61b 63b E2 EM116 M

Kb

485 436,5 388 339,5 291 242,5 194 145,5 97 45,5 23,1 9,42

Fig. 1. Analysis of L. casei isolates by pulsed field gel electrophoresis. A) Macrorestriction profiles generated by SfiI PFGE. B) Plasmid profiles visualized by S1-PFGE. The Lambda ladder PFG marker and Low Range PFG marker (New England BioLabs) were used as size standards.

4aEM76). Combinations of BA-degrading and BA-producing strains were used in cheese manufacture. L. casei EM116 was used as a non-degrading control strain. Samples were collected just after inoculation with the corresponding microorganisms (T0) and after 1 (T1), 2 (T2), 3 (T3) and 4 (T4) months of ripening, and analysed for their BA content by RP-HPLC. The growth of BA-producing and degrading strains was monitored by qPCR. None of the control cheeses, i.e., those containing only the starter and the mould, or containing only the non-degrading control strain L. casei EM116, or those to which only a BA-degrading strain (L. casei 4aEM76 or L. casei 5b) had been added, accumulated any histamine or tyramine (data not shown). As expected, in the cheese made with the tyramine producer E. durans 655, an increase in tyramine concentration was seen between T0 and T4 (from undetectable to 1.7 mM) (Fig. 2A). In contrast, those cheeses made with E. durans 655 plus a BAdegrading strain (L. casei 4aEM76 or L. casei 5b) showed lower tyramine concentrations (0.088 and 0.015 mM respectively) at T4 (Fig. 2A). This reduction was not observed in the cheeses in which both E. durans 655 and the non-degrading control strain L. casei EM116 were present. For the latter combination, the tyramine concentration evolved in the same way over time as in the cheeses containing only BA producers, and reached a similar final concentration at T4 of 1.6 mM (Fig. 2A). A

similar situation was seen for the cheeses containing the histamine producer L. parabuchneri DSM 5987. The concentration of histamine increased from undetectable at T0 to 5.1 mM at T4 in the absence of BA-degraders (Fig. 2B). In the cheeses containing one of the BAdegrading strains, the concentration of histamine at T4 was much lower (Fig. 2B). As expected, the presence of the non-degrading control strain L. casei EM116 plus L. parabuchneri DSM 5987 led to no reduction in the final histamine concentration (4.7 mM) (Fig. 2B). To exclude the possibility that the observed reduction in the final cheese BA concentration might be due to the growth of the BAproducing strains being inhibited by the BA-degrading isolates, the growth of BA producers was monitored by qPCR over ripening. The results showed there to be no significant difference between the growth of E. durans 655 alone or in combination with a BA-degrading or the non-degrading control strain (Fig. 3A). Equivalent results were obtained when the growth of the histamine producer L. parabuchneri DSM 5987 in the presence of a BA-degrading or the non-degrading control strain was followed (Fig. 3B). As for E. durans 655, growth was not altered when a BA-degrading or the control strain was present. To monitor the growth of the BA-degrading strains, samples from cheeses containing the strains L. casei 4aEM76 (degrading) or L. casei EM116 (non-degrading control) were collected T0–T4 and processed

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Time (months) Fig. 2. Analysis of the BA content by RP-HPLC in mini-cheeses during the ripening period. A) Monitoring of tyramine concentration in mini-cheeses made with E. durans 655. B) Monitoring of histamine concentration in mini-cheeses made with L. parabuchneri DSM 5987. (Adjunct strain indicated in brackets).

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Time (months) Fig. 3. Growth of the BA-degrading and BA-producing isolates during the ripening period, as shown by qPCR. A) Growth of the tyramine producer E. durans 655. B) Growth of the histamine producer L. parabuchneri DSM 5987. C) Growth of the L. casei 4aEM76. D) Growth of the non-degrading (control) strain L. casei EM116. (Adjunct strain indicated in brackets).

for DNA extraction and qPCR analysis. The results showed both strains to follow the same behaviour (Fig. 3C, 3D). Thus, the growth of these strains was similar in all cheeses, independent of the presence/absence of a BA-producing strain. 4. Discussion Cheese, especially that made from raw milk, is undoubtedly a complex ecosystem involving many different microorganisms with different metabolic machineries. The transformation of milk into the great cheese diversity of different flavour and other organoleptic characteristics requires the participation of many and diverse enzymatic activities. Amino acids provide carbon, nitrogen and energy sources for bacterial cells and play an important role in the development of flavour in cheeses (Fernández and Zuñiga, 2006). Some amino acids can be decarboxylated leading to the formation of BAs, the accumulation of which in food is undesirable. However, BAs can be broken down by catabolic enzymes; indeed, amino-oxidase activity is a major factor in their degradation (Ladero et al., 2010c), an activity that higher organisms, yeast, fungi and also some bacteria possess. In bacteria, such activity could allow BAs to be used as the sole nitrogen source. The screening method described in the present work is based on this. Of the 300 isolates initially collected, 17 LAB strains were found to be able to degrade tyramine and histamine in broth culture while showing no capacity to produce BAs. Selected strains were surprisingly capable of degrading both histamine and tyramine, although they were initially selected by their ability to use only one of these BA. Zamorano cheese was the main source of these BA-degrading strains (12 out of 17 isolates). Only one BA-degrading strain was isolated from Emmental, although the selection process for this cheese only

involved the ability to break down histamine (the most abundant BA in this cheese type) (Ladero et al., 2009). All 17 BA-degrading strains were identified as L. casei based on 16S rRNA gene sequencing — just one species, even though different cheese types were used and different BA breakdown capacity was sought. L. casei is commonly found in food matrices such as fish, meat and silage (Fadda et al., 2001; García-Ruiz et al., 2011; Nishino et al., 2007; Rabie et al., 2011; Zhong-Yi et al., 2010). The differences between the isolates, and the existence of L. casei strains that showed no BA-degrading ability indicate this to be a strain-dependent trait. The high L. casei strain differentiation efficiency of SfiI PFGE analysis has been previously demonstrated (Cai et al., 2007). The results of the present PFGE analysis showed high heterogenicity among the 17 BA-degrading strains, with nine different SfiI PFGE profiles and nine S1 plasmid profiles detected. Apart from the characterization of the strains, these results provide other valuable information. For example, they show that L. casei E2 differs from the non-degrading strain L. casei EM116 by the presence of a plasmid. The latter may encode the enzymes involved in BA degradation, opening up new possibilities of study. Two of the 17 L. casei strains that showed strong tyramine and histamine degrading capacity were further assessed for their potential to reduce the BA content in experimental mini-cheeses. When controlled bacteriological conditions are needed, pilot plant experiments can be difficult to perform. Thus, in the present work, a protocol for a laboratory scale cheese model was used (Hynes et al., 2000), with some modifications, to produce blue mini-cheeses. Strict hygiene conditions during cheese manufacture and ripening were imposed in order to avoid contaminations that might confound the results. A blue Cabrales-like cheese was used as the model since blue cheeses in general, and Cabrales in particular, accumulate high BA concentrations (Fernández

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et al., 2007a). Although higher BA concentrations are usually detected in cheeses made from raw milk (as is traditional Cabrales cheese) than in those made from pasteurised milk, the mini-cheeses were manufactured using pasteurised milk in order to homogenize the microbiota present. A crucial factor in BA production in cheese is the presence of microorganisms with decarboxylation activity (Linares et al., 2011). Enterococcus and Lactobacillus are mainly responsible for BA production in cheeses, with enterococci the predominant bacteria in raw milk cheeses ripened over long periods (Burdychova and Komprda, 2007; Foulquié Moreno et al., 2006; Ladero et al., 2012). Moreover, in Cabrales cheese it has been shown that tyramine-producing enterococci are present in the raw milk used to make it, and that they are mainly responsible for the accumulation of tyramine (Ladero et al., 2010a). Thus, to ensure BA production, E. durans 655, an extensively characterized tyramine producer (Fernández et al., 2004; Linares et al., 2009), and the histamine producer strain L. parabuchneri DSM 5987 (unpublished) were used in the mini-cheese manufacturing experiments. The Cabralesspecific industrial starter known as CABI, which is composed of several strains of L. lactis was used as a starter culture, and a mould (P. roqueforti 1AMB) isolated from Cabrales cheese (B. Mayo personal communication and a kind gift) was used as an adjunct culture. The presence of producer microorganisms alone, however, is not enough for BAs to accumulate in cheese: proteolysis must also occur, the pH must be correct, and the ripening period must be long enough (Fernández et al., 2007a). Indeed, the proteolytic role of the mould might be essential for BA accumulation via its release of amino acid substrates (tyrosine or histidine) to the decarboxylation enzymes (Fernández et al., 2007a; FernándezGarcía et al., 2000; Novella-Rodríguez et al., 2004; Ordóñez et al., 1997). This would explain the high BA concentration commonly detected in blue cheeses. In contrast, it has been recently shown, that some species of fungi are able to degrade BA in wine, suggesting their potential application for BA removal (Cueva et al., 2012). The length of the ripening period is also important; the highest BA concentrations are usually detected in cheeses ripened the longest (Fernández et al., 2007a). In the present work, the longest ripening period was 4 months, and, as shown in Fig. 2, the greatest BA accumulation occurred at this time. Once BA production was optimised in the mini-cheese model, a great reduction in tyramine and histamine accumulation over ripening was seen in those cheeses in which L. casei 4aEM76 or L. casei 5b were present (i.e., compared to those inoculated with only the producer strain). These results confirm the BA-reducing potential of these strains. It might be argued that their effect could be owed to inhibition of the growth of BA-producing strains. Indeed, L. casei strains can reduce the tyramine concentration in cheese by inhibiting the growth of enterococci (Bachmann et al., 1997). However, monitoring of the growth of the BA producer strains showed them not to be inhibited by any of the BA-degrading strains nor by the non-degrading L. casei strain used as a control (Fig. 3A, B). In fact, the histamine producer L. parabuchneri increased in numbers during ripening, with similar final populations reached in all cheeses. These results suggest that the assayed L. casei strains are able to degrade tyramine and histamine. With regard to the enzymatic activity responsible for this, only amino-oxidase activity (present in certain microorganisms) has so far been described (Corpillo et al., 2003; Leuschner et al., 1998). In all other studies the mechanism involved in BA reduction has been unknown. The present work provides no conclusive evidence to confirm that the BA-degrading strains show amino-oxidase activity. The reduction of the histamine and tyramine concentrations observed in the presence of the L. casei strains, together with the latters' stable growth (from T2 onwards) during ripening, suggests them as suitable for use in cheesemaking as adjunct cultures to prevent histamine and tyramine accumulation. Such a strategy might be particularly useful when making cheeses from raw milk in which a specific non-starter microbiota is essential for the organoleptic characteristic of the final product. Therefore, it would be very useful to have BAdegrading LAB at our disposal to avoid the accumulation of BAs.

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High concentrations of BAs could thus be prevented, meeting the goal set of diminishing the health threat they pose (ten Brink et al., 1990; EFSA, 2011). In conclusion, the present work identifies LAB strains that could be used as highly competitive adjunct cultures capable of reducing the concentrations of tyramine and histamine, the two most common and toxic BAs, in cheese. The use of such cultures together with high quality raw materials and appropriate manufacturing practices might afford the best way of making products with reduced BAassociated health risks.

Acknowledgements This research was funded by the Spanish Ministry of Science and Innovation (AGL2010-18430) and the European Community's Seventh Framework Programme (BIAMFOOD project no. 211441). N. Martínez is the beneficiary of an I3P-CSIC contract financed by the European Social Fund, and M. Diaz is the recipient of a fellowship from the Spanish Ministry of Science and Innovation. The authors thank Isabel Cuesta for excellent technical assistance, Adrian Burton for linguistic assistance, Baltasar Mayo and Ana Rodriguez for valuable advice regarding the optimisation of the cheese model, and Baltasar Mayo once again for the kind gifts of the CABI starter and P. roqueforti 1AMB.

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