International Journal of Food Microbiology 191 (2014) 144–148
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Selection of potential probiotic Enterococcus faecium isolated from Portuguese fermented food Joana Barbosa, Sandra Borges, Paula Teixeira ⁎ CBQF-Centro de Biotecnologia e Química Fina — Laboratório Associado, Escola Superior de Biotecnologia, Universidade Católica Portuguesa/Porto, Rua Dr. António Bernardino Almeida, 4200-072 Porto, Portugal
a r t i c l e
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Article history: Received 10 February 2014 Received in revised form 28 August 2014 Accepted 14 September 2014 Available online 19 September 2014 Keywords: Enterococcus faecium Fermented product Probiotic Antimicrobial activity Gastrointestinal tract survival
a b s t r a c t Four Enterococcus faecium strains isolated from fermented products were evaluated for potential use as probiotic strains. In addition to efaAfm gene, commonly found in E. faecium food isolates, none of the isolates possessed virulence genes and none had positive reactions for the production of tyramine, histamine, putrescine and cadaverine in the screening medium used. All of these four isolates proved to be resistant to 65 °C. E. faecium 119 did not show antimicrobial activity against any of the target bacteria investigated. E. faecium 85 and 101 inhibited Listeria innocua and E. faecium DSMZ 13590. The strain E. faecium 120 inhibited seven target bacteria (Listeria monocytogenes 7946, L. monocytogenes 7947, L. innocua 2030c, L. innocua NCTC 11286, E. faecium DSMZ 13590, Enterococcus faecalis ATCC 29212 and Staphylococcus aureus ATCC 29213) and was chosen as the representative to assess the ability to survive gastrointestinal tract passage simulation, as well as the protective role of two food matrices (skim milk and Alheira) during its passage. For both matrices used, no significant differences (p b 0.05) were obtained between the types of digestion — quick and slow passage simulation. In the skim milk matrix the isolate was reduced to values below the detection limit of the enumeration technique by the end of the two digestions, in contrast to the Alheira matrix, for which isolate 120 showed a reduction of only ca. 1 log CFU/ml. The E. faecium strain 120 was shown to be a potential candidate for further investigations as a potential probiotic culture. © 2014 Published by Elsevier B.V.
1. Introduction Although being ubiquitous microorganisms, enterococci have as their main habitat the gastrointestinal tract of humans and warm-blooded animals (Murray, 1990). A small number of enterococci are also present in oropharyngeal secretions, in vaginal secretions and on the skin, especially in the perineal area and, consequently, they can be considered as normal inhabitants of the human organism (Kayser, 2003). Enterococci are involved in the development of the typical organoleptic characteristics of a variety of fermented foods such as cheeses, fermented sausages and vegetables where, in some cases, they are the predominant lactic acid bacteria (Franz et al., 2011; Macedo et al., 1995; Suzzi et al., 2000). In Portuguese fermented meat products the numbers of enterococci ranged from 104 to 108 CFU/g in Alheira and from 104 to 107 CFU/g in Chouriça (Ferreira et al., 2006, 2007), because of their tolerance to low pH, sodium chloride and nitrite (Martin et al., 2005). Various studies have commented on the benefits of using Enterococcus, particularly Enterococcus faecium strains, as adjunct cultures in fermented foods, because of their ability to inhibit the growth of food-borne
⁎ Corresponding author. Tel.: +351 22 5580001; fax: +351 22 5090351. E-mail address: [email protected] (P. Teixeira).
http://dx.doi.org/10.1016/j.ijfoodmicro.2014.09.009 0168-1605/© 2014 Published by Elsevier B.V.
pathogens, commonly present in these kinds of products (Bouton et al., 2009; Callewaert et al., 2000; Sarantinopoulos et al., 2002). According to FAO/WHO (2002), probiotics are defined as “live microorganisms which when administered in adequate amounts confer a health benefit on the host”. A probiotic food is a processed product containing viable probiotic microorganisms in amounts of about 106–107 CFU/g, which are able to maintain their characteristics during the production and commercialization of the product, as well as to survive during passage in the gastrointestinal tract of the consumer (FAO/WHO, 2002). The consumption of probiotics has beneficial effects, including balancing of colonic microbiota, protection of the normal intestinal microbiota and prevention of gastrointestinal disorders, reduction of serum cholesterol, antagonism against food-borne pathogens and improvement in the nutritional value of foods (Hlivak et al., 2005; Huang and Zheng, 2009; Pascual et al., 2010; Salminem et al., 1996). Several studies have mentioned the use of E. faecium as probiotic cultures (Ahmadova et al., 2013; Gardiner et al., 1999; Lund et al., 2000; Matijašic´ et al., 2010; Saavedra et al., 2003). Their ability to survive and compete in the gastrointestinal tract allows their successful use. The objective of this work was to evaluate four E. faecium isolated from fermented products for potential use as probiotic strains, concerning their antimicrobial activity, their ability to survive at mild hot temperatures of cooking and their survival during simulated
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gastrointestinal tract passage. Furthermore, we tested the protective role of different matrices during simulated gastrointestinal tract passage. 2. Materials and methods 2.1. Origin of isolates Four isolates (85, 101, 119, 120) belonging to E. faecium species, from a collection of 182 enterococci isolated from various stages of production (in the processing plants) and final products of traditional fermented meat products (Barbosa et al., 2009; Ferreira et al., 2006, 2007) were selected. They presented only one virulence factor, efaAfm, commonly found in E. faecium food isolates (Barbosa et al., 2010), and some intermediate antibiotic susceptibilities determined by agar microdilution method (Barbosa et al., 2009). The isolates 85 and 101, both from the same producer, were collected from Alheira paste before stuffing in March 2005 and Alheira (final product) in May 2005, respectively. The isolates 119 and 120 were both collected from Chouriça paste before stuffing in March 2005 from another producer. 2.2. Growth and storage conditions All the isolates used in this work were grown on Tryptic Soy Agar (Pronadisa, Madrid, Spain) supplemented with 0.6% (w/v) of Yeast Extract (Lab M, Lancashire, United Kingdom) (TSAYE) at 37 °C for 24 h and stored at − 80 °C in Tryptic Soy Broth (TSB) containing 30% (v/v) of glycerol (Sigma, Steinheim, Germany), and sub-cultured twice before use in assays. 2.3. Amino acid decarboxylase detection The detection of amino acid decarboxylase was assessed according to Bover-Cid and Holzapfel (1999). The enterococcal isolates were subcultured seven times in Brain Heart Infusion Broth (BHIB, Lab M) containing 0.1% of each precursor amino acid (all from Sigma): tyrosine free base for tyramine, histidine monohydrochloride for histamine, ornithine monohydrochloride for putrescine and lysine monohydrochloride for cadaverine, and supplemented with 0.005% of pyridoxal-5-phosphate (Fluka, Steinheim, Germany), with the purpose of promoting enzyme induction. Each isolate was then spotted in duplicate on the agar medium, developed by the same authors, with each amino acid and incubated, under aerobic conditions, at 37 °C for 4 days. Plates without amino acid were used as controls. Positive reaction was confirmed when a purple color occurred or tyrosine precipitate disappeared around the colonies (Bover-Cid and Holzapfel, 1999).
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24 h, subsequently, one colony of each isolate was transferred to 10 ml of TSBYE and incubated overnight at 37 °C. Suspensions of each target bacterial culture were spread onto TSAYE plates and 10-μL drops of each Enterococcus isolate were spotted on the lawns of target bacteria and incubated overnight at 37 °C. All the tests were carried out in duplicate and Pediococcus acidilactici HA-6111-2 was used as the positive control (Albano et al., 2007). Clear halo zones observed around the spots were registered as positive for antimicrobial activity. In order to determine the nature of inhibition for those isolates which showed antimicrobial activity, the Enterococcus isolates suspensions were centrifuged at 8877 ×g for 10 min at 4 °C (Rotina 35R, Hettich, Germany), and the supernatants were adjusted to pH 6.0 with sterile 1 M of NaOH (Pronalab, Lisbon, Portugal) and aliquots then treated for 1 h with 0.1 mg/ml of catalase and 0.1 mg/ml of trypsin (both from Sigma). After these treatments, supernatants were spotted against target bacteria. 2.5. Heat resistance of isolates 2.5.1. Inoculum From TSAYE incubated at 37 °C for 24 h, one colony of each Enterococcus isolate was transferred to 10 ml of TSBYE and incubated in the same conditions. For the final inoculum, 0.1 ml of the last culture was transferred to 10 ml of TSBYE (1:100) and incubated at 37 °C for 24 h to reach stationary phase. Each isolate was harvested by centrifugation (8877 ×g, 10 min, 37 °C; Rotina 35R), re-suspended in 10 ml of sterile quarter strength Ringer's solution (Lab M) and mixed to obtain an inoculum of approximately 107 CFU/ml. 2.5.2. Heat resistance experiment Aliquots of 0.5 ml of inoculum were transferred to glass flasks with 49.5 ml of Buffered Peptone Water (BPW, LabM) at pH 7.0. The glass flasks were kept at 65 °C and samples were taken at time 0 (time of inoculation), 5, 10, 15, 20, 30, 40, 50 and 60 min. All assays were done in duplicate. For each experiment an aliquot of 0.5 ml of inoculum was placed into glass flasks with: 49.5 ml of BPW at pH 7.0 and 37 °C, and used as control. Serial decimal dilutions of each sample were made in sterile quarter strength Ringer's solution and plated for enumeration by the drop count technique (Miles and Misra, 1938). Each dilution was plated on TSAYE in duplicate. After incubation at 37 °C for 48 h, the colonies were counted and the CFU/ml was calculated. 2.6. Gastrointestinal tract simulation 2.6.1. Inoculum The inoculum was prepared as described in Section 2.5.1.
2.4. Antimicrobial activity 2.6.2. Different matrices used 2.4.1. Target bacteria To evaluate the antimicrobial activity of Enterococcus isolates, the following target bacteria were used: five Gram positive isolates – Bacillus cereus, Bacillus subtilis, Listeria monocytogenes 7946, L. monocytogenes 7947 and Listeria innocua 2030c – and five Gram negative isolates – Salmonella Typhimurium, Salmonella enteritidis, Klebsiella pneumoniae, Proteus vulgaris and Pseudomonas aeruginosa – all isolated from foods and belonging to Escola Superior de Biotecnologia culture collection and 8 reference strains — L. innocua NCTC 11286, Enterococcus faecalis ATCC 29212, E. faecalis DSMZ 12956, E. faecium DSMZ 13590, Enterococcus gallinarum DSMZ 20628, Enterococcus flavescens DSMZ 7370, Enterococcus casseliflavus DSMZ 20680 and S. aureus ATCC 29213. 2.4.2. Antimicrobial activity screening The agar spot test method was used. Each target bacterium and Enterococcus isolates were grown on TSAYE and incubated at 37 °C for
2.6.2.1. Skim milk. Each isolate grown in TSBYE (1:10) was harvested by centrifugation (8877 ×g, 10 min, 37 °C; Rotina 35R) and re-suspended in 0.5 ml of skim milk (11% w/v, Sigma), being 10 min in contact before use in following tests. 2.6.2.2. Alheira. Each isolate grown in TSBYE (1:10) was harvested by centrifugation (8877 ×g, 10 min, 37 °C), re-suspended in 0.5 ml of sterile quarter strength Ringer's solution and mixed with 5 g of Alheira, being in contact for 10 min before use in following tests. 2.6.3. Simulated gastrointestinal conditions The simulation was achieved according to Barbosa et al. (2012). Aliquots of 0.5 ml of inoculum (re-suspended in skim milk or placed in 5 g of Alheira) were placed into glass flasks with 49.5 ml of BPW adjusted to pH 2.5 with hydrochloric acid (1 M HCl, Pronalab) and
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Fig. 1. Logarithmic reduction obtained during the temperature treatment of four Enterococcus faecium isolates: (
with 1000 units/ml of a filter sterilized solution of pepsin (Sigma). Also an aliquot of 0.5 ml of inocula was placed into a glass flask with 49.5 ml of BPW (without pH adjustment or pepsin) as a matrix control. The glass flasks were kept at 37 °C and samples were taken at time 0 (time of inoculation) and 30 min until a total of 60 min (quick gastric transit simulation) or for a total of 120 min (slow gastric transit simulation). After this, a filter sterilized solution of sodium hydroxide (1 M NaOH, Pronalab) was added to each glass flask in order to increase the pH from 2.5 to 7.0 and a sterile solution of bile salts (Pronadisa) was also added to achieve a final concentration of 0.3% (w/v). The flasks were held at 37 °C and samples were taken at time 0 (time of bile salts addition) and every 30 min for a total of 60 min (quick digestion simulation) or for a total of 120 min (slow digestion simulation). All assays were done in triplicate. Serial decimal dilutions of each sample were made in sterile quarter strength Ringer's solution and plated for enumeration. For each experiment five controls were done: an aliquot of 0.5 ml of inoculum was placed into glass flasks with: 49.5 ml of BPW at pH 7.0; 49.5 ml of BPW at pH 2.5; 49.5 ml of BPW at pH 7.0 with 1000 units/ml of pepsin; and two glass flasks with 49.5 ml of BPW at pH 7.0; after 60 min, a bile salt solution was added (0.3% (w/v) final concentration) to one, and
Table 1 Antimicrobial activity screening of Enterococcus isolates against some target bacteria used (before the determination of the nature of inhibition). Target bacteria
Enterococcus isolate 85
101
119
120
L. monocytogenes 7946 L. monocytogenes 7947 E. faecalis ATCC 29212 E. faecium DSMZ 13590 L. innocua 2030c L. innocua NTCT 11286 S. aureus ATCC 29213
− − − ± ± − −
− − − ± ± ± −
− − − − − − −
++ ++ ± ++ ++ + +
−: Absence of halo (0 mm). ±: Thin halo (b2 mm). +: Median halo (2–4 mm). ++: Large halo (N4 mm).
) control (inoculum in BPW at 37 °C), (
) BPW at 65 °C.
after 120 min, a bile salt solution was added to the other. All controls were performed in duplicate. Enumeration was done as described above. 2.6.4. Statistical analysis Three replicates were conducted for each experiment. Microbial counts were transformed to logarithmic reduction using the equation: log (N / N0), where N is the microbial cell density at a particular sampling time and N0 is the initial cell density. An analysis of variance (one-way ANOVA) was performed to assess any significant effects of different matrices used as well as of slow and quick gastric transit simulations and digestions on the survival of Enterococcus isolates in simulated GI tract. Multiple comparisons were evaluated by Tukey's post-hoc test. All calculations were carried out using the software Kaleidagraph (version 4.4, Synergy Software, Reading, USA). 3. Results and discussion Four E. faecium strains isolated from fermented products were chosen as not possessing virulence genes, in addition to efaAfm gene, commonly found in E. faecium food isolates (Barbosa et al., 2010). Strains of Enterococcus without hemolytic activity and the absence of cytolysin and vancomycin resistance genes may be considered as safe and can be used as starter cultures or probiotics (De Vuyst et al., 2003). In the assays for amino acid decarboxylase, none of the isolates had positive reactions for tyramine, histamine, putrescine and cadaverine in the screening medium used. Sarantinopoulos et al. (2001) also found E. faecium strains which did not produce biogenic amines. Bover-Cid et al. (2001) obtained similar results for enterococci isolated from fermented pork sausages, with the exception of positive reactions for tyramine. Since the ingestion of these amines has various toxicological implications, being considered potentially dangerous to human health (Ferreira and Pinho, 2006; Las Rivas et al., 2006), the inability of these isolates to produce biogenic amines is, together with their absence of virulence factors, an advantage. It is important to emphasize that the present study was based on the results obtained by in vitro microbiological methods. To evaluate the potential use of these isolates as probiotic cultures a preliminary test was done to assess their survival at the temperature of
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Fig. 2. Logarithmic reduction of E. faecium strain 120 through quick ( ) and slow ( ) digestions simulation for each matrix. The dotted lines correspond to the controls used for the different matrices: pH 7.0 (●); pH 7.0 with pepsin ( ); pH 2.5 without pepsin (▲); pH 7.0 and bile salts added after 60 min (♦) and pH 7.0 and bile salts added after 120 min ( ). The gray lines mean that the isolate was reduced to values below the detection limit of the enumeration technique.
65 °C. This temperature was chosen because in a previous study, which monitored the internal temperature of Alheira during real scenarios of cooking, namely electric grilling, frying and roasting in a gas oven as well as a wood-fired oven, the highest temperature achieved was 65 °C (Felício et al., 2011). According to the obtained results, after 1 h of exposure at a temperature of 65°C, there was a decrease between 1.0 and 2.7 log-units (Fig. 1), being the isolate 119 which had a smaller reduction at the end of the exposure time. It is important to highlight that 1 h is an exaggerated time of exposure, since in the absence of a timetemperature set for this type of product, generally the cooking time is much less, approximately 15 min. Over 15 min at 65 °C there were no significant differences in viability of the isolates 85 (p = 0.90), 101 (p = 0.76) and 119 (p = 0.78), with logarithmic reductions of 0.2, 0.3 and 0.2, respectively. For the isolate 120 the reduction was greater with 1.2 log-unit reduction (p b 0.05). Considering these low reduction values and that reduction in food matrices would be even lower, we assume that all of the four isolates are resistant to cooking at these conditions. Since the '80s, enterococci have been recognized as among the most thermotolerant non-spore forming bacteria (Magnus et al., 1988; Sanz Perez et al., 1982).
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Table 1 shows the results for antimicrobial activity of each isolate against some target bacteria used. Among 18 target bacteria used in this study, the growth of only seven strains, belonging to five different species, was inhibited. Isolate 119 did not show antimicrobial activity against any of the target bacteria used, in contrast to isolate 120 which inhibited all seven target bacteria. Growth inhibition can result from competition and production of lactic acid, hydrogen peroxide or bacteriocins. According to the screening method used, isolates 85, 101 and 120, produced proteinaceous compounds that exhibited antimicrobial activity (activity was lost only after the addition of trypsin). This suggests that the activity was caused by a bacteriocin. In the case of S. aureus, growth inhibition was caused by the production of lactic acid (activity was lost after the neutralization of culture supernatant). E. faecium 120 inhibited important foodborne pathogens such as L. monocytogenes and S. aureus. These are commonly found in fermented foods rendering these products as unacceptable/potentially hazardous (Ferreira et al., 2007). Many authors have demonstrated the bacteriocinogenic activity of E. faecium against food pathogens (Ibarguren et al., 2010; Pinto et al., 2009; Vera Pingitore et al., 2012). Inhibition of some of the target enterococci investigated was also observed for E. faecium 120. Enterococcus spp. are present in high values in fermented foods and their inhibition may be important, since some strains of this genus possesses virulence factors (Barbosa et al., 2010; Semedo et al., 2003; Valenzuela et al., 2008). The inhibition of pathogens' growth suggests that E. faecium isolates may have potential application in food preservation. Taking into account that isolate 120 survived a temperature of 65 °C and showed antimicrobial activity against important pathogenic organisms, it was selected to assess its survival in simulated gastrointestinal tract passage. Furthermore, this isolate has important characteristics which allow considering as safe, such as the absence of virulence factors and the absence of antibiotic resistances — just with an intermediate susceptibility to ciprofloxacin and nitrofurantoin (Barbosa et al., 2009). These results are not a reason of concern, since it is not considered an antibiotic resistance and, furthermore, the microbiological cut-off values for these two antibiotics are not defined into the Panel on Additives and Products or Substances in Animal Feed (FEEDAP) of European Food Safety Authority (EFSA, 2012). As this strain was isolated from a fermented meat product, we decided to use Alheira as matrix to assess this ability. A high stability of enterococci in Alheira is expected since it was demonstrated that they are normally present in this sausage in values higher than 6.5 log CFU/g (Ferreira et al., 2006). Strains of this genus are frequently isolated from fermented sausages where no competitive starter cultures are used such as Alheira (Hugas et al., 2003). Additionally, skim milk was assessed as a suitable matrix since many probiotics are added to dairy products. Fig. 2 shows the results obtained for the survival of E. faecium strain 120 through simulated quick and slow digestion, for both matrices and controls. For both matrices used, no significant differences (p b 0.05) were obtained between the two types of digestion — quick and slow. When pH 2.5 with pepsin (gastric transit) was applied as the gastrointestinal tract simulation, there was a greater reduction in viable cells in the skim milk matrix (4.67 log reduction in quick digestion and 6.78 log reduction in long digestion), than in the Alheira matrix (only 0.17 log reduction in quick digestion and 0.58 log reduction in slow digestion). These differences may be explained by the different compositions of the matrices. Alheira, with its high fat content (18.4%), may confer a protective effect to the isolate when it is in contact with the acid. Also the fact that this isolate was collected from a fermented meat product, it may have adapted it to this environment (Ferreira et al., 2006). When bile salts were added (simulated small intestine digestion), for both types of digestion, unlike the skim milk (for which the isolate was reduced to values below the detection limited of the enumeration technique), there was a further reduction of only approximately 1 log for the Alheira matrix. Once again, the Alheira conferred protection on this Enterococcus isolate.
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Several studies have demonstrated that many Enterococcus strains, including E. faecium species, are resistant to the conditions that simulate the gastrointestinal tract (Lund et al., 2002; Marciňáková et al., 2010; Nueno-Palop and Narbad, 2011). Our results allowed demonstrating this Enterococcus resistance, and also that this resistance is influenced by the matrix of the product. Therefore, the choice of the food product is crucial to ensure maximum stability of probiotic culture, so that it exerts a beneficial effect on the consumer. 4. Conclusion Some crucial characteristics such as the absence of virulence factors, the absence of antibiotic resistances – just with an intermediate susceptibility to ciprofloxacin and nitrofurantoin (Barbosa et al., 2009) – the ability to survive exposure to high temperatures, to inhibit the growth of food-borne pathogens and to survive through gastrointestinal tract passage, make the E. faecium isolate 120 studied in this work a potential candidate for further investigations concerning its use as a probiotic culture. Acknowledgments This work was supported by the National Funds from FCT — Fundação para a Ciência e a Tecnologia through projects PEst-OE/EQB/ LA0016/2013 and POCTI/AGG/39587/2001. The financial support for author J. Barbosa was provided by the PhD fellowship, SFRH/BD/48894/2008 (FCT). The authors would also like to acknowledge Dr. Paul Gibbs for editing the English language. References Ahmadova, A., Todorov, S.D., Choiset, Y., Rabesona, H., Zadi, T.M., Kuliyev, A., Franco, B.D.G.M., Chobert, J.M., Haertlé, T., 2013. Evaluation of antimicrobial activity, probiotic properties and safety of wild strain Enterococcus faecium AQ71 isolated from Azerbaijani Motal cheese. Food Control 30, 631–641. Albano, H., Todorov, S.D., van Reenen, C.A., Hogg, T., Dicks, L.M., Teixeira, P., 2007. Characterization of two bacteriocins produced by Pediococcus acidilactici isolated from “Alheira”, a fermented sausage traditionally produced in Portugal. Int. J. Food Microbiol. 116, 239–247. Barbosa, J., Ferreira, V., Teixeira, P., 2009. Antibiotic susceptibility of enterococci isolated from traditional fermented meat products. Food Microbiol. 26, 527–532. Barbosa, J., Gibbs, P.A., Teixeira, P., 2010. Virulence factors among enterococci isolated from traditional fermented meat products produced in the North of Portugal. Food Control 21, 651–656. Barbosa, J., Borges, S., Magalhães, R., Ferreira, V., Santos, I., Silva, J., Almeida, G., Gibbs, P., Teixeira, P., 2012. Behaviour of Listeria monocytogenes isolates through gastro-intestinal tract passage simulation, before and after two sub-lethal stresses. Food Microbiol. 30, 24–28. Bouton, Y., Buchin, S., Pochet, S., Beuvier, E., 2009. Effect of mesophilic lactobacilli and enterococci adjunct cultures on the final characteristics of a microfiltered milk Swisstype cheese. Food Microbiol. 26, 183–191. Bover-Cid, S., Holzapfel, W.H., 1999. Improved screening procedure for biogenic amine production by lactic acid bacteria. Int. J. Food Microbiol. 53, 33–41. Bover-Cid, S., Hugas, M., Izquierdo-Pulido, M., Vidal-Carou, M.C., 2001. Amino acid-decarboxylase activity of bacteria isolated from fermented pork sausages. Int. J. Food Microbiol. 66, 185–189. Callewaert, R., Hugas, M., De Vuyst, L., 2000. Competitiveness and bacteriocin production of enterococci in the production of Spanish-style dry fermented sausages. Int. J. Food Microbiol. 57, 33–42. De Vuyst, L., Foulquié Moreno, M.R., Revets, H., 2003. Screening for enterocins and detection of hemolysin and vancomycin resistance in enterococci of different origins. Int. J. Food Microbiol. 84, 299–318. European Food Safety Authority, 2012. Guidance on the assessment of bacterial susceptibility to antimicrobials of human and veterinary importance by EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP). EFSA J. 10, 2740–2749. Felício, M.T.S., Ramalheira, R., Ferreira, V., Brandão, T., Silva, J., Hogg, T., Teixeira, P., 2011. Thermal inactivation of Listeria monocytogenes from Alheiras, traditional Portuguese sausage during cooking. Food Control 22, 1960–1964. Ferreira, I.M.P.L.V.O., Pinho, O., 2006. Biogenic amines in Portuguese traditional foods and wines. J. Food Prot. 69, 2293–2303. Ferreira, V., Barbosa, J., Vendeiro, S., Mota, A., Silva, F., Monteiro, M.J., Hogg, T., Gibbs, P., Teixeira, P., 2006. Chemical and microbiological characterization of Alheira: a typical Portuguese fermented sausage with particular reference to factors relating to food safety. Meat Sci. 73, 570–575.
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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
Selection of potential probiotic Enterococcus faecium isolated from Portuguese fermented food Joana Barbosa, Sandra Borges, Paula Teixeira ⁎ CBQF-Centro de Biotecnologia e Química Fina — Laboratório Associado, Escola Superior de Biotecnologia, Universidade Católica Portuguesa/Porto, Rua Dr. António Bernardino Almeida, 4200-072 Porto, Portugal
a r t i c l e
i n f o
Article history: Received 10 February 2014 Received in revised form 28 August 2014 Accepted 14 September 2014 Available online 19 September 2014 Keywords: Enterococcus faecium Fermented product Probiotic Antimicrobial activity Gastrointestinal tract survival
a b s t r a c t Four Enterococcus faecium strains isolated from fermented products were evaluated for potential use as probiotic strains. In addition to efaAfm gene, commonly found in E. faecium food isolates, none of the isolates possessed virulence genes and none had positive reactions for the production of tyramine, histamine, putrescine and cadaverine in the screening medium used. All of these four isolates proved to be resistant to 65 °C. E. faecium 119 did not show antimicrobial activity against any of the target bacteria investigated. E. faecium 85 and 101 inhibited Listeria innocua and E. faecium DSMZ 13590. The strain E. faecium 120 inhibited seven target bacteria (Listeria monocytogenes 7946, L. monocytogenes 7947, L. innocua 2030c, L. innocua NCTC 11286, E. faecium DSMZ 13590, Enterococcus faecalis ATCC 29212 and Staphylococcus aureus ATCC 29213) and was chosen as the representative to assess the ability to survive gastrointestinal tract passage simulation, as well as the protective role of two food matrices (skim milk and Alheira) during its passage. For both matrices used, no significant differences (p b 0.05) were obtained between the types of digestion — quick and slow passage simulation. In the skim milk matrix the isolate was reduced to values below the detection limit of the enumeration technique by the end of the two digestions, in contrast to the Alheira matrix, for which isolate 120 showed a reduction of only ca. 1 log CFU/ml. The E. faecium strain 120 was shown to be a potential candidate for further investigations as a potential probiotic culture. © 2014 Published by Elsevier B.V.
1. Introduction Although being ubiquitous microorganisms, enterococci have as their main habitat the gastrointestinal tract of humans and warm-blooded animals (Murray, 1990). A small number of enterococci are also present in oropharyngeal secretions, in vaginal secretions and on the skin, especially in the perineal area and, consequently, they can be considered as normal inhabitants of the human organism (Kayser, 2003). Enterococci are involved in the development of the typical organoleptic characteristics of a variety of fermented foods such as cheeses, fermented sausages and vegetables where, in some cases, they are the predominant lactic acid bacteria (Franz et al., 2011; Macedo et al., 1995; Suzzi et al., 2000). In Portuguese fermented meat products the numbers of enterococci ranged from 104 to 108 CFU/g in Alheira and from 104 to 107 CFU/g in Chouriça (Ferreira et al., 2006, 2007), because of their tolerance to low pH, sodium chloride and nitrite (Martin et al., 2005). Various studies have commented on the benefits of using Enterococcus, particularly Enterococcus faecium strains, as adjunct cultures in fermented foods, because of their ability to inhibit the growth of food-borne
⁎ Corresponding author. Tel.: +351 22 5580001; fax: +351 22 5090351. E-mail address: [email protected] (P. Teixeira).
http://dx.doi.org/10.1016/j.ijfoodmicro.2014.09.009 0168-1605/© 2014 Published by Elsevier B.V.
pathogens, commonly present in these kinds of products (Bouton et al., 2009; Callewaert et al., 2000; Sarantinopoulos et al., 2002). According to FAO/WHO (2002), probiotics are defined as “live microorganisms which when administered in adequate amounts confer a health benefit on the host”. A probiotic food is a processed product containing viable probiotic microorganisms in amounts of about 106–107 CFU/g, which are able to maintain their characteristics during the production and commercialization of the product, as well as to survive during passage in the gastrointestinal tract of the consumer (FAO/WHO, 2002). The consumption of probiotics has beneficial effects, including balancing of colonic microbiota, protection of the normal intestinal microbiota and prevention of gastrointestinal disorders, reduction of serum cholesterol, antagonism against food-borne pathogens and improvement in the nutritional value of foods (Hlivak et al., 2005; Huang and Zheng, 2009; Pascual et al., 2010; Salminem et al., 1996). Several studies have mentioned the use of E. faecium as probiotic cultures (Ahmadova et al., 2013; Gardiner et al., 1999; Lund et al., 2000; Matijašic´ et al., 2010; Saavedra et al., 2003). Their ability to survive and compete in the gastrointestinal tract allows their successful use. The objective of this work was to evaluate four E. faecium isolated from fermented products for potential use as probiotic strains, concerning their antimicrobial activity, their ability to survive at mild hot temperatures of cooking and their survival during simulated
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gastrointestinal tract passage. Furthermore, we tested the protective role of different matrices during simulated gastrointestinal tract passage. 2. Materials and methods 2.1. Origin of isolates Four isolates (85, 101, 119, 120) belonging to E. faecium species, from a collection of 182 enterococci isolated from various stages of production (in the processing plants) and final products of traditional fermented meat products (Barbosa et al., 2009; Ferreira et al., 2006, 2007) were selected. They presented only one virulence factor, efaAfm, commonly found in E. faecium food isolates (Barbosa et al., 2010), and some intermediate antibiotic susceptibilities determined by agar microdilution method (Barbosa et al., 2009). The isolates 85 and 101, both from the same producer, were collected from Alheira paste before stuffing in March 2005 and Alheira (final product) in May 2005, respectively. The isolates 119 and 120 were both collected from Chouriça paste before stuffing in March 2005 from another producer. 2.2. Growth and storage conditions All the isolates used in this work were grown on Tryptic Soy Agar (Pronadisa, Madrid, Spain) supplemented with 0.6% (w/v) of Yeast Extract (Lab M, Lancashire, United Kingdom) (TSAYE) at 37 °C for 24 h and stored at − 80 °C in Tryptic Soy Broth (TSB) containing 30% (v/v) of glycerol (Sigma, Steinheim, Germany), and sub-cultured twice before use in assays. 2.3. Amino acid decarboxylase detection The detection of amino acid decarboxylase was assessed according to Bover-Cid and Holzapfel (1999). The enterococcal isolates were subcultured seven times in Brain Heart Infusion Broth (BHIB, Lab M) containing 0.1% of each precursor amino acid (all from Sigma): tyrosine free base for tyramine, histidine monohydrochloride for histamine, ornithine monohydrochloride for putrescine and lysine monohydrochloride for cadaverine, and supplemented with 0.005% of pyridoxal-5-phosphate (Fluka, Steinheim, Germany), with the purpose of promoting enzyme induction. Each isolate was then spotted in duplicate on the agar medium, developed by the same authors, with each amino acid and incubated, under aerobic conditions, at 37 °C for 4 days. Plates without amino acid were used as controls. Positive reaction was confirmed when a purple color occurred or tyrosine precipitate disappeared around the colonies (Bover-Cid and Holzapfel, 1999).
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24 h, subsequently, one colony of each isolate was transferred to 10 ml of TSBYE and incubated overnight at 37 °C. Suspensions of each target bacterial culture were spread onto TSAYE plates and 10-μL drops of each Enterococcus isolate were spotted on the lawns of target bacteria and incubated overnight at 37 °C. All the tests were carried out in duplicate and Pediococcus acidilactici HA-6111-2 was used as the positive control (Albano et al., 2007). Clear halo zones observed around the spots were registered as positive for antimicrobial activity. In order to determine the nature of inhibition for those isolates which showed antimicrobial activity, the Enterococcus isolates suspensions were centrifuged at 8877 ×g for 10 min at 4 °C (Rotina 35R, Hettich, Germany), and the supernatants were adjusted to pH 6.0 with sterile 1 M of NaOH (Pronalab, Lisbon, Portugal) and aliquots then treated for 1 h with 0.1 mg/ml of catalase and 0.1 mg/ml of trypsin (both from Sigma). After these treatments, supernatants were spotted against target bacteria. 2.5. Heat resistance of isolates 2.5.1. Inoculum From TSAYE incubated at 37 °C for 24 h, one colony of each Enterococcus isolate was transferred to 10 ml of TSBYE and incubated in the same conditions. For the final inoculum, 0.1 ml of the last culture was transferred to 10 ml of TSBYE (1:100) and incubated at 37 °C for 24 h to reach stationary phase. Each isolate was harvested by centrifugation (8877 ×g, 10 min, 37 °C; Rotina 35R), re-suspended in 10 ml of sterile quarter strength Ringer's solution (Lab M) and mixed to obtain an inoculum of approximately 107 CFU/ml. 2.5.2. Heat resistance experiment Aliquots of 0.5 ml of inoculum were transferred to glass flasks with 49.5 ml of Buffered Peptone Water (BPW, LabM) at pH 7.0. The glass flasks were kept at 65 °C and samples were taken at time 0 (time of inoculation), 5, 10, 15, 20, 30, 40, 50 and 60 min. All assays were done in duplicate. For each experiment an aliquot of 0.5 ml of inoculum was placed into glass flasks with: 49.5 ml of BPW at pH 7.0 and 37 °C, and used as control. Serial decimal dilutions of each sample were made in sterile quarter strength Ringer's solution and plated for enumeration by the drop count technique (Miles and Misra, 1938). Each dilution was plated on TSAYE in duplicate. After incubation at 37 °C for 48 h, the colonies were counted and the CFU/ml was calculated. 2.6. Gastrointestinal tract simulation 2.6.1. Inoculum The inoculum was prepared as described in Section 2.5.1.
2.4. Antimicrobial activity 2.6.2. Different matrices used 2.4.1. Target bacteria To evaluate the antimicrobial activity of Enterococcus isolates, the following target bacteria were used: five Gram positive isolates – Bacillus cereus, Bacillus subtilis, Listeria monocytogenes 7946, L. monocytogenes 7947 and Listeria innocua 2030c – and five Gram negative isolates – Salmonella Typhimurium, Salmonella enteritidis, Klebsiella pneumoniae, Proteus vulgaris and Pseudomonas aeruginosa – all isolated from foods and belonging to Escola Superior de Biotecnologia culture collection and 8 reference strains — L. innocua NCTC 11286, Enterococcus faecalis ATCC 29212, E. faecalis DSMZ 12956, E. faecium DSMZ 13590, Enterococcus gallinarum DSMZ 20628, Enterococcus flavescens DSMZ 7370, Enterococcus casseliflavus DSMZ 20680 and S. aureus ATCC 29213. 2.4.2. Antimicrobial activity screening The agar spot test method was used. Each target bacterium and Enterococcus isolates were grown on TSAYE and incubated at 37 °C for
2.6.2.1. Skim milk. Each isolate grown in TSBYE (1:10) was harvested by centrifugation (8877 ×g, 10 min, 37 °C; Rotina 35R) and re-suspended in 0.5 ml of skim milk (11% w/v, Sigma), being 10 min in contact before use in following tests. 2.6.2.2. Alheira. Each isolate grown in TSBYE (1:10) was harvested by centrifugation (8877 ×g, 10 min, 37 °C), re-suspended in 0.5 ml of sterile quarter strength Ringer's solution and mixed with 5 g of Alheira, being in contact for 10 min before use in following tests. 2.6.3. Simulated gastrointestinal conditions The simulation was achieved according to Barbosa et al. (2012). Aliquots of 0.5 ml of inoculum (re-suspended in skim milk or placed in 5 g of Alheira) were placed into glass flasks with 49.5 ml of BPW adjusted to pH 2.5 with hydrochloric acid (1 M HCl, Pronalab) and
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Fig. 1. Logarithmic reduction obtained during the temperature treatment of four Enterococcus faecium isolates: (
with 1000 units/ml of a filter sterilized solution of pepsin (Sigma). Also an aliquot of 0.5 ml of inocula was placed into a glass flask with 49.5 ml of BPW (without pH adjustment or pepsin) as a matrix control. The glass flasks were kept at 37 °C and samples were taken at time 0 (time of inoculation) and 30 min until a total of 60 min (quick gastric transit simulation) or for a total of 120 min (slow gastric transit simulation). After this, a filter sterilized solution of sodium hydroxide (1 M NaOH, Pronalab) was added to each glass flask in order to increase the pH from 2.5 to 7.0 and a sterile solution of bile salts (Pronadisa) was also added to achieve a final concentration of 0.3% (w/v). The flasks were held at 37 °C and samples were taken at time 0 (time of bile salts addition) and every 30 min for a total of 60 min (quick digestion simulation) or for a total of 120 min (slow digestion simulation). All assays were done in triplicate. Serial decimal dilutions of each sample were made in sterile quarter strength Ringer's solution and plated for enumeration. For each experiment five controls were done: an aliquot of 0.5 ml of inoculum was placed into glass flasks with: 49.5 ml of BPW at pH 7.0; 49.5 ml of BPW at pH 2.5; 49.5 ml of BPW at pH 7.0 with 1000 units/ml of pepsin; and two glass flasks with 49.5 ml of BPW at pH 7.0; after 60 min, a bile salt solution was added (0.3% (w/v) final concentration) to one, and
Table 1 Antimicrobial activity screening of Enterococcus isolates against some target bacteria used (before the determination of the nature of inhibition). Target bacteria
Enterococcus isolate 85
101
119
120
L. monocytogenes 7946 L. monocytogenes 7947 E. faecalis ATCC 29212 E. faecium DSMZ 13590 L. innocua 2030c L. innocua NTCT 11286 S. aureus ATCC 29213
− − − ± ± − −
− − − ± ± ± −
− − − − − − −
++ ++ ± ++ ++ + +
−: Absence of halo (0 mm). ±: Thin halo (b2 mm). +: Median halo (2–4 mm). ++: Large halo (N4 mm).
) control (inoculum in BPW at 37 °C), (
) BPW at 65 °C.
after 120 min, a bile salt solution was added to the other. All controls were performed in duplicate. Enumeration was done as described above. 2.6.4. Statistical analysis Three replicates were conducted for each experiment. Microbial counts were transformed to logarithmic reduction using the equation: log (N / N0), where N is the microbial cell density at a particular sampling time and N0 is the initial cell density. An analysis of variance (one-way ANOVA) was performed to assess any significant effects of different matrices used as well as of slow and quick gastric transit simulations and digestions on the survival of Enterococcus isolates in simulated GI tract. Multiple comparisons were evaluated by Tukey's post-hoc test. All calculations were carried out using the software Kaleidagraph (version 4.4, Synergy Software, Reading, USA). 3. Results and discussion Four E. faecium strains isolated from fermented products were chosen as not possessing virulence genes, in addition to efaAfm gene, commonly found in E. faecium food isolates (Barbosa et al., 2010). Strains of Enterococcus without hemolytic activity and the absence of cytolysin and vancomycin resistance genes may be considered as safe and can be used as starter cultures or probiotics (De Vuyst et al., 2003). In the assays for amino acid decarboxylase, none of the isolates had positive reactions for tyramine, histamine, putrescine and cadaverine in the screening medium used. Sarantinopoulos et al. (2001) also found E. faecium strains which did not produce biogenic amines. Bover-Cid et al. (2001) obtained similar results for enterococci isolated from fermented pork sausages, with the exception of positive reactions for tyramine. Since the ingestion of these amines has various toxicological implications, being considered potentially dangerous to human health (Ferreira and Pinho, 2006; Las Rivas et al., 2006), the inability of these isolates to produce biogenic amines is, together with their absence of virulence factors, an advantage. It is important to emphasize that the present study was based on the results obtained by in vitro microbiological methods. To evaluate the potential use of these isolates as probiotic cultures a preliminary test was done to assess their survival at the temperature of
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Fig. 2. Logarithmic reduction of E. faecium strain 120 through quick ( ) and slow ( ) digestions simulation for each matrix. The dotted lines correspond to the controls used for the different matrices: pH 7.0 (●); pH 7.0 with pepsin ( ); pH 2.5 without pepsin (▲); pH 7.0 and bile salts added after 60 min (♦) and pH 7.0 and bile salts added after 120 min ( ). The gray lines mean that the isolate was reduced to values below the detection limit of the enumeration technique.
65 °C. This temperature was chosen because in a previous study, which monitored the internal temperature of Alheira during real scenarios of cooking, namely electric grilling, frying and roasting in a gas oven as well as a wood-fired oven, the highest temperature achieved was 65 °C (Felício et al., 2011). According to the obtained results, after 1 h of exposure at a temperature of 65°C, there was a decrease between 1.0 and 2.7 log-units (Fig. 1), being the isolate 119 which had a smaller reduction at the end of the exposure time. It is important to highlight that 1 h is an exaggerated time of exposure, since in the absence of a timetemperature set for this type of product, generally the cooking time is much less, approximately 15 min. Over 15 min at 65 °C there were no significant differences in viability of the isolates 85 (p = 0.90), 101 (p = 0.76) and 119 (p = 0.78), with logarithmic reductions of 0.2, 0.3 and 0.2, respectively. For the isolate 120 the reduction was greater with 1.2 log-unit reduction (p b 0.05). Considering these low reduction values and that reduction in food matrices would be even lower, we assume that all of the four isolates are resistant to cooking at these conditions. Since the '80s, enterococci have been recognized as among the most thermotolerant non-spore forming bacteria (Magnus et al., 1988; Sanz Perez et al., 1982).
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Table 1 shows the results for antimicrobial activity of each isolate against some target bacteria used. Among 18 target bacteria used in this study, the growth of only seven strains, belonging to five different species, was inhibited. Isolate 119 did not show antimicrobial activity against any of the target bacteria used, in contrast to isolate 120 which inhibited all seven target bacteria. Growth inhibition can result from competition and production of lactic acid, hydrogen peroxide or bacteriocins. According to the screening method used, isolates 85, 101 and 120, produced proteinaceous compounds that exhibited antimicrobial activity (activity was lost only after the addition of trypsin). This suggests that the activity was caused by a bacteriocin. In the case of S. aureus, growth inhibition was caused by the production of lactic acid (activity was lost after the neutralization of culture supernatant). E. faecium 120 inhibited important foodborne pathogens such as L. monocytogenes and S. aureus. These are commonly found in fermented foods rendering these products as unacceptable/potentially hazardous (Ferreira et al., 2007). Many authors have demonstrated the bacteriocinogenic activity of E. faecium against food pathogens (Ibarguren et al., 2010; Pinto et al., 2009; Vera Pingitore et al., 2012). Inhibition of some of the target enterococci investigated was also observed for E. faecium 120. Enterococcus spp. are present in high values in fermented foods and their inhibition may be important, since some strains of this genus possesses virulence factors (Barbosa et al., 2010; Semedo et al., 2003; Valenzuela et al., 2008). The inhibition of pathogens' growth suggests that E. faecium isolates may have potential application in food preservation. Taking into account that isolate 120 survived a temperature of 65 °C and showed antimicrobial activity against important pathogenic organisms, it was selected to assess its survival in simulated gastrointestinal tract passage. Furthermore, this isolate has important characteristics which allow considering as safe, such as the absence of virulence factors and the absence of antibiotic resistances — just with an intermediate susceptibility to ciprofloxacin and nitrofurantoin (Barbosa et al., 2009). These results are not a reason of concern, since it is not considered an antibiotic resistance and, furthermore, the microbiological cut-off values for these two antibiotics are not defined into the Panel on Additives and Products or Substances in Animal Feed (FEEDAP) of European Food Safety Authority (EFSA, 2012). As this strain was isolated from a fermented meat product, we decided to use Alheira as matrix to assess this ability. A high stability of enterococci in Alheira is expected since it was demonstrated that they are normally present in this sausage in values higher than 6.5 log CFU/g (Ferreira et al., 2006). Strains of this genus are frequently isolated from fermented sausages where no competitive starter cultures are used such as Alheira (Hugas et al., 2003). Additionally, skim milk was assessed as a suitable matrix since many probiotics are added to dairy products. Fig. 2 shows the results obtained for the survival of E. faecium strain 120 through simulated quick and slow digestion, for both matrices and controls. For both matrices used, no significant differences (p b 0.05) were obtained between the two types of digestion — quick and slow. When pH 2.5 with pepsin (gastric transit) was applied as the gastrointestinal tract simulation, there was a greater reduction in viable cells in the skim milk matrix (4.67 log reduction in quick digestion and 6.78 log reduction in long digestion), than in the Alheira matrix (only 0.17 log reduction in quick digestion and 0.58 log reduction in slow digestion). These differences may be explained by the different compositions of the matrices. Alheira, with its high fat content (18.4%), may confer a protective effect to the isolate when it is in contact with the acid. Also the fact that this isolate was collected from a fermented meat product, it may have adapted it to this environment (Ferreira et al., 2006). When bile salts were added (simulated small intestine digestion), for both types of digestion, unlike the skim milk (for which the isolate was reduced to values below the detection limited of the enumeration technique), there was a further reduction of only approximately 1 log for the Alheira matrix. Once again, the Alheira conferred protection on this Enterococcus isolate.
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Several studies have demonstrated that many Enterococcus strains, including E. faecium species, are resistant to the conditions that simulate the gastrointestinal tract (Lund et al., 2002; Marciňáková et al., 2010; Nueno-Palop and Narbad, 2011). Our results allowed demonstrating this Enterococcus resistance, and also that this resistance is influenced by the matrix of the product. Therefore, the choice of the food product is crucial to ensure maximum stability of probiotic culture, so that it exerts a beneficial effect on the consumer. 4. Conclusion Some crucial characteristics such as the absence of virulence factors, the absence of antibiotic resistances – just with an intermediate susceptibility to ciprofloxacin and nitrofurantoin (Barbosa et al., 2009) – the ability to survive exposure to high temperatures, to inhibit the growth of food-borne pathogens and to survive through gastrointestinal tract passage, make the E. faecium isolate 120 studied in this work a potential candidate for further investigations concerning its use as a probiotic culture. Acknowledgments This work was supported by the National Funds from FCT — Fundação para a Ciência e a Tecnologia through projects PEst-OE/EQB/ LA0016/2013 and POCTI/AGG/39587/2001. The financial support for author J. Barbosa was provided by the PhD fellowship, SFRH/BD/48894/2008 (FCT). The authors would also like to acknowledge Dr. Paul Gibbs for editing the English language. References Ahmadova, A., Todorov, S.D., Choiset, Y., Rabesona, H., Zadi, T.M., Kuliyev, A., Franco, B.D.G.M., Chobert, J.M., Haertlé, T., 2013. Evaluation of antimicrobial activity, probiotic properties and safety of wild strain Enterococcus faecium AQ71 isolated from Azerbaijani Motal cheese. Food Control 30, 631–641. Albano, H., Todorov, S.D., van Reenen, C.A., Hogg, T., Dicks, L.M., Teixeira, P., 2007. Characterization of two bacteriocins produced by Pediococcus acidilactici isolated from “Alheira”, a fermented sausage traditionally produced in Portugal. Int. J. Food Microbiol. 116, 239–247. Barbosa, J., Ferreira, V., Teixeira, P., 2009. Antibiotic susceptibility of enterococci isolated from traditional fermented meat products. Food Microbiol. 26, 527–532. Barbosa, J., Gibbs, P.A., Teixeira, P., 2010. Virulence factors among enterococci isolated from traditional fermented meat products produced in the North of Portugal. Food Control 21, 651–656. Barbosa, J., Borges, S., Magalhães, R., Ferreira, V., Santos, I., Silva, J., Almeida, G., Gibbs, P., Teixeira, P., 2012. Behaviour of Listeria monocytogenes isolates through gastro-intestinal tract passage simulation, before and after two sub-lethal stresses. Food Microbiol. 30, 24–28. Bouton, Y., Buchin, S., Pochet, S., Beuvier, E., 2009. Effect of mesophilic lactobacilli and enterococci adjunct cultures on the final characteristics of a microfiltered milk Swisstype cheese. Food Microbiol. 26, 183–191. Bover-Cid, S., Holzapfel, W.H., 1999. Improved screening procedure for biogenic amine production by lactic acid bacteria. Int. J. Food Microbiol. 53, 33–41. Bover-Cid, S., Hugas, M., Izquierdo-Pulido, M., Vidal-Carou, M.C., 2001. Amino acid-decarboxylase activity of bacteria isolated from fermented pork sausages. Int. J. Food Microbiol. 66, 185–189. Callewaert, R., Hugas, M., De Vuyst, L., 2000. Competitiveness and bacteriocin production of enterococci in the production of Spanish-style dry fermented sausages. Int. J. Food Microbiol. 57, 33–42. De Vuyst, L., Foulquié Moreno, M.R., Revets, H., 2003. Screening for enterocins and detection of hemolysin and vancomycin resistance in enterococci of different origins. Int. J. Food Microbiol. 84, 299–318. European Food Safety Authority, 2012. Guidance on the assessment of bacterial susceptibility to antimicrobials of human and veterinary importance by EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP). EFSA J. 10, 2740–2749. Felício, M.T.S., Ramalheira, R., Ferreira, V., Brandão, T., Silva, J., Hogg, T., Teixeira, P., 2011. Thermal inactivation of Listeria monocytogenes from Alheiras, traditional Portuguese sausage during cooking. Food Control 22, 1960–1964. Ferreira, I.M.P.L.V.O., Pinho, O., 2006. Biogenic amines in Portuguese traditional foods and wines. J. Food Prot. 69, 2293–2303. Ferreira, V., Barbosa, J., Vendeiro, S., Mota, A., Silva, F., Monteiro, M.J., Hogg, T., Gibbs, P., Teixeira, P., 2006. Chemical and microbiological characterization of Alheira: a typical Portuguese fermented sausage with particular reference to factors relating to food safety. Meat Sci. 73, 570–575.
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