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The antimicrobial effect of wine on Bacillus cereus in simulated gastro-intestinal conditions
Food Control 28 (2012) 230e236
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The antimicrobial effect of wine on Bacillus cereus in simulated gastro-intestinal conditions Miguel Vaz, Tim Hogg, José António Couto* CBQF/Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Rua Dr. António Bernardino de Almeida, 4200-072 Porto, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 December 2011 Received in revised form 4 May 2012 Accepted 15 May 2012
This study aimed to evaluate the antimicrobial activity of wine against Bacillus cereus vegetative cells and spores. The results clearly show that wine exerts a strong inactivation effect against vegetative cells of B. cereus. The red wine tested inactivated stationary phase cultures to undetectable numbers in less than 10 s. Thus, further inactivation assays were carried out with wine diluted with water (1:4 and 1:8). Diluted wine 1:4 caused a reduction of approximately 5 log cycles on viable cell counts in 20 s. On the other hand, B. cereus spores were found to be highly resistant to wine exposure. The influence of wine components (organic acids, ethanol and phenolic compounds) was investigated on vegetative cells. The wine organic acids tested exhibited a strong inactivation effect, and, when combined with ethanol, a slight synergistic effect was observed. The wine phenolic compounds assayed displayed no activity against the vegetative cells at the concentrations tested. At the simulated gastric conditions studied (in the presence of food), wine diminished considerably the number of B. cereus viable cells in addition to the effect of the synthetic gastric fluid. The behaviour of B. cereus spores under gastro-intestinal conditions was also evaluated. In a consumption-like scenario, the addition of wine led to lower total counts (vegetative cells þ spores) of B. cereus in the simulated intestine conditions, showing that wine inhibits the proliferation of the vegetative cells obtained from the germination of spores. This work provides evidence that consumption of wine during a meal may diminish the number of viable cells of B. cereus and reduces the impact of the germination of spores that may occur in the small intestine, thus lowering the risk of toxi-infection that may be caused by this pathogen. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Bacillus cereus Wine Cell inactivation Gastro-intestinal conditions
1. Introduction Several studies demonstrate the antibacterial properties of wine against relevant food-borne pathogenic bacteria (Carneiro, Couto, Mena, Queiroz, & Hogg, 2008; Correia et al., 2003; Fernandes, Gomes, Couto, & Hogg, 2007; Moretro & Daeschel, 2004; SugitaKonishi, Hara-Kudo, Iwamoto, & Kondo, 2001; Waite & Daeschel, 2007; Weisse, Eberly, & Person, 1995). Weisse et al. (1995) reported that red and white wines were able to reduce the viability of several bacteria responsible for traveller’s diarrhoea by 5e6 log cycles in 20 min. Similar inactivation patterns were obtained by Sugita-Konishi et al. (2001) for Salmonella enteritidis, Escherichia coli O157:H7 and Vibrio parahaemolyticus. Moretro and Daeschel (2004) have shown that wine has bactericidal activity against E. coli, Listeria monocytogenes, Salmonella typhimurium and Staphylococcus aureus. More recently, Carneiro et al. (2008) and
* Corresponding author. Tel.: þ351 225580027; fax: þ351 225090351. E-mail address: [email protected] (J.A. Couto). 0956-7135/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2012.05.034
Fernandes et al. (2007) have focused their studies on the activity of wine against the important food-borne pathogens Listeria and Campylobacter jejuni, respectively. The exact mechanisms responsible for the antimicrobial effect of wine are, however, not fully understood. Wine is a beverage with some unique characteristics. It possesses a relatively high ethanol content in addition to other antimicrobial agents like organic acids, low pH, polyphenol compounds and preservatives (Just & Daeschel, 2003). In vitro studies indicate that, for a given ethanol concentration, wine has a more potent antibacterial activity than other alcoholic beverages. This potency has been partly attributed to the combination of ethanol and organic acids (Just & Daeschel, 2003; Weisse et al., 1995). Malic and tartaric acids are the most abundant organic acids in wine, and their antimicrobial effects are well known, especially at low pH conditions, such as those found in wines (Hsiao & Siebert, 1999; Ricke, 2003). The importance of ethanol to this overall antimicrobial activity is illustrated by the findings of Just and Daeschel (2003) who showed that a grape juice had a very little antimicrobial activity against E. coli O157:H7 and Salmonella spp., whereas wine made from the same juice demonstrated
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considerable activity in this respect. The bactericidal effect of ethanol alone, in concentrations generally encountered in wine (between 10 and 13% v/v) is low, when compared with the bactericidal effect of wine itself (Just & Daeschel, 2003; Marimon, Bujanda, Gutierrez-Stampa, Cosme, & Arenas, 1998; Weisse et al., 1995). Moretro and Daeschel (2004) found that a combination of ethanol with organic acids, at pH 3.0, had the strongest bactericidal effect. Carneiro et al. (2008) reported that ethanol and certain organic acids act synergistically and represent the major components for the bactericidal effects of wine on Campylobacter. Studies incorporating wine as a food additive in the form of marinades provide further evidence of its protective role (Friedman, Henika, Levin, & Mandrell, 2006). Friedman et al. (2007) have evaluated the bactericidal activity of several wine recipes against Bacillus cereus, E. coli O157:H7, L. monocytogenes, and Salmonella enterica. The wine marinades were highly effective against the four pathogens. The extremely low pH of the stomach (wpH 1e2) is considered a major defence barrier against food-borne infection, this being recognized for various bacterial pathogens, such as Vibrio, Salmonella and Campylobacter (Barmpalia-Davis, Geornaras, Kendall, & Sofos, 2008; Tamplin, 2005). It’s not easy to model the survival of bacteria in the human stomach due to the huge diversity of food matrices and individual human physiological characteristics. However, it is possible to study pathogen behaviour under controlled conditions that approximate gastric conditions. For example, Clavel, Carlin, Lairon, Nguyen-The, and Schmitt (2004) studied the survival of B. cereus spores and vegetative cells in acid media simulating the human stomach. Wijnands, Pielaat, Dufrenne, Zwietering, and van Leusden (2009) modelled the number of viable vegetative cells of B. cereus passing through the stomach. B. cereus is a Gram positive spore-forming bacterium that is a common contaminant in a wide variety of foods, such as pasta, rice, dairy products, dried foodstuffs, vegetables, fruit, seafood, meat and poultry (Schoeni & Wong, 2005). Two distinct food-borne disease types, emetic and diarrhoeal, are associated with B. cereus. The intoxication, or emetic syndrome, is caused by a single peptide toxin produced in the food prior to consumption (Agata, Ohta, Mori, & Isobe, 1995; Ehling-Schulz, Fricker, & Scherer, 2004); the toxicinfection, or diarrhoeal syndrome, is caused by enterotoxins produced in the small intestine, including two three-component toxins (haemolysin BL and non haemolytic enterotoxin), and a single protein toxin (cytotoxin K) (Beecher, Schoeni, & Lee Wong, 1995; Granum, O’Sullivan, & Lund, 1999; Lund, DeBuyser, & Granum, 2000). Bacterial spores and/or vegetative cells may survive the stomach’s acidic conditions and germinate within the small intestine (Wijnands, Dufrenne, van Leusden, & Abee, 2007). B. cereus is an important cause of food-borne disease worldwide (Clavel et al., 2007; Granum, 2007), although it is probably highly under-reported in official lists of food-borne disease causes. In the European Union, Bacillus species (including non-cereus) were reported to be responsible for 1.4% of food-borne outbreaks in 2005 (Anonymous, 2006). Between 1993 and 1998 in the Netherlands, B. cereus accounted for 12% of food-borne disease outbreaks where a causative agent was identified (Schmidt, 2001). In this work, we aimed to evaluate the effect of red wine on B. cereus vegetative cells and spores. Firstly, we have characterised the effect of direct exposure of cells and spores to wine and the antimicrobial activity of selected wine components (organic acids, ethanol, low pH and phenolic compounds) in separate and in combination. Then, the kinetics of inactivation of B. cereus vegetative cells in simulated gastric conditions, with and without wine, in food consumption scenarios (wine/food/bacteria) was evaluated. Finally, we studied the behaviour of spores on simulated gastric and intestinal conditions, in food matrices with and without wine. This
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is, to our knowledge, the first report where the behaviour of vegetative cells and spores of B. cereus is characterized in direct wine immersions and under simulated gastro-intestinal conditions. The results obtained are discussed in a risk assessment context. 2. Materials and methods 2.1. Bacterial strains and growth media Two B. cereus strains from the American Type Culture Collection (ATCC) were used in this work (B. cereus ATCC 11778 and B. cereus ATCC 14579). The cultures were preserved in slants of Tryptone Soy Agar (TSA) medium (Pronadisa, Madrid, Spain) and kept at 4 C. Stock cultures of vegetative cells were kept at 80 C in Tryptone Soy Broth (TSB) medium (Pronadisa, Madrid, Spain) with glycerol 20% (v/v). For each assay, inoculated TSB medium was incubated at 30 C for 16 h to attain the stationary growth phase. 2.2. Spore production Pure B. cereus strains were grown on TSA for 24 h at 30 C. These cells were then used to inoculate TSA plates supplemented with 40 mg L1 MnSO4 (Merck, Darmstad, Germany) and 100 mg L1 CaCl2 (Merck) to stimulate sporulation. TSA plates were incubated at 30 C for 7 days. Sporulation was checked by microscopic visualization using malachite green for spore staining. Spores were collected by scraping colonies from the plates, suspended in sterile phosphate buffer at pH 7.0 and washed by centrifugation (10,000 g for 10 min). Washings were performed until the suspension was milky white. After the final centrifugation, the pellet was suspended in 10 mL of a 50% (v/v) ethanol solution and the suspension was kept at 4 C for 12 h, in order to eliminate vegetative cells, and washed again three times in sterile distilled water. The final suspension (approximately 1010 spores per mL) was distributed in sterile Eppendorf microtubes and kept at 20 C. 2.3. Wine and wine components A red wine, from the Douro demarcated region (Portugal, 2008) with an ethanol concentration of 13% (v/v) was used. The wine was filter sterilized using 0.45 mm cellulose acetate membranes (Orange Scientific, Brain L’ Alleud, Belgium) and was kept at 4 C in 1 L sterile Schott flasks until used. A mixture of organic acids was made containing the final concentrations of 2 g L1 lactic acid, 5.5 g L1 tartaric acid, 0.5 g L1 acetic and 0.5 g L1 citric acid, in the presence or absence of 13% (v/ v) ethanol and with pH adjusted to 3.3 with 1 M HCl (Pronalab, Lisbon, Portugal). Tartaric and lactic acids were obtained from Sigma (St. Louis, USA), while acetic and citric acids were obtained from Merck. A solution of 0.15 M KH2PO4 at pH 7.0 was used as control. The individual effect of each organic acid was also tested. Phenolic compounds solutions were prepared at the following concentrations: 1 mg L1 of resveratrol, 5 mg L1 of ferulic acid, 5 mg L1 of p-coumaric acid, 2 mg L1 of kaempferol and 10 mg L1 of quercetin, with pH adjusted to 3.3. All the solutions were filter sterilized using 0.45 mm cellulose acetate membranes (Orange Scientific). 2.4. Survival of B. cereus vegetative cells and spores in wine and wine components Suspensions of stationary phase vegetative cells grown in TSB medium for 16 h at 30 C and spores of B. cereus ATCC 11778 and B. cereus ATCC 14579 were used in the inactivation experiments. For the experiments with wine components, only vegetative cells were
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used. One mL of each suspension was transferred onto 100 mL Erlenmeyer flasks containing 49 mL of wine, solutions of wine components, or a control solution (0.15 M KH2PO4, pH 7.0), all at a final volume of 50 mL. These flasks were immersed in a thermostatic water bath at 37 C and stirred magnetically (200 rpm). At selected times, 1 mL samples (vegetative cells or spores) were collected and serially diluted in 9 mL of Ringer solution (Lab M, Lancashire, United Kingdom). Counts were made by the drop count technique (Miles & Misra, 1938) by plating, in triplicate, 20 mL of each ten-fold dilution onto TSA medium and incubating at 30 C for 24 h. Culture densities were expressed as colony forming units per millilitre (CFU mL1). The detection limit was taken as one colony on the lowest dilution plate, i.e. 500 CFU mL1. 2.5. Synthetic gastric and intestinal fluids The synthetic gastric fluid (SGF) was prepared following an adapted formulation from Beumer, de Vries, and Rombouts (1992) consisting of NaCl (2.05 g), KH2PO4 (0.6 g), CaCl2 (0.11 g), KCl (0.37 g) and pepsin (0.133 g) in 1 L of deionised water. The SGF was adjusted to pH 1.5 with 1 M HCl and filter sterilized using 0.20 mm cellulose acetate membranes (Orange Scientific). The synthetic intestinal fluid (SIF) was prepared following an adapted formulation from Rotard, Christmann, Knoth, and Mailahn (1995) consisting of NaCl (6.58 g), KH2PO4 (0.66 g), KCl (0.043 g), MgCl2 (0.17 g), CaCl2 (0.060 g), pancreatin (2.25 g) and bile salts (1.5 g) in 1 L of deionised water. The pH was adjusted to 7.5 with 0.5 M NaOH and the solution was sterilized by filtration using 0.20 mm cellulose acetate membranes. Both SGF and SIF were prepared fresh daily. KH2PO, MgCl2 and CaCl2 were obtained from Merck; NaCl, KCl, pepsin and pancreatin were purchased from Sigma and bile salts from Difco (Detroit, USA). 2.6. Survival of B. cereus vegetative cells in simulated gastric conditions Vegetative cells of B. cereus ATCC 14579 were suspended in 50 mL of SGF and in 43 mL of SGF supplemented with 7 mL of sterile wine for 4 min in 100 mL Erlenmeyer flasks stirred magnetically (200 rpm) and immersed in a thermostatted water bath at 37 C. 1 mL samples were taken at selected times and ten-fold dilutions were prepared. Counts were made by the drop count technique as described above. The behaviour of B. cereus vegetative cells was also studied in the presence of a food matrix. 7 mL of sterile wine and 21.5 mL of SGF were added to 21.5 g of solid food matrix (sterile chicken-rice baby meal [Nestlé Portugal, Linda-a-Velha, Portugal] and pasteurized fresh cheese [Santiago & Santiago, Lisbon, Portugal]) to a final volume of approximately 50 mL. Control solutions consisted of 28.5 mL sterile water þ 21.5 g food and 7 mL sterile water þ 21.5 mL SGF þ 21.5 g food. In proportion to the amount of food used in these experiments, the volume of wine tested corresponds, approximately, to a glass of wine ingested in a regular meal (100e150 mL). Samples were collected and plated as described above. 2.7. Behaviour of B. cereus spores in simulated gastro-intestinal conditions Experiments were carried out with spore suspensions of B. cereus ATCC 14579. The solutions tested (involving a food matrix) were prepared as described above (Section 2.6) for the vegetative cells. Spores were exposed to SGF and SIF for 60 and 240 min, respectively, as described by Wijnands, Dufrenne, Zwietering, and van Leusden (2006). After 60 min in SGF at 37 C, 1 mL of the suspensions were transferred to 49 mL of SIF and then incubated at
the same temperature for 240 min. The 50-fold dilution upon this transfer was compensated when making the calculations of the simulated intestinal data. Total counts, i.e. total number of CFU (vegetative cells þ spores) and spore counts (after heat-treatment of the ten-fold serial diluted samples at 80 C, for 10 min) were determined. 2.8. Data analysis The survival of B. cereus strains was calculated by enumeration on TSA plates and the counts were converted to log10 CFU mL1. The experiments were performed in triplicate and the results were expressed as the mean value standard error. Statistical analysis (ANOVA), significance level: P < 0.05 was recorded in an Excel spread-sheet (Microsoft Corporation, Redmond, WA, USA). 3. Results and discussion 3.1. Survival of B. cereus vegetative cells and spores in wine and wine components The red wine tested was found to be strongly effective against stationary phase vegetative cells of B. cereus ATCC 11778 and ATCC 14579, whereas spores showed a noticeable resistance to the wine exposure. Wine treatment decreased initial vegetative cell counts from 107e108 CFU mL1 to no detectable levels (<500 CFU mL1) in less than 10 s (data not shown). Therefore, further inactivation experiments were carried out with diluted wine, 1:4 and 1:8. A reduction of around 5 log cycles in the cell viability of both strains was obtained in the cultures exposed to diluted wine 1:4 in 20 s (Fig. 1). No colonies were detected (<500 CFU mL1) in the following sampling times. The wine dilution 1:8 provoked a 5 log cycles reduction on the viable cell counts of ATCC 14579 strain in 40 s, and a similar reduction was noticed in ATCC 11778 strain in 30 s of exposure. The initial populations of vegetative cells in the control solution (KH2PO4) remained constant until the end of the assay, even when a phosphate solution at pH 3.3 was tested (data not shown). Concerning the spores, only a slight decrease, less than 1 log cycle, was observed throughout the 180 min of exposure to undiluted wine (Fig. 2). Activity of grape juice and wine marinades against B. cereus has been reported previously (Friedman et al., 2007; Rhodes, Mitchell, Wilson, & Melton, 2006). Rhodes et al. (2006) found a reduction of 1 log cycle within 10 min of contact with the grape juice. Friedman et al. (2007) have evaluated the bactericidal activity of several wine recipes consisting of red and white wine extracts of oregano leaves with added garlic juice and oregano oil against B. cereus, E. coli O157:H7, L. monocytogenes, and S. enterica. The wine marinades were highly effective against the four pathogens. In comparison with published data, our work shows that vegetative cells of B. cereus are considerably sensitive to wine. Weisse et al. (1995) showed that wine reduced the viable numbers of S. enteritidis, Shigella sonnei and E. coli, 5e6 log cycles, in 20 min. The same extent of inactivation was obtained for Salmonella spp. and E. coli in 5e30 min and of 20e60 min, respectively (Harding & Maidment, 1996; Just & Daeschel, 2003; Marimon et al., 1998). Moretro and Daeschel (2004) investigated the bactericidal effect of the wine on E. coli O157:H7, L. monocytogenes, S. typhimurium and S. aureus and concluded that S. typhimurium was the most sensitive species, with a reduction of 6 log cycles after 10 min of exposure. Bacterial inactivation experiments depend on the exposure conditions (ex: variability of wine composition) making direct comparisons difficult. However, the results presented here suggest that B. cereus vegetative cells are, amongst the food-borne pathogens studied, some of the most sensitive to exposure to wine.
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Fig. 1. The effect of wine, dilutions 1:4 and 1:8, on the vegetative cells of (A) B. cereus ATCC 11778 and (B) B. cereus ATCC 14579. Error bars represent the standard deviation of the mean of three replications.
No significant differences were found between the behaviour of the two strains (P > 0.05), both for vegetative cells and spores. The subsequent experiments were performed only with strain ATCC 14579. The contribution of certain wine components (organic acids, low pH, ethanol and phenolic compounds) to the antibacterial effect of wine on this strain was studied. The main organic acids of red wine (tartaric, acetic, lactic and citric acids) (Jackson, 2000) with and without ethanol, at concentrations commonly found in wines, were tested. The solutions of organic acids were diluted 1:4 and the pH was adjusted to 3.3 (pH of the wine tested) to make meaningful comparisons with the antimicrobial activity of
Fig. 2. The effect of wine on spores of B. cereus ATCC 11778 and ATCC 14579. Error bars represent the standard deviation of the mean of three replications.
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Fig. 3. The effect of wine organic acids (5.5 g L1 tartaric acid, 0.5 g L1 acetic acid, 2 g L1 lactic acid and 0.5 g L1 citric acid) with and without ethanol 13% (v/v), diluted 1:4, in (A) combination and (B) separated. All solutions were adjusted to pH 3.3. Error bars represent the standard deviation of the mean of three replications.
the diluted wine. As can be seen in Fig. 3A, the mixture of organic acids with ethanol displayed a higher bactericidal effect than the mixture of acids alone, but the difference is not statistically significant (P > 0.05). It can be assumed that the organic acids mixture contributes notably to the antimicrobial effect of wine, but not to the same extent as the wine itself (Fig. 1). The effect of each organic acid was studied in separate. Fig. 3B shows that, at the concentrations tested, lactic acid was found to be the most effective in the inhibition of B. cereus ATCC 14579 vegetative cells, being responsible for a 3.6 log cycles reduction in 40 s, whereas citric acid was the least effective. Various in vitro studies indicated that the potency of wine as an antibacterial agent was higher than ethanolic solutions at concentrations equivalent to the wine (Just & Daeschel, 2003; Weisse et al., 1995). Fernandes et al. (2007) reported that malic acid and lactic acid were amongst the most effective organic acids in the inactivation of Listeria inocua NCTC 11288. Moretro and Daeschel (2004) found that the combination of organic acids (malic and tartaric) with ethanol (15%) and low pH (3.0) had significantly stronger antimicrobial activity than the sum of the individual effects of these components against various food-borne pathogens, indicating potential synergistic interactions. Fernandes et al. (2007) also demonstrated a synergistic effect when using a combination of organic acids with ethanol. Changes in cell membrane permeability caused by ethanol (Barker & Park, 2001) may lead to enhanced efficacy of organic acids and may partly explain the difference in antimicrobial activity between grape juice and wine (Barker & Park, 2001; Harding & Maidment,
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1996; Just & Daeschel, 2003). The synergism between ethanol and organic acids in cell inactivation seems less relevant in B. cereus than in other bacteria. The wine phenolic compounds assayed in this work (resveratrol, ferulic acid, p-coumaric acid, kaempferol and quercetin) displayed no activity, at the concentrations tested, against B. cereus vegetative cells (data not shown). Wine phenolics have been found, however, to be active against other microorganisms (Gañan, MartínezRodríguez, & Carrascosa, 2009). The higher levels of phenolics generally found in red wines have been used to explain the higher inactivation effectiveness often seen with red wines compared to white wines (Moretro & Daeschel, 2004; Papadopoulou, Soulti, & Roussis, 2005; Vaquero, Alberto, & Manca de Nadra, 2007). Mahady and Pendland (2000) and Mahady, Pendland, and Chadwick (2003) found a MIC50 value of 12.5 mg mL1 for resveratrol against Helicobacter pylori strains using an agar disk diffusion assay. Chan (2002) used a broth dilution assay to determine the MIC of resveratrol against S. aureus, Enterococcus faecalis, and Pseudomonas aeruginosa to be 171e342 mg mL1. Aziz, Farak, Mousa, and Abo-Zaid (1998) investigated the inhibitory effect of several phenolic compounds against E. coli, Klebsiella pneumoniae and B. cereus using a suspension test. Caffeic acid and protocatechuic acid were effective at inhibiting the growth of E. coli and K. pneumoniae at levels of 0.3 mg mL1. Vanillic acid and p-coumaric acid were capable of inhibiting growth of E. coli, K. pneumoniae, and B. cereus at levels of 0.4 mg mL1. The reason why the phenolic compounds tested in this work did not exhibit activity against B. cereus vegetative cells might be related with the concentrations used. The efficacy of phenolic compounds that has been observed in other works against various bacterial species was attained using concentrations 10e1000 times higher than those normally found in wines. 3.2. Survival of B. cereus vegetative cells in simulated gastric conditions As shown in Fig. 4, the exposure of cells to synthetic gastric fluid (SGF) led to a significant decrease in the viability of B. cereus ATCC 14579. It is noteworthy that no significant differences were found (P > 0.05) between the effect of SGF alone and SGF combined with wine. The inhibitory action of SGF and wine against B. cereus vegetative cells under a simulated food consumption scenario was then studied using sterile chicken-rice baby or pasteurized fresh cheese as food matrix. As depicted in Fig. 5A, the number of viable cells in the control (Food þ Water) remained constant until the end of the 60 min time exposure. In the presence of SGF
Fig. 4. The effect of synthetic gastric fluid (SGF) and of SGF combined with wine on the vegetative cells of B. cereus ATCC 14579. Error bars represent the standard deviation of the mean of three replications.
Fig. 5. The effect of different treatments on the vegetative cells of B. cereus ATCC 14579 in a model stomach system in the presence of (A) chicken-rice baby meal and (B) pasteurized fresh cheese as food matrix. Error bars represent the standard deviation of the mean of three replications.
(Food þ SGF þ Water), B. cereus ATCC 14579 cells suffered an almost 2 log reduction in 20 min, and an almost 4 log reduction in 60 min, much less than when SGF was used in the absence of food (Fig. 4). During the consumption of food, the pH in the stomach varies and may reach values up to 5, a condition allowing the survival of B. cereus vegetative cells (Dressman et al., 1990; Kramer & Gilbert, 1989). In this work, the pH of the mixtures Food þ SGF reached the values 4.4 and 4.5 for fresh cheese and chicken-rice meal, respectively. When wine was added, the pH decreased to 4.3 and 4.4. It has been already described that the bactericidal activity of stomach is predominantly pH (HCl) dependent (Just & Daeschel, 2003). Waterman and Small (1998) showed the survival of S. typhimurium, Shigella flexneri and E. coli on beef particles and suggested this could be due to the raise of pH in the microenvironment of the bacteria. A much stronger inactivation effect (P < 0.05) was found when wine was added (Food þ SGF þ Wine), causing a reduction on the viable cell counts of 4.5 log cycles in 6 min. No viable cells were detected (<500 CFU mL1) from this time on. Fig. 5B shows the results obtained with different food matrix (pasteurized fresh cheese). The assay Food þ SGF þ Water led to an inactivation of the vegetative cells, but to a lesser extent than the inactivation obtained with the chicken-rice meal. With the addition of wine (Food þ SGF þ Wine), a significant decrease (P < 0.05) of 3.7 log cycles in the cell survival was noticed after 20 min. Duplicating the volume of wine (Food þ SGF þ Wine2x), a 4 log reduction was achieved in 3.5 min with no subsequent detectable colonies in the following exposure times (<500 CFU mL1). It can be seen that the pasteurized fresh cheese exhibits a higher protective effect on the vegetative cells of
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B. cereus ATCC 14579 than the chicken-rice meal. Clavel et al. (2004), when using a gastric electrolyte solution (GM) and food, showed that survival of B. cereus cells was higher in GM-milk than in the other GM combinations. The protective role of dairy products against inactivation by low pH has been previously observed for some microorganisms such as on Salmonella in cheese (D’Aoust, 1985). In comparison to the SGF, recognized as a bactericidal barrier against the ingested pathogens, the presence of wine, in an equivalent amount to a glass of wine in a meal (proportionally to the food in the model stomach), led to a significant additional cell inactivation effect. 3.3. Behaviour of B. cereus spores in simulated gastro-intestinal passage
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The infective dose of B. cereus is highly variable ranging from 105 to 108 cells per g of food. This is dependent on a number of factors such as the presence of viable cells or spores in the food, the amounts of enterotoxin(s) produced, and the susceptibility of individuals (Anonymous, 2005). The ingestion of wine with food may be expected to, in some instances, reduce the number of viable B. cereus to levels below the infectious dose. 4. Conclusions The results of this work clearly show that wine exerts a strong inactivation effect against vegetative cells of two B. cereus strains. Regarding the data obtained in simulated gastric conditions, we can conclude that the ingestion of wine during a meal substantially diminishes the number of B. cereus vegetative cells in addition to the effect of the gastric fluid. The rate and extent of inactivation depends, however, in the type of food present due to the known protective role of certain food components. We also evaluated the behaviour of B. cereus spores under gastro-intestinal conditions. In a consumption-like scenario, the treatment SGFeSIF þ Food þ Wine led to lower total counts then in the absence of wine. This is, to our knowledge, the first study reporting the antimicrobial activity of wine against B. cereus in food consumption scenarios. This work provides evidence suggesting that drinking wine with meals may lead to a reduction of the number of vegetative cells in the stomach and reduce the impact of the germination of spores that may occur in the small intestine, thus lowering the risk of toxiinfection. The antimicrobial effect of wine in marinades can be expected to be high, due to the relatively long exposition times (one to several hours), and to the synergistic effect with other substances (spices, vinegar, etc) commonly used in these preparations. Wine, used as a beverage or as a marinade, may be expected to diminish the incidence of B. cereus illnesses.
The experiments were carried out with spore suspensions that were first incubated in SGF and subsequently transferred to SIF. Spore and the total counts (vegetative cells þ spores) of B. cereus ATCC 14579 were obtained from experiments using two different food matrices: chicken-rice baby meal and fresh cheese. Since the effect of the two food matrices were similar, only the results from chicken-rice baby meal are shown (Fig. 6). In the SGFeSIF þ Food þ Water treatment, there was a decrease in the spore counts (Fig. 6A), mostly under the simulated intestine conditions, which can be explained by spore germination. At the same time, an increase of around 2 log cycles in the total counts can be observed (Fig. 6B), certainly due to the growth of the germinated vegetative cells. Remarkably, the treatment SGFeSIF þ Food þ Wine led to a lower increase (approximately 1 log cycle less) in the total counts in comparison to the treatment without wine. The results suggest that wine might hinder the growth and/or inactivate the vegetative cells of B. cereus originating from the germination of the spores in the small intestine. Once again, pasteurized fresh cheese appears to be more protective to the B. cereus cells than the chicken-rice food (data not shown).
References
Fig. 6. The effect of different treatments on the (A) spore counts and (B) total counts of B. cereus ATCC 14579 in simulated gastric and intestinal conditions using chicken-rice baby meal as food matrix. Error bars represent the standard deviation of the mean of three replications. SGF: synthetic gastric fluid, SIF: synthetic intestinal fluid.
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Dressman, J. B., Berardi, R. R., Dermentzoglou, L. C., Russell, T. L., Schmaltz, S. P., Barnett, J. L., et al. (1990). Upper gastro-intestinal (GI) pH in young, healthy men and women. Pharmaceutical Research, 7, 756e761. Ehling-Schulz, M., Fricker, M., & Scherer, S. (2004). Bacillus cereus, the causative agent of an emetic type of foodborne illness. Molecular Nutrition & Food Research, 48, 479e487. Fernandes, J., Gomes, F., Couto, J. A., & Hogg, T. (2007). The antimicrobial effect of wine on Listeria innocua in a model stomach system. Food Control, 18, 1477e1483. Friedman, M., Henika, P. R., Levin, C. E., & Mandrell, R. E. (2006). Antimicrobial wine formulations active against the foodborne pathogens Escherichia coli O157:H7, and Salmonella enterica. Journal of Food Science, 71, M245eM251. Friedman, M., Henika, P. R., Levin, C. E., & Mandrell, R. E. (2007). Recipes for antimicrobial wine marinades against Bacillus cereus, Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella enterica. Journal of Food Science, 72, M207eM213. Gañan, M., Martínez-Rodríguez, A. J., & Carrascosa, A. V. (2009). Antimicrobial activity of phenolic compounds of wine against Campylobacter jejuni. Food Control, 20, 739e742. Granum, P. E. (2007). Bacillus cereus. In M. P. Doyle, & L. R. Beuchat (Eds.), Food microbiology: fundamentals and frontiers (pp. 445e455). Washington, DC: ASM Press. Granum, P. E., O’Sullivan, K., & Lund, T. (1999). The sequence of the non-haemolytic enterotoxin operon from Bacillus cereus. FEMS Microbiology Letters, 177, 225e229. Harding, C., & Maidment, C. (1996). An investigation into the anti-bacterial effects of wine and other beverages. Journal of Biological Education, 30, 237e239. Hsiao, C. P., & Siebert, K. J. (1999). Modeling the inhibitory effects of organic acids on bacteria. International Journal of Food Microbiology, 47, 189e201. Jackson, R. S. (2000). Wine science: Principles, practice, perception. New York, NY, USA: Academic Press. Just, J. R., & Daeschel, M. A. (2003). Antimicrobial effects of wine on Escherichia coli O157:H7 and Salmonella typhimurium in a model stomach system. Journal of Food Science, 68, 285e290. Kramer, J. M., & Gilbert, R. J. (1989). Bacillus cereus and other Bacillus species. In M. P. Doyle (Ed.), Foodborne bacterial pathogens (pp. 21e70). New York: Marcel Dekker. Lund, T., DeBuyser, M.-L., & Granum, P. E. (2000). A new cytotoxin from Bacillus cereus that may cause necrotic enteritis. Molecular Microbiology, 38, 254e261. Mahady, G. B., & Pendland, S. L. (2000). Resveratrol inhibits the growth of Helicobacter pylori in vitro. American Journal of Gastroenterology, 95, 1849. Mahady, G. B., Pendland, S. L., & Chadwick, L. R. (2003). Resveratrol and red wine extracts inhibit the growth of CagA þstrains of Helicobacter pylori in vitro. American Journal of Gastroenterology, 98, 1440e1441. Marimon, J. M., Bujanda, L., Gutierrez-Stampa, M. A., Cosme, A., & Arenas, J. I. (1998). Antibacterial activity of wine against Salmonella enteritidis pH or alcohol? Journal of Clinical Gastroenterology, 27, 179e180.
Miles, A., & Misra, S. (1938). The estimation of the bactericidal power of blood. Journal of Hygiene, 38, 732e749. Moretro, T., & Daeschel, M. A. (2004). Wine is bactericidal to foodborne pathogens. Journal of Food Science, 69, 251e257. Papadopoulou, C., Soulti, K., & Roussis, I. G. (2005). Potential antimicrobial activity of red and white wine phenolic extracts against strains of Staphylococcus aureus, Escherichia coli and Candida albicans. Food Technology and Biotechnology, 43, 41e46. Rhodes, P. L., Mitchell, J. W., Wilson, M. W., & Melton, L. D. (2006). Antilisterial activity of grape juice and grape extracts derived from Vitis vinifera variety Ribier. International Journal of Food Microbiology, 107, 281e286. Ricke, S. C. (2003). Perspectives on the use of organic acids and short chain fatty acids as antimicrobials. Poultry Science, 82, 632e639. Rotard, W., Christmann, W., Knoth, W., & Mailahn, W. (1995). Bestimmung der resorptionsverfügbaren PCDD⁄ PCDF aus Kieselrot. Z UmweltchemÖkotoxikol, 7, 3e9. Schmidt, K. (Ed.). (2001). WHO surveillance programmme for control of foodborne infections and intoxications in Europe. Seventh report. Berlin, Germany: FAO/WHO Collaborating Centre for Research and Training in Food Hygiene and Zoonoses. Schoeni, J. L., & Wong, A. C. (2005). Bacillus cereus food poisoning and its toxins. Journal of Food Protection, 68, 636e648. Sugita-Konishi, Y., Hara-Kudo, Y., Iwamoto, T., & Kondo, K. (2001). Wine has activity against entero-pathogenic bacteria in vitro but not in vivo. Bioscience Biotechnology and Biochemistry, 65, 954e957. Tamplin, M. L. (2005). Inactivation of Escherichia coli O157:H7 in simulated human gastric fluid. Applied and Environmental Microbiology, 69, 320e325. Vaquero, M. J. R., Alberto, M. R., & Manca de Nadra, M. C. (2007). Antibacterial effect of phenolic compounds from different wines. Food Control, 18, 93e101. Waite, J. G., & Daeschel, M. A. (2007). Contribution of wine components to inactivation of food-borne pathogens. Journal of Food Science, 72, M286eM291. Waterman, S. R., & Small, P. L. C. (1998). Acid-sensitive enteric pathogens are protected from killing under extremely acidic conditions of pH 2.5 when they are inoculated onto certain solid food sources. Applied and Environmental Microbiology, 64, 3882e3886. Weisse, M. E., Eberly, B., & Person, D. A. (1995). Wine as a digestive aid: comparative antimicrobial effects of bismuth salicylate and red and white wines. British Medical Journal, 311, 1657e1660. Wijnands, L. M., Dufrenne, J. B., van Leusden, F. M., & Abee, T. (2007). Germination of Bacillus cereus spores is induced by germinants from differentiated Caco-2 cells, a human cell line mimicking the epithelial cells of the small intestine. Applied and Environmental Microbiology, 73, 5052e5054. Wijnands, L. M., Dufrenne, J., Zwietering, M., & van Leusden, F. (2006). Spores from mesophilic Bacillus cereus strains germinate better and grow faster in simulated gastro-conditions that spores from psychrothrophic strains. International Journal of Food Microbiology, 112, 120e128. Wijnands, L. M., Pielaat, A., Dufrenne, J. B., Zwietering, M. H., & van Leusden, F. M. (2009). Modelling the number of viable vegetative cells of Bacillus cereus passing through the stomach. Journal of Applied Microbiology, 106, 258e267.
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The antimicrobial effect of wine on Bacillus cereus in simulated gastro-intestinal conditions Miguel Vaz, Tim Hogg, José António Couto* CBQF/Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Rua Dr. António Bernardino de Almeida, 4200-072 Porto, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 December 2011 Received in revised form 4 May 2012 Accepted 15 May 2012
This study aimed to evaluate the antimicrobial activity of wine against Bacillus cereus vegetative cells and spores. The results clearly show that wine exerts a strong inactivation effect against vegetative cells of B. cereus. The red wine tested inactivated stationary phase cultures to undetectable numbers in less than 10 s. Thus, further inactivation assays were carried out with wine diluted with water (1:4 and 1:8). Diluted wine 1:4 caused a reduction of approximately 5 log cycles on viable cell counts in 20 s. On the other hand, B. cereus spores were found to be highly resistant to wine exposure. The influence of wine components (organic acids, ethanol and phenolic compounds) was investigated on vegetative cells. The wine organic acids tested exhibited a strong inactivation effect, and, when combined with ethanol, a slight synergistic effect was observed. The wine phenolic compounds assayed displayed no activity against the vegetative cells at the concentrations tested. At the simulated gastric conditions studied (in the presence of food), wine diminished considerably the number of B. cereus viable cells in addition to the effect of the synthetic gastric fluid. The behaviour of B. cereus spores under gastro-intestinal conditions was also evaluated. In a consumption-like scenario, the addition of wine led to lower total counts (vegetative cells þ spores) of B. cereus in the simulated intestine conditions, showing that wine inhibits the proliferation of the vegetative cells obtained from the germination of spores. This work provides evidence that consumption of wine during a meal may diminish the number of viable cells of B. cereus and reduces the impact of the germination of spores that may occur in the small intestine, thus lowering the risk of toxi-infection that may be caused by this pathogen. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Bacillus cereus Wine Cell inactivation Gastro-intestinal conditions
1. Introduction Several studies demonstrate the antibacterial properties of wine against relevant food-borne pathogenic bacteria (Carneiro, Couto, Mena, Queiroz, & Hogg, 2008; Correia et al., 2003; Fernandes, Gomes, Couto, & Hogg, 2007; Moretro & Daeschel, 2004; SugitaKonishi, Hara-Kudo, Iwamoto, & Kondo, 2001; Waite & Daeschel, 2007; Weisse, Eberly, & Person, 1995). Weisse et al. (1995) reported that red and white wines were able to reduce the viability of several bacteria responsible for traveller’s diarrhoea by 5e6 log cycles in 20 min. Similar inactivation patterns were obtained by Sugita-Konishi et al. (2001) for Salmonella enteritidis, Escherichia coli O157:H7 and Vibrio parahaemolyticus. Moretro and Daeschel (2004) have shown that wine has bactericidal activity against E. coli, Listeria monocytogenes, Salmonella typhimurium and Staphylococcus aureus. More recently, Carneiro et al. (2008) and
* Corresponding author. Tel.: þ351 225580027; fax: þ351 225090351. E-mail address: [email protected] (J.A. Couto). 0956-7135/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2012.05.034
Fernandes et al. (2007) have focused their studies on the activity of wine against the important food-borne pathogens Listeria and Campylobacter jejuni, respectively. The exact mechanisms responsible for the antimicrobial effect of wine are, however, not fully understood. Wine is a beverage with some unique characteristics. It possesses a relatively high ethanol content in addition to other antimicrobial agents like organic acids, low pH, polyphenol compounds and preservatives (Just & Daeschel, 2003). In vitro studies indicate that, for a given ethanol concentration, wine has a more potent antibacterial activity than other alcoholic beverages. This potency has been partly attributed to the combination of ethanol and organic acids (Just & Daeschel, 2003; Weisse et al., 1995). Malic and tartaric acids are the most abundant organic acids in wine, and their antimicrobial effects are well known, especially at low pH conditions, such as those found in wines (Hsiao & Siebert, 1999; Ricke, 2003). The importance of ethanol to this overall antimicrobial activity is illustrated by the findings of Just and Daeschel (2003) who showed that a grape juice had a very little antimicrobial activity against E. coli O157:H7 and Salmonella spp., whereas wine made from the same juice demonstrated
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considerable activity in this respect. The bactericidal effect of ethanol alone, in concentrations generally encountered in wine (between 10 and 13% v/v) is low, when compared with the bactericidal effect of wine itself (Just & Daeschel, 2003; Marimon, Bujanda, Gutierrez-Stampa, Cosme, & Arenas, 1998; Weisse et al., 1995). Moretro and Daeschel (2004) found that a combination of ethanol with organic acids, at pH 3.0, had the strongest bactericidal effect. Carneiro et al. (2008) reported that ethanol and certain organic acids act synergistically and represent the major components for the bactericidal effects of wine on Campylobacter. Studies incorporating wine as a food additive in the form of marinades provide further evidence of its protective role (Friedman, Henika, Levin, & Mandrell, 2006). Friedman et al. (2007) have evaluated the bactericidal activity of several wine recipes against Bacillus cereus, E. coli O157:H7, L. monocytogenes, and Salmonella enterica. The wine marinades were highly effective against the four pathogens. The extremely low pH of the stomach (wpH 1e2) is considered a major defence barrier against food-borne infection, this being recognized for various bacterial pathogens, such as Vibrio, Salmonella and Campylobacter (Barmpalia-Davis, Geornaras, Kendall, & Sofos, 2008; Tamplin, 2005). It’s not easy to model the survival of bacteria in the human stomach due to the huge diversity of food matrices and individual human physiological characteristics. However, it is possible to study pathogen behaviour under controlled conditions that approximate gastric conditions. For example, Clavel, Carlin, Lairon, Nguyen-The, and Schmitt (2004) studied the survival of B. cereus spores and vegetative cells in acid media simulating the human stomach. Wijnands, Pielaat, Dufrenne, Zwietering, and van Leusden (2009) modelled the number of viable vegetative cells of B. cereus passing through the stomach. B. cereus is a Gram positive spore-forming bacterium that is a common contaminant in a wide variety of foods, such as pasta, rice, dairy products, dried foodstuffs, vegetables, fruit, seafood, meat and poultry (Schoeni & Wong, 2005). Two distinct food-borne disease types, emetic and diarrhoeal, are associated with B. cereus. The intoxication, or emetic syndrome, is caused by a single peptide toxin produced in the food prior to consumption (Agata, Ohta, Mori, & Isobe, 1995; Ehling-Schulz, Fricker, & Scherer, 2004); the toxicinfection, or diarrhoeal syndrome, is caused by enterotoxins produced in the small intestine, including two three-component toxins (haemolysin BL and non haemolytic enterotoxin), and a single protein toxin (cytotoxin K) (Beecher, Schoeni, & Lee Wong, 1995; Granum, O’Sullivan, & Lund, 1999; Lund, DeBuyser, & Granum, 2000). Bacterial spores and/or vegetative cells may survive the stomach’s acidic conditions and germinate within the small intestine (Wijnands, Dufrenne, van Leusden, & Abee, 2007). B. cereus is an important cause of food-borne disease worldwide (Clavel et al., 2007; Granum, 2007), although it is probably highly under-reported in official lists of food-borne disease causes. In the European Union, Bacillus species (including non-cereus) were reported to be responsible for 1.4% of food-borne outbreaks in 2005 (Anonymous, 2006). Between 1993 and 1998 in the Netherlands, B. cereus accounted for 12% of food-borne disease outbreaks where a causative agent was identified (Schmidt, 2001). In this work, we aimed to evaluate the effect of red wine on B. cereus vegetative cells and spores. Firstly, we have characterised the effect of direct exposure of cells and spores to wine and the antimicrobial activity of selected wine components (organic acids, ethanol, low pH and phenolic compounds) in separate and in combination. Then, the kinetics of inactivation of B. cereus vegetative cells in simulated gastric conditions, with and without wine, in food consumption scenarios (wine/food/bacteria) was evaluated. Finally, we studied the behaviour of spores on simulated gastric and intestinal conditions, in food matrices with and without wine. This
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is, to our knowledge, the first report where the behaviour of vegetative cells and spores of B. cereus is characterized in direct wine immersions and under simulated gastro-intestinal conditions. The results obtained are discussed in a risk assessment context. 2. Materials and methods 2.1. Bacterial strains and growth media Two B. cereus strains from the American Type Culture Collection (ATCC) were used in this work (B. cereus ATCC 11778 and B. cereus ATCC 14579). The cultures were preserved in slants of Tryptone Soy Agar (TSA) medium (Pronadisa, Madrid, Spain) and kept at 4 C. Stock cultures of vegetative cells were kept at 80 C in Tryptone Soy Broth (TSB) medium (Pronadisa, Madrid, Spain) with glycerol 20% (v/v). For each assay, inoculated TSB medium was incubated at 30 C for 16 h to attain the stationary growth phase. 2.2. Spore production Pure B. cereus strains were grown on TSA for 24 h at 30 C. These cells were then used to inoculate TSA plates supplemented with 40 mg L1 MnSO4 (Merck, Darmstad, Germany) and 100 mg L1 CaCl2 (Merck) to stimulate sporulation. TSA plates were incubated at 30 C for 7 days. Sporulation was checked by microscopic visualization using malachite green for spore staining. Spores were collected by scraping colonies from the plates, suspended in sterile phosphate buffer at pH 7.0 and washed by centrifugation (10,000 g for 10 min). Washings were performed until the suspension was milky white. After the final centrifugation, the pellet was suspended in 10 mL of a 50% (v/v) ethanol solution and the suspension was kept at 4 C for 12 h, in order to eliminate vegetative cells, and washed again three times in sterile distilled water. The final suspension (approximately 1010 spores per mL) was distributed in sterile Eppendorf microtubes and kept at 20 C. 2.3. Wine and wine components A red wine, from the Douro demarcated region (Portugal, 2008) with an ethanol concentration of 13% (v/v) was used. The wine was filter sterilized using 0.45 mm cellulose acetate membranes (Orange Scientific, Brain L’ Alleud, Belgium) and was kept at 4 C in 1 L sterile Schott flasks until used. A mixture of organic acids was made containing the final concentrations of 2 g L1 lactic acid, 5.5 g L1 tartaric acid, 0.5 g L1 acetic and 0.5 g L1 citric acid, in the presence or absence of 13% (v/ v) ethanol and with pH adjusted to 3.3 with 1 M HCl (Pronalab, Lisbon, Portugal). Tartaric and lactic acids were obtained from Sigma (St. Louis, USA), while acetic and citric acids were obtained from Merck. A solution of 0.15 M KH2PO4 at pH 7.0 was used as control. The individual effect of each organic acid was also tested. Phenolic compounds solutions were prepared at the following concentrations: 1 mg L1 of resveratrol, 5 mg L1 of ferulic acid, 5 mg L1 of p-coumaric acid, 2 mg L1 of kaempferol and 10 mg L1 of quercetin, with pH adjusted to 3.3. All the solutions were filter sterilized using 0.45 mm cellulose acetate membranes (Orange Scientific). 2.4. Survival of B. cereus vegetative cells and spores in wine and wine components Suspensions of stationary phase vegetative cells grown in TSB medium for 16 h at 30 C and spores of B. cereus ATCC 11778 and B. cereus ATCC 14579 were used in the inactivation experiments. For the experiments with wine components, only vegetative cells were
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used. One mL of each suspension was transferred onto 100 mL Erlenmeyer flasks containing 49 mL of wine, solutions of wine components, or a control solution (0.15 M KH2PO4, pH 7.0), all at a final volume of 50 mL. These flasks were immersed in a thermostatic water bath at 37 C and stirred magnetically (200 rpm). At selected times, 1 mL samples (vegetative cells or spores) were collected and serially diluted in 9 mL of Ringer solution (Lab M, Lancashire, United Kingdom). Counts were made by the drop count technique (Miles & Misra, 1938) by plating, in triplicate, 20 mL of each ten-fold dilution onto TSA medium and incubating at 30 C for 24 h. Culture densities were expressed as colony forming units per millilitre (CFU mL1). The detection limit was taken as one colony on the lowest dilution plate, i.e. 500 CFU mL1. 2.5. Synthetic gastric and intestinal fluids The synthetic gastric fluid (SGF) was prepared following an adapted formulation from Beumer, de Vries, and Rombouts (1992) consisting of NaCl (2.05 g), KH2PO4 (0.6 g), CaCl2 (0.11 g), KCl (0.37 g) and pepsin (0.133 g) in 1 L of deionised water. The SGF was adjusted to pH 1.5 with 1 M HCl and filter sterilized using 0.20 mm cellulose acetate membranes (Orange Scientific). The synthetic intestinal fluid (SIF) was prepared following an adapted formulation from Rotard, Christmann, Knoth, and Mailahn (1995) consisting of NaCl (6.58 g), KH2PO4 (0.66 g), KCl (0.043 g), MgCl2 (0.17 g), CaCl2 (0.060 g), pancreatin (2.25 g) and bile salts (1.5 g) in 1 L of deionised water. The pH was adjusted to 7.5 with 0.5 M NaOH and the solution was sterilized by filtration using 0.20 mm cellulose acetate membranes. Both SGF and SIF were prepared fresh daily. KH2PO, MgCl2 and CaCl2 were obtained from Merck; NaCl, KCl, pepsin and pancreatin were purchased from Sigma and bile salts from Difco (Detroit, USA). 2.6. Survival of B. cereus vegetative cells in simulated gastric conditions Vegetative cells of B. cereus ATCC 14579 were suspended in 50 mL of SGF and in 43 mL of SGF supplemented with 7 mL of sterile wine for 4 min in 100 mL Erlenmeyer flasks stirred magnetically (200 rpm) and immersed in a thermostatted water bath at 37 C. 1 mL samples were taken at selected times and ten-fold dilutions were prepared. Counts were made by the drop count technique as described above. The behaviour of B. cereus vegetative cells was also studied in the presence of a food matrix. 7 mL of sterile wine and 21.5 mL of SGF were added to 21.5 g of solid food matrix (sterile chicken-rice baby meal [Nestlé Portugal, Linda-a-Velha, Portugal] and pasteurized fresh cheese [Santiago & Santiago, Lisbon, Portugal]) to a final volume of approximately 50 mL. Control solutions consisted of 28.5 mL sterile water þ 21.5 g food and 7 mL sterile water þ 21.5 mL SGF þ 21.5 g food. In proportion to the amount of food used in these experiments, the volume of wine tested corresponds, approximately, to a glass of wine ingested in a regular meal (100e150 mL). Samples were collected and plated as described above. 2.7. Behaviour of B. cereus spores in simulated gastro-intestinal conditions Experiments were carried out with spore suspensions of B. cereus ATCC 14579. The solutions tested (involving a food matrix) were prepared as described above (Section 2.6) for the vegetative cells. Spores were exposed to SGF and SIF for 60 and 240 min, respectively, as described by Wijnands, Dufrenne, Zwietering, and van Leusden (2006). After 60 min in SGF at 37 C, 1 mL of the suspensions were transferred to 49 mL of SIF and then incubated at
the same temperature for 240 min. The 50-fold dilution upon this transfer was compensated when making the calculations of the simulated intestinal data. Total counts, i.e. total number of CFU (vegetative cells þ spores) and spore counts (after heat-treatment of the ten-fold serial diluted samples at 80 C, for 10 min) were determined. 2.8. Data analysis The survival of B. cereus strains was calculated by enumeration on TSA plates and the counts were converted to log10 CFU mL1. The experiments were performed in triplicate and the results were expressed as the mean value standard error. Statistical analysis (ANOVA), significance level: P < 0.05 was recorded in an Excel spread-sheet (Microsoft Corporation, Redmond, WA, USA). 3. Results and discussion 3.1. Survival of B. cereus vegetative cells and spores in wine and wine components The red wine tested was found to be strongly effective against stationary phase vegetative cells of B. cereus ATCC 11778 and ATCC 14579, whereas spores showed a noticeable resistance to the wine exposure. Wine treatment decreased initial vegetative cell counts from 107e108 CFU mL1 to no detectable levels (<500 CFU mL1) in less than 10 s (data not shown). Therefore, further inactivation experiments were carried out with diluted wine, 1:4 and 1:8. A reduction of around 5 log cycles in the cell viability of both strains was obtained in the cultures exposed to diluted wine 1:4 in 20 s (Fig. 1). No colonies were detected (<500 CFU mL1) in the following sampling times. The wine dilution 1:8 provoked a 5 log cycles reduction on the viable cell counts of ATCC 14579 strain in 40 s, and a similar reduction was noticed in ATCC 11778 strain in 30 s of exposure. The initial populations of vegetative cells in the control solution (KH2PO4) remained constant until the end of the assay, even when a phosphate solution at pH 3.3 was tested (data not shown). Concerning the spores, only a slight decrease, less than 1 log cycle, was observed throughout the 180 min of exposure to undiluted wine (Fig. 2). Activity of grape juice and wine marinades against B. cereus has been reported previously (Friedman et al., 2007; Rhodes, Mitchell, Wilson, & Melton, 2006). Rhodes et al. (2006) found a reduction of 1 log cycle within 10 min of contact with the grape juice. Friedman et al. (2007) have evaluated the bactericidal activity of several wine recipes consisting of red and white wine extracts of oregano leaves with added garlic juice and oregano oil against B. cereus, E. coli O157:H7, L. monocytogenes, and S. enterica. The wine marinades were highly effective against the four pathogens. In comparison with published data, our work shows that vegetative cells of B. cereus are considerably sensitive to wine. Weisse et al. (1995) showed that wine reduced the viable numbers of S. enteritidis, Shigella sonnei and E. coli, 5e6 log cycles, in 20 min. The same extent of inactivation was obtained for Salmonella spp. and E. coli in 5e30 min and of 20e60 min, respectively (Harding & Maidment, 1996; Just & Daeschel, 2003; Marimon et al., 1998). Moretro and Daeschel (2004) investigated the bactericidal effect of the wine on E. coli O157:H7, L. monocytogenes, S. typhimurium and S. aureus and concluded that S. typhimurium was the most sensitive species, with a reduction of 6 log cycles after 10 min of exposure. Bacterial inactivation experiments depend on the exposure conditions (ex: variability of wine composition) making direct comparisons difficult. However, the results presented here suggest that B. cereus vegetative cells are, amongst the food-borne pathogens studied, some of the most sensitive to exposure to wine.
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Fig. 1. The effect of wine, dilutions 1:4 and 1:8, on the vegetative cells of (A) B. cereus ATCC 11778 and (B) B. cereus ATCC 14579. Error bars represent the standard deviation of the mean of three replications.
No significant differences were found between the behaviour of the two strains (P > 0.05), both for vegetative cells and spores. The subsequent experiments were performed only with strain ATCC 14579. The contribution of certain wine components (organic acids, low pH, ethanol and phenolic compounds) to the antibacterial effect of wine on this strain was studied. The main organic acids of red wine (tartaric, acetic, lactic and citric acids) (Jackson, 2000) with and without ethanol, at concentrations commonly found in wines, were tested. The solutions of organic acids were diluted 1:4 and the pH was adjusted to 3.3 (pH of the wine tested) to make meaningful comparisons with the antimicrobial activity of
Fig. 2. The effect of wine on spores of B. cereus ATCC 11778 and ATCC 14579. Error bars represent the standard deviation of the mean of three replications.
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Fig. 3. The effect of wine organic acids (5.5 g L1 tartaric acid, 0.5 g L1 acetic acid, 2 g L1 lactic acid and 0.5 g L1 citric acid) with and without ethanol 13% (v/v), diluted 1:4, in (A) combination and (B) separated. All solutions were adjusted to pH 3.3. Error bars represent the standard deviation of the mean of three replications.
the diluted wine. As can be seen in Fig. 3A, the mixture of organic acids with ethanol displayed a higher bactericidal effect than the mixture of acids alone, but the difference is not statistically significant (P > 0.05). It can be assumed that the organic acids mixture contributes notably to the antimicrobial effect of wine, but not to the same extent as the wine itself (Fig. 1). The effect of each organic acid was studied in separate. Fig. 3B shows that, at the concentrations tested, lactic acid was found to be the most effective in the inhibition of B. cereus ATCC 14579 vegetative cells, being responsible for a 3.6 log cycles reduction in 40 s, whereas citric acid was the least effective. Various in vitro studies indicated that the potency of wine as an antibacterial agent was higher than ethanolic solutions at concentrations equivalent to the wine (Just & Daeschel, 2003; Weisse et al., 1995). Fernandes et al. (2007) reported that malic acid and lactic acid were amongst the most effective organic acids in the inactivation of Listeria inocua NCTC 11288. Moretro and Daeschel (2004) found that the combination of organic acids (malic and tartaric) with ethanol (15%) and low pH (3.0) had significantly stronger antimicrobial activity than the sum of the individual effects of these components against various food-borne pathogens, indicating potential synergistic interactions. Fernandes et al. (2007) also demonstrated a synergistic effect when using a combination of organic acids with ethanol. Changes in cell membrane permeability caused by ethanol (Barker & Park, 2001) may lead to enhanced efficacy of organic acids and may partly explain the difference in antimicrobial activity between grape juice and wine (Barker & Park, 2001; Harding & Maidment,
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1996; Just & Daeschel, 2003). The synergism between ethanol and organic acids in cell inactivation seems less relevant in B. cereus than in other bacteria. The wine phenolic compounds assayed in this work (resveratrol, ferulic acid, p-coumaric acid, kaempferol and quercetin) displayed no activity, at the concentrations tested, against B. cereus vegetative cells (data not shown). Wine phenolics have been found, however, to be active against other microorganisms (Gañan, MartínezRodríguez, & Carrascosa, 2009). The higher levels of phenolics generally found in red wines have been used to explain the higher inactivation effectiveness often seen with red wines compared to white wines (Moretro & Daeschel, 2004; Papadopoulou, Soulti, & Roussis, 2005; Vaquero, Alberto, & Manca de Nadra, 2007). Mahady and Pendland (2000) and Mahady, Pendland, and Chadwick (2003) found a MIC50 value of 12.5 mg mL1 for resveratrol against Helicobacter pylori strains using an agar disk diffusion assay. Chan (2002) used a broth dilution assay to determine the MIC of resveratrol against S. aureus, Enterococcus faecalis, and Pseudomonas aeruginosa to be 171e342 mg mL1. Aziz, Farak, Mousa, and Abo-Zaid (1998) investigated the inhibitory effect of several phenolic compounds against E. coli, Klebsiella pneumoniae and B. cereus using a suspension test. Caffeic acid and protocatechuic acid were effective at inhibiting the growth of E. coli and K. pneumoniae at levels of 0.3 mg mL1. Vanillic acid and p-coumaric acid were capable of inhibiting growth of E. coli, K. pneumoniae, and B. cereus at levels of 0.4 mg mL1. The reason why the phenolic compounds tested in this work did not exhibit activity against B. cereus vegetative cells might be related with the concentrations used. The efficacy of phenolic compounds that has been observed in other works against various bacterial species was attained using concentrations 10e1000 times higher than those normally found in wines. 3.2. Survival of B. cereus vegetative cells in simulated gastric conditions As shown in Fig. 4, the exposure of cells to synthetic gastric fluid (SGF) led to a significant decrease in the viability of B. cereus ATCC 14579. It is noteworthy that no significant differences were found (P > 0.05) between the effect of SGF alone and SGF combined with wine. The inhibitory action of SGF and wine against B. cereus vegetative cells under a simulated food consumption scenario was then studied using sterile chicken-rice baby or pasteurized fresh cheese as food matrix. As depicted in Fig. 5A, the number of viable cells in the control (Food þ Water) remained constant until the end of the 60 min time exposure. In the presence of SGF
Fig. 4. The effect of synthetic gastric fluid (SGF) and of SGF combined with wine on the vegetative cells of B. cereus ATCC 14579. Error bars represent the standard deviation of the mean of three replications.
Fig. 5. The effect of different treatments on the vegetative cells of B. cereus ATCC 14579 in a model stomach system in the presence of (A) chicken-rice baby meal and (B) pasteurized fresh cheese as food matrix. Error bars represent the standard deviation of the mean of three replications.
(Food þ SGF þ Water), B. cereus ATCC 14579 cells suffered an almost 2 log reduction in 20 min, and an almost 4 log reduction in 60 min, much less than when SGF was used in the absence of food (Fig. 4). During the consumption of food, the pH in the stomach varies and may reach values up to 5, a condition allowing the survival of B. cereus vegetative cells (Dressman et al., 1990; Kramer & Gilbert, 1989). In this work, the pH of the mixtures Food þ SGF reached the values 4.4 and 4.5 for fresh cheese and chicken-rice meal, respectively. When wine was added, the pH decreased to 4.3 and 4.4. It has been already described that the bactericidal activity of stomach is predominantly pH (HCl) dependent (Just & Daeschel, 2003). Waterman and Small (1998) showed the survival of S. typhimurium, Shigella flexneri and E. coli on beef particles and suggested this could be due to the raise of pH in the microenvironment of the bacteria. A much stronger inactivation effect (P < 0.05) was found when wine was added (Food þ SGF þ Wine), causing a reduction on the viable cell counts of 4.5 log cycles in 6 min. No viable cells were detected (<500 CFU mL1) from this time on. Fig. 5B shows the results obtained with different food matrix (pasteurized fresh cheese). The assay Food þ SGF þ Water led to an inactivation of the vegetative cells, but to a lesser extent than the inactivation obtained with the chicken-rice meal. With the addition of wine (Food þ SGF þ Wine), a significant decrease (P < 0.05) of 3.7 log cycles in the cell survival was noticed after 20 min. Duplicating the volume of wine (Food þ SGF þ Wine2x), a 4 log reduction was achieved in 3.5 min with no subsequent detectable colonies in the following exposure times (<500 CFU mL1). It can be seen that the pasteurized fresh cheese exhibits a higher protective effect on the vegetative cells of
M. Vaz et al. / Food Control 28 (2012) 230e236
B. cereus ATCC 14579 than the chicken-rice meal. Clavel et al. (2004), when using a gastric electrolyte solution (GM) and food, showed that survival of B. cereus cells was higher in GM-milk than in the other GM combinations. The protective role of dairy products against inactivation by low pH has been previously observed for some microorganisms such as on Salmonella in cheese (D’Aoust, 1985). In comparison to the SGF, recognized as a bactericidal barrier against the ingested pathogens, the presence of wine, in an equivalent amount to a glass of wine in a meal (proportionally to the food in the model stomach), led to a significant additional cell inactivation effect. 3.3. Behaviour of B. cereus spores in simulated gastro-intestinal passage
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The infective dose of B. cereus is highly variable ranging from 105 to 108 cells per g of food. This is dependent on a number of factors such as the presence of viable cells or spores in the food, the amounts of enterotoxin(s) produced, and the susceptibility of individuals (Anonymous, 2005). The ingestion of wine with food may be expected to, in some instances, reduce the number of viable B. cereus to levels below the infectious dose. 4. Conclusions The results of this work clearly show that wine exerts a strong inactivation effect against vegetative cells of two B. cereus strains. Regarding the data obtained in simulated gastric conditions, we can conclude that the ingestion of wine during a meal substantially diminishes the number of B. cereus vegetative cells in addition to the effect of the gastric fluid. The rate and extent of inactivation depends, however, in the type of food present due to the known protective role of certain food components. We also evaluated the behaviour of B. cereus spores under gastro-intestinal conditions. In a consumption-like scenario, the treatment SGFeSIF þ Food þ Wine led to lower total counts then in the absence of wine. This is, to our knowledge, the first study reporting the antimicrobial activity of wine against B. cereus in food consumption scenarios. This work provides evidence suggesting that drinking wine with meals may lead to a reduction of the number of vegetative cells in the stomach and reduce the impact of the germination of spores that may occur in the small intestine, thus lowering the risk of toxiinfection. The antimicrobial effect of wine in marinades can be expected to be high, due to the relatively long exposition times (one to several hours), and to the synergistic effect with other substances (spices, vinegar, etc) commonly used in these preparations. Wine, used as a beverage or as a marinade, may be expected to diminish the incidence of B. cereus illnesses.
The experiments were carried out with spore suspensions that were first incubated in SGF and subsequently transferred to SIF. Spore and the total counts (vegetative cells þ spores) of B. cereus ATCC 14579 were obtained from experiments using two different food matrices: chicken-rice baby meal and fresh cheese. Since the effect of the two food matrices were similar, only the results from chicken-rice baby meal are shown (Fig. 6). In the SGFeSIF þ Food þ Water treatment, there was a decrease in the spore counts (Fig. 6A), mostly under the simulated intestine conditions, which can be explained by spore germination. At the same time, an increase of around 2 log cycles in the total counts can be observed (Fig. 6B), certainly due to the growth of the germinated vegetative cells. Remarkably, the treatment SGFeSIF þ Food þ Wine led to a lower increase (approximately 1 log cycle less) in the total counts in comparison to the treatment without wine. The results suggest that wine might hinder the growth and/or inactivate the vegetative cells of B. cereus originating from the germination of the spores in the small intestine. Once again, pasteurized fresh cheese appears to be more protective to the B. cereus cells than the chicken-rice food (data not shown).
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Fig. 6. The effect of different treatments on the (A) spore counts and (B) total counts of B. cereus ATCC 14579 in simulated gastric and intestinal conditions using chicken-rice baby meal as food matrix. Error bars represent the standard deviation of the mean of three replications. SGF: synthetic gastric fluid, SIF: synthetic intestinal fluid.
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