In Vitro Evaluation of the Probiotic Potential of Bacteriocin Producer Lactobacillus Sakei 1

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Journal of Food Protection, Vol. 75, No. 6, 2012, Pages 1083–1089 doi:10.4315/0362-028X.JFP-11-523 Copyright G, International Association for Food Protection

In Vitro Evaluation of the Probiotic Potential of Bacteriocin Producer Lactobacillus sakei 1

BRUNA C. GOMES,* MARINA R. RODRIGUES, LIZZIANE K. WINKELSTRO¨ TER, AURO NOMIZO, AND ELAINE C. P. DE MARTINIS

Faculdade de Cieˆncias Farmaceûticas de Ribeiraõ Preto–Universidade de Saõ Paulo, Av. do Cafe´ s/n, CEP 14040-903, Ribeiraõ Preto/SP, Brazil Downloaded from http://meridian.allenpress.com/jfp/article-pdf/75/6/1083/1683422/0362-028x_jfp-11-523.pdf by guest on 28 September 2021

MS 11-523: Received 28 November 2011/Accepted 10 February 2012

ABSTRACT Lactobacillus sakei 1 is a food isolate that produces a heat-stable antimicrobial peptide (sakacin 1, a class IIa bacteriocin) inhibitory to the opportunistic pathogen Listeria monocytogenes. Bacterial isolates with antimicrobial activity may be useful for food biopreservation and also for developing probiotics. To evaluate the probiotic potential of L. sakei 1, it was tested for (i) in vitro gastric resistance (with synthetic gastric juice adjusted to pH 2.0, 2.5, or 3.0); (ii) survival and bacteriocin production in the presence of bile salts and commercial prebiotics (inulin and oligofructose); (iii) adhesion to Caco-2 cells; and (iv) effect on the adhesion of L. monocytogenes to Caco-2 cells and invasion of these cells by the organism. The results showed that L. sakei 1 survival in gastric environment varied according to pH, with the maximum survival achieved at pH 3.0, despite a 4-log reduction of the population after 3 h. Regarding the bile salt tolerance and influence of prebiotics, it was observed that L. sakei 1 survival rates were similar (P . 0.05) for all de Man Rogosa Sharpe (MRS) broth formulations when tests were done after 4 h of incubation. However, after incubation for 24 h, the survival of L. sakei 1 in MRS broth was reduced by 1.8 log (P ,0.001), when glucose was replaced by either inulin or oligofructose (without Oxgall). L. sakei 1 was unable to deconjugate bile salts, and there was a significant decrease (1.4 log) of the L. sakei 1 population in regular MRS broth plus Oxgall (P , 0.05). In spite of this, tolerance levels of L. sakei 1 to bile salts were similar in regular MRS broth and in MRS broth with oligofructose. Lower bacteriocin production was observed in MRS broth when inulin (3,200 AU/ml) or oligofructose (2,400 AU/ml) was used instead of glucose (6,400 AU/ml). L. sakei 1 adhered to Caco-2 cells, and its cell-free pH-neutralized supernatant containing sakacin 1 led to a significant reduction of in vitro listerial invasion of human intestinal Caco-2 cells.

The development of products containing starter cultures evaluation of (i) gastrointestinal resistance, (ii) capability to with probiotic potential is a very promising approach in the hydrolyze bile salts, (iii) adhesion to intestinal epithelial field of functional foods. Lactobacillus sakei is a lactic acid cells, (iv) inhibition of pathogens’ adhesion, and (v) bacterium (LAB) originally isolated from rice wine and is antimicrobial action against pathogenic bacteria. The last used as starter culture for meat fermentation, with potential property is important for the protection of GIT from to be applied in food biopreservation (3, 29, 41). There is microbial infection, and it has been the focus of many also interest in the application of L. sakei as a probiotic with studies (27, 31, 35). potential for health benefit. Recently, Woo et al. (44) Recently, Santos et al. (34) reported that Lactobacillus studied the effect of the intake of L. sakei KCTC 10755BP delbrueckii UFV-H2b20 protected germ-free mice against by children with atopic eczema-dermatitis syndrome and Listeria monocytogenes infection by favoring effector showed a positive correlation between ingestion of LAB and responses and preventing immunopathological consequenc- alleviation of the clinical severity of atopic eczema- es. Listeriosis is of special concern for food safety because it dermatitis syndrome, as well as decreased chemokine levels is transmitted mainly by foods and may cause high fatality (44). rates among neonates, the elderly, and immunocompromised The ability to resist low pH, digestive enzymes, and persons. The GIT health may also be improved by the bile salts (32, 36, 40) enables probiotic organisms to reach consumption of prebiotics, such as inulin and oligofructose. the site of action in the intestine and to survive in the Prebiotics are defined as nondigestible soluble carbohydrates gastrointestinal tract (GIT). The World Health Organization with ability to selectively stimulate the activity, survival, or (14) recommends the selection of probiotic cultures by even the colonization of gut by beneficial microorganisms in humans and animals (16, 33). Lactobacillus and Bifidobac- * Author for correspondence. Present address: Escola de Engenharia de Saõ terium are the main genera of colonic microorganisms Carlos–USP, Departamento de Hidraúlica e Saneamento, Laborato´rio de stimulated by prebiotic compounds (16, 37). Processos Biolo´gicos, Av. Joaõ Dagnone, 1100, CEP:13563-120, Saõ Carlos/SP, Brazil. Tel: z55 16 3602 4267; Fax: z55 16 3602 4725; The purpose of the present study was to evaluate the E-mail: [email protected]. probiotic potential of L. sakei 1, an isolate from fresh pork 1084 GOMES ET AL. J. Food Prot., Vol. 75, No. 6

TABLE 1. Description of the commercial prebiotics used in this study Prebiotic Source Commercial name Compositiona

Inulin Extracted from chicory root Beneo HPX–Orafti Inulin (99.5%) Oligofructose Partial enzymatic hydrolysis of inulin Beneo P95–Orafti Oligofructose (93.2%) a Data from the manufacturer, BENEO-Orafti Active Food Ingredients, Tienen, Belgium. sausage that produces a bacteriocin of 4.4 kDa (sakacin 1), was inoculated into 9 ml of MRS broth containing prebiotics inulin with antilisterial activity and potential to inhibit early stages or oligofructose at 20 mg ml21. Both these cultures also contained 21 of biofilm formation by L. monocytogenes on an abiotic bile salts at 3 mg ml (Oxgall, Sigma). Regular MRS broth (20 21 surface (1, 10–12, 26, 43). Experiments were conducted mg ml glucose) containing bile salts was used as control in these with L. sakei 1 to evaluate in vitro gastric survival and the experiments. Samples of each broth (0.1 ml) were withdrawn after 4 and 24 h, and viable cells were enumerated by surface plating on influence of commercial prebiotics on bile salt tolerance and Downloaded from http://meridian.allenpress.com/jfp/article-pdf/75/6/1083/1683422/0362-028x_jfp-11-523.pdf by guest on 28 September 2021 MRS agar following incubation at 37uC for 48 h (22, 38). bacteriocin production. The adhesion ability of L. sakei 1to Caco-2 cells was evaluated, and the effect of its bacteriocin Bacteriocin production by L. sakei 1 in broths containing on adhesion and invasion of Caco-2 cells by L. monocyto- prebiotics. Bacteriocin was quantified by critical dilution assay genes was also studied. (28) after overnight growth of L. sakei 1 in MRS broths containing 20 mg of prebiotics (inulin or oligofructose) per ml and regular MATERIALS AND METHODS MRS broth (control). Briefly, the cell-free pH-neutralized super- Bacterial strains and culture media. Bacteriocin producer natants of L. sakei 1 (CFNS-S1) from each broth formulation were L. sakei 1 was previously isolated from Brazilian fresh pork serially diluted with PBS (10 mM, pH 7.0). Each dilution (10 ml) was spotted onto BHI agar plates previously overlaid with 7 ml sausage and produces sakacin 1 (10, 12). L. monocytogenes ATCC 5 6 19115 was used as indicator strain in agar antagonism tests (19). of soft BHI agar inoculated with ca. 10 to 10 CFU of L. Working cultures were prepared (1%, vol/vol) in de Man monocytogenes per ml. Plates were incubated at 37uC for 24 h, and Rogosa Sharpe broth (MRS; Oxoid, Basingstoke, UK) for LAB and results were expressed in arbitrary units per milliliter (AU/ml), in brain heart infusion (BHI) broth (Oxoid) for L. monocytogenes defined as the reciprocal of the highest dilution that presented an and incubated at 25 and 37uC, respectively, for 24 h. Stock cultures inhibition zone. were kept at 280uC in appropriate liquid media containing 20% (vol/vol) glycerol (Synth, Diadema, Brazil). Prebiotics inulin Caco-2 cell culture. Human intestinal epithelial Caco-2 cells (ATCC HBT37) were obtained from the Rio de Janeiro Cell Bank (Orafti-HP, BENEO-Orafti, Tienen, Belgium) and oligofructose 2 (Orafti-P95, BENEO-Orafti) were used at 20 mg ml21 (Table 1). In (UFRJ, Rio de Janeiro, Brazil) and routinely cultivated in 75-cm selected experiments, glucose from MRS broth was replaced by tissue culture flasks (TPP, Trasadingen, Switzerland) at 37uC under inulin (MRS plus inulin) or oligofructose (MRS plus oligofructose). 5% CO2, humidified atmosphere (Forma Scientific, Marietta, OH), in complete RPMI medium, composed of RPMI 1640 medium In vitro survival of L. sakei 1 in gastric juice. Synthetic (Gibco, Gaithersburg, MD) supplemented with 2.4 g of HEPES per gastric juice was prepared by addition of 3.0 g liter21 pepsin liter (Invitrogen, Carlsbad, CA), 2.0 g of sodium bicarbonate per (Sigma, St. Louis, MO) in sodium chloride solution (0.85%, wt/ liter, 100 g of heat-inactivated fetal bovine serum per liter vol) and adjusted to pH 2.0, 2.5, or 3.0 with 0.1 N HCl (6). (Invitrogen), 100 U of streptomycin per ml, and 100 mgof Populations of L. sakei 1 in overnight cultures of MRS broth were penicillin per ml (Sigma). The medium was replaced every 2 days enumerated by surface plating (0.1 ml) of appropriate dilutions on until a confluent monolayer was achieved. The Caco-2 cell culture 5 MRS agar before challenge and incubated at 37uC for 48 h. The was trypsinized, transferred (10 cells per well) to 24-well tissue remaining broth (ca. 30 ml) was centrifuged (6,000 | g, 20 min, culture plates (TPP) containing 12-mm-diameter glass coverslips 5uC; Sorvall Legend Mach 1.6R, Wilmington, DE), washed twice (30), and incubated under 5% CO2 in complete RPMI medium for with phosphate-buffered saline (PBS), pH 7.4, and the pellet was 24 h. The medium was removed, adhered Caco-2 cells were suspended in 3.0 ml of the same buffer. One milliliter of the washed twice with PBS, and 1 ml of RPMI incomplete (without suspension was centrifuged (12,000 | g, 5 min, 5uC), and L. sakei antibiotics) was added (17, 30). 1 cells were resuspended in 10 ml of the synthetic gastric juice. The suspension was kept at 37uC, and populations were L. sakei 1 adhesion to Caco-2 cells. Cultures of Caco-2 cells enumerated at 0, 1, 2, and 3 h by surface plating on MRS agar were prepared as previously described, inoculated with 100 mlofan 6 followed by incubation at 37uC for 48 h. The results were active culture of L. sakei 1 (ca. 10 bacteria per well), and incubated expressed as log CFU per milliliter (6, 42). at 37uC for 2 h under 5% CO2. The coverslips were gently washed three times with PBS (pH 7.4) to remove nonadherent bacterial Bile salt hydrolysis activity. Overnight culture of L. sakei 1 cells, fixed with methanol, stained with May-Grunwald-Giemsa in MRS broth was spotted (10 ml) on the surface of MRS agar (Laborclin, Pinhais, Brazil), and observed under oil immersion supplemented with 0.5% (wt/vol) taurodeoxycholic acid (Sigma) lens with a magnification of |1,000 (Axiolab-Zeiss, Stuttgart, plus 0.37 g of CaCl2 per liter (Vetec, Rio de Janeiro, Brazil) and Germany). incubated at 37uC in anaerobic jars (Anaerogen, Oxoid). Bile salt hydrolysis activity was scored by measuring precipitation zones Preparation of CFNS-S1. An overnight culture of L. sakei 1 around colonies as per the method of Franz et al. (15). was inoculated in MRS broth (1%, vol/vol) and incubated at 37uC for 24 h. The broth culture was centrifuged at 4uC at 6,720 | g for Effect of commercial prebiotics on tolerance of L. sakei 1 25 min (Sorvall RC Plus, Du Pont, Wilmington, DE), the pH of to bile salts. Overnight culture of L. sakei 1 in MRS broth (1 ml) supernatant was adjusted to pH 7.0, and it was filter sterilized J. Food Prot., Vol. 75, No. 6 PROBIOTIC POTENTIAL OF L. SAKEI 1 1085

FIGURE 1. Survival of L. sakei 1 during the simulated gastric digestion (0.3% [wt/vol] pepsin) at pH 2.0, 2.5, and 3.0, incubated at 37uC for up to 3 h. Data represent the means of three independent replicates ¡ standard deviations. } BC (before challenge), initial inocula of L. sakei 1 in MRS used to prepare experiments of gastric challenge at different pHs; * below the detection limit of the method (10 CFU/ml). Downloaded from http://meridian.allenpress.com/jfp/article-pdf/75/6/1083/1683422/0362-028x_jfp-11-523.pdf by guest on 28 September 2021 through 0.22-mm-pore-size membranes (Millipore, Sa˜o Paulo, the log of the total number of bacteria inoculated, multiplied by Brazil). The titer of sakacin 1 was determined by the critical 100 (4, 31). Results are presented as averages of three independent dilution assay (26, 28) using L. monocytogenes as the indicator assays done in triplicate. microorganism. The CFNS-S1 containing the semipurified sakacin 1 was diluted in incomplete RPMI at final concentrations of 0, 10, Statistical analysis. The data presented are the means (¡ 25, 50, 75, and 90% (vol/vol) and immediately used in adhesion standard deviations) of three independent replicates. The one-way and invasion assays. analysis of variance with significance level set at P values of ,0.05 was used to evaluate the differences among the treatments Adhesion of L. monocytogenes to Caco-2 cells in the studied. The Student-Newman-Keuls test or Tukey’s multiple presence of CFNS-S1. Caco-2 cells were cultivated up to comparisons test was applied to evaluate significant differences. semiconfluent growth in 24-well tissue culture plates with no Analyses were performed using the software package Sigma Stat coverslips. Overnight culture of L. monocytogenes (1 ml) grown in 3.1 (Systat Software, Inc., Richmond, CA). BHI broth at 37uC was diluted to 107 CFU/ml in RPMI incomplete medium, added to the wells to obtain the initial multiplicity of RESULTS AND DISCUSSION infection of 100:1, and incubated for 30 min at 37 C under 5 u % L. sakei 1 survival under conditions simulating CO . Cells were washed twice with PBS buffer to eliminate 2 gastric environment. It is desirable that microorganisms nonadherent bacteria, trypsinized, and centrifuged at 10,000 | g for 5 min. The pellet was suspended in 1 ml of PBS, serial dilutions intended to be used as probiotic cultures be able to survive were prepared, and each dilution was surface plated on tryptic soy under the adverse conditions of the GIT (13, 14, 32). agar supplemented with 6 g of yeast extract per liter (TSAYE; Although discrepancies can be observed between in vitro Oxoid) and incubated for 24 h at 37uC. The percentage of adhesion and in vivo assays (42), in vitro tests still constitute an was defined as the log of the number of adherent bacterial cells important tool for screening probiotic strains. (log CFU) divided by the log of the total number of bacteria Results of tests from the present study on bile salt inoculated, multiplied by 100 (2, 31). hydrolysis showed no precipitation halos in agar plates To evaluate the effect of the bacteriocin sakacin 1 in L. containing taurodeoxycholic acid, which indicated L. sakei monocytogenes adhesion to Caco-2 cells, different dilutions of CFNS- 1 did not deconjugate bile salts (data not shown). However, S1 (0, 10, 25, 50, 75, and 90%) were used in the wells of the tissue Vinderola and Reinheimer (42) reported that it is possible culture plate, either at the same time as the addition of L. monocytogenes that probiotic strains present good growth rate in media or 30 min before or 30 min after inoculation. All adhesion assays were containing bile salts, despite being unable to produce bile performed independently at least three times, in triplicate. salt hydrolases. Influence of CFNS-S1 on L. monocytogenes invasion of In Figure 1 it is shown that the L. sakei 1 population Caco-2 cells. L. monocytogenes was inoculated in Caco-2 cell was below the detection limit after 1 h of exposure to cultures as described in the previous section and kept for 60 min at synthetic gastric juice at pH values of 2.0 and 2.5. However, 37uCin5% CO2 atmosphere. To kill noninternalized bacteria, cells at pH 3.0, L. sakei 1 presented a slower death rate and it were washed twice with PBS and incubated in 250 mlof remained viable for up to 3 h, despite a 4-log reduction of incomplete RPMI medium containing 250 mg of gentamicin per the population. In a study by Charteris et al. (6), it was ml for 60 min at 37uCin5% CO2 atmosphere. Cells were washed observed that most strains of Lactobacillus spp. and with PBS, and Caco-2 cells were lysed with 0.1% (vol/vol) Triton Bifidobacterium spp. evaluated died after exposure to X-100 (Sigma). Cell lysates were serially diluted in PBS buffer and synthetic gastric juice (pH 2.0, 0.3% pepsin, 0.5% sodium plated onto TSAYE agar (0.1 ml) to enumerate intracellular chloride). In contrast, it has been demonstrated that LAB bacteria. To evaluate the possible protective role of sakacin 1 against L. monocytogenes invasion, these assays were repeated isolated from diverse niches tolerated gastrointestinal using incomplete RPMI medium containing CFNS-S1 at 0, 10, conditions well in the presence of foods due to a buffering 25, 50, 75, and 90% dilutions, concomitant to addition of L. effect, reinforcing the hypothesis that the food matrix may monocytogenes. improve tolerance of probiotic bacteria to gastrointestinal The invasion efficiency (percentage) was calculated as the log conditions (5, 23, 24). Vinderola and Reinheimer (42) of the number of intracellular bacterial cells (log CFU) divided by highlighted that gastric tolerance is a strain-specific 1086 GOMES ET AL. J. Food Prot., Vol. 75, No. 6

FIGURE 2. Populations of L. sakei 1 after 4 h (A) and 24 h (B) of exposure to bile salts (3 mg ml21 Oxgall) in MRS broth in the presence of prebiotics (inulin and oligofructose), incubated at 37uC. Data represent means of three independent replicates ¡ standard deviations. Different letters represent statistically significant differences at a P value of ,0.001. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/75/6/1083/1683422/0362-028x_jfp-11-523.pdf by guest on 28 September 2021

characteristic and affirmed that low gastric resistance of fructoligosaccharides, and trehalose (7, 39). According to LAB should not exclude a strain from probiotic classifica- Leroy and De Vuyst (21), the conditions for production of tion if it exhibits other properties of interest. bacteriocins are not easily predicted because the regulation of the synthesis of these antimicrobial peptides depends on Effect of commercial prebiotics on bacteriocin complex parameters. production by L. sakei 1. In this research, a high production of sakacin 1 was observed in standard MRS Effect of commercial prebiotics on tolerance of L. medium (6,400 AU/ml), while with the prebiotics inulin and sakei 1 to bile salts. At 4 h, there were no significant oligofructose the titers of bacteriocin were only 3,200 and differences in L. sakei 1 viable counts (P . 0.05) for any 2,400 AU/ml, respectively. Our results are in accordance MRS formulations tested (Fig. 2A). However, after 24 h, with those reported by Vamanu and Vamanu (39), who the viability of L. sakei 1 in MRS broth was 1.8 log lower (P analyzed bacteriocin production by Lactobacillus paracasei , 0.001) when glucose was replaced by either inulin or CMGB16 using the prebiotics inulin, raffinose, and oligofructose (without Oxgall). A statistically significant lactulose and found that inulin did not increase bacteriocin decrease (1.4 log) of the L. sakei 1 population in regular production (24-h growth). MRS broth plus Oxgall (Fig. 2B) after 24 h was also On the other hand, in the literature there are several observed. The replacement of glucose as the main reports on the improvement of bacteriocin production when carbohydrate source by inulin or oligofructose did not LAB were grown in the presence of raffinose, lactulose, improve (P , 0.05) tolerance of L. sakei 1 to bile salts J. Food Prot., Vol. 75, No. 6 PROBIOTIC POTENTIAL OF L. SAKEI 1 1087 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/75/6/1083/1683422/0362-028x_jfp-11-523.pdf by guest on 28 September 2021

FIGURE 3. Photomicrography showing adhesion of L. sakei 1in Caco-2 epithelial cells at 2 h, stained with May-Grunwald-Giemsa. The image was obtained with an oil immersion lens (magnification, |1,000). Bar, 5 mm.

(Fig. 2B). However, the survival of L. sakei 1 was statistically similar when broths containing bile salts were formulated with either glucose or oligofructose. This may represent an advantage for the development of symbiotic food formulations, since it has been demonstrated that some prebiotics may protect probiotic strains during passage through GIT models (25), besides the intrinsic prebiotic effect of selective stimulation of beneficial bacteria from the gut residential microbiota.

Inhibition of L. monocytogenes adhesion to and invasion of Caco-2 cells. The microphotography presented in Figure 3 shows L. sakei 1 adhered to Caco-2 cells, an important property for a transient colonization of GIT, for stimulation of the immune system, and for antagonistic FIGURE 4. Adhesion (in percentages) of L. monocytogenes in activity against other enteropathogens (9, 20, 45). Haller Caco-2 cells when incubated with different concentrations of the et al. (18) showed the adhesion of several food-fermenting cell-free neutralized supernatant of L. sakei 1 containing sakacin bacterial strains to Caco-2 cells and observed that soluble 1. (A) CFNS-S1 added 30 min before L. monocytogenes; (B) factors from spent culture supernatants of lactobacilli CFNS-S1 added concomitantly during incubation; (C) CFNS-S1 added 30 min after addition of L. monocytogenes. Results are cultures promoted adhesion to the eukaryotic cells. means of triplicates ¡ standard deviations. An asterisk (*) Culture supernatant of L. sakei 1 (CFNS-S1) influenced indicates statistically significant difference from control. L. monocytogenes adhesion to Caco-2 cells (Fig. 4). Application of 90% CFNS-S1 (1,440 AU/ml) for 30 min before or during inoculation of L. monocytogenes led to Our results of L. monocytogenes invasion to Caco-2 significant reduction (P , 0.05) of L. monocytogenes cells (37.91% ¡ 3.74%) were comparable to the results of adhesion to Caco-2 cells (Fig. 4A and 4B). However, Moroni et al. (31), who used L. monocytogenes LSD348 in a addition (Fig. 4C) of the CFNS-S1 after 30 min of similar in vitro model (44.65%). The present results (Fig. 5) incubation did not affect L. monocytogenes adhesion to also revealed that L. monocytogenes was unable to invade Caco-2 cells. Similar results were reported by Coconnier Caco-2 cells when 90 and 75% CFNS-S1 were added to the et al. (8), who found that pretreatment of Salmonella cultures (P , 0.05). The addition of 50 and 25% CFNS-S1 enterica serovar Typhimurium SL1344 cells with spent also resulted in reduced invasion, while 10% CFNS-S1 did culture supernatant of Lactobacillus acidophilus strain LB- not modify invasion of L. monocytogenes compared to the SCS inhibited the infection and decreased the number of control without CFNS-S1. intracellular viable cells in cultured human intestinal Caco-2 Botes et al. (2) reported that cell-free neutralized cells. The protective effect was not observed when LB-SCS supernatants of Lactobacillus plantarum 423 (plantaricin was added after Salmonella infection to Caco-2 cells, 423) and Enterococcus mundtii ST4SA (peptide ST4SA) indicating that its mechanism of action involved interaction did not prevent adhesion of L. monocytogenes to Caco-2 with the pathogen (8). cells but did inhibit invasion of L. monocytogenes strain 1088 GOMES ET AL. J. Food Prot., Vol. 75, No. 6

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Downloaded from http://meridian.allenpress.com/jfp/article-pdf/75/6/1083/1683422/0362-028x_jfp-11-523.pdf by guest on 28 September 2021 FIGURE 5. Invasion (in percentages) of L. monocytogenes in Spadaro, and E. C. P. De Martinis. 2009. Purification, characterization and N-terminal amino acid sequencing of sakacin 1, a bacteriocin Caco-2 cells incubated with different concentrations of the CFNS- produced by Lactobacillus sakei 1. J. Food Saf. 29:636–649. S1 containing sakacin 1 at 37 C for 1 h. Results are means of u 11. De Martinis, E. C. P., and F. Z. Freitas. 2003. Screening of lactic acid ¡ triplicates standard deviations. A single asterisk (*) indicates bacteria from Brazilian meats for bacteriocin formation. Food Control that counts were below the detection limit of the method (10 CFU/ 14:197–200. ml); a double asterisk (**) indicates the estimated counts (1 to 12. De Martinis, E. C. P., M. R. P. Publio, P. R. Santarosa, and F. Z. 25 CFU per plate). Freitas. 2001. 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Available at: ftp: //ftp.fao.org/docrep/fao/009/a0512e/a0512e00.pdf. Accessed 14 were similar in the presence of glucose and of oligofructose. March 2012. The CFNS-S1 presented a protective effect for intestinal 15. Franz, C. M. A. P., I. Specht, P. Haberer, and W. H. Holzapfel. 2001. Caco-2 cells against invasion of L. monocytogenes, thus Bile salt hydrolase activity of enterococci isolated from food: suggesting that bacteriocins may influence the dynamic of screening and quantitative determination. J. Food Prot. 64:725–729. GIT colonization by pathogens. 16. Gibson, G. R., and M. B. Roberfroid. 1995. Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. J. Nutr. 125:1401–1412. ACKNOWLEDGMENTS 17. Gomes, B. C., C. T. Esteves, I. C. V. Palazzo, A. L. C. Darini, G. E. This research was supported by Sa˜o Paulo Research Foundation Felis, L. A. Sechi, B. D. G. M. Franco, and E. C. P. De Martinis. 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