14 Inhibition of Bacillus Cereus and Bacillus in Raw Vegetables by Application of Washing Solutions Containing Enterocin as-48 Alone

ARTICLE IN PRESSFood Microbiology 25 (2008) 762– 770 Contents lists available at ScienceDirect Food Microbiology journal homepage: www.elsevier.com/locate/fm Inhibition of Bacillus cereus and Bacillus weihenstephanensis in raw vegetables by application of washing solutions containing enterocin AS-48 alone and in combination with other antimicrobials Antonio Cobo Molinos a, Hikmate Abriouel a, Rosario Lucas Lo´pez a, Nabil Ben Omar a, Eva Valdivia b,c, Antonio Ga´lvez a, ´ rea de Microbiologı´a, Departamento de Ciencias de la Salud, Facultad de Ciencias Experimentales, Universidad de Jae´n, 23071 Jae´n, Spain A Departamento de Microbiologı´a, Facultad de Ciencias, Universidad de Granada, 18071 Granada, Spain c Instituto de Biotecnologı´a, Facultad de Ciencias, Universidad de Granada, 18071 Granada, Spain a b a r t i c l e in f o a b s t r a c t Article history: Received 11 February 2008 Received in revised form 29 April 2008 Accepted 4 May 2008 Available online 13 May 2008 Enterocin AS-48 is a broad-spectrum cyclic antimicrobial peptide produced by Enterococcus faecalis. In the present study, the bacteriocin was tested alone and in combination with other antimicrobials for decontamination of Bacillus inoculated on alfalfa, soybean sprouts and green asparagus. Washing with enterocin AS-48 solutions reduced viable cell counts of Bacillus cereus and Bacillus weihenstephanensis by 1.0–1.5 and by 1.5–2.38 log units right after application of treatment, respectively. In both cases, the bacteriocin was effective in reducing the remaining viable population below detection levels during further storage of the samples at 6 1C, but failed to prevent regrowth in samples stored at 15 or 22 1C. Application of washing treatments containing enterocin AS-48 in combination with several other antimicrobials and sanitizers (cinnamic and hydrocinnamic acids, carvacrol, polyphosphoric acid, peracetic acid, hexadecylpyridinium chloride and sodium hypochlorite) greatly enhanced the bactericidal effects. The combinations of AS-48 and sodium hypochlorite, peracetic acid or hexadecylpyridinium chloride provided the best results. After application of the combined treatments on alfalfa sprouts contaminated with B. cereus or with B. weihenstephanensis, viable bacilli were not detected or remained at very low concentrations in the treated samples during a 1-week storage period at 15 1C. Inhibition of B. cereus by in situ produced bacteriocin was tested by cocultivation with the AS-48 producer strain E. faecalis A-48-32 inoculated on soybean sprouts. Strain A-48-32 was able to grow and produce bacteriocin on sprouts both at 15 and 22 1C. At 15 1C, growth of B. cereus was completely inhibited in the cocultures, while a much more limited effect was observed at 22 1C. The results obtained for washing treatments are very encouraging for the application of enterocin AS-48 in the decontamination of sprouts. Application of washing treatments containing AS-48 alone can serve to reduce viable cell counts of bacilli in samples stored under refrigeration, while application of combined treatments should be recommended to avoid proliferation of the surviving bacilli under temperatureabuse conditions. & 2008 Elsevier Ltd. All rights reserved. Keywords: Bacillus cereus Bacillus weihenstephanensis Bacteriocins Biopreservation Vegetable foods Sprouts 1. Introduction Endospore-forming bacteria of the Bacillus cereus group are often considered hazardous to food safety because of their capacity to produce enterotoxins, together with their wide distribution in nature and frequent contamination of raw materials used in food production. B. cereus is a common soil inhabitant that is often present in a variety of foods, such as rice, spices, milk and dairy Corresponding author. Tel.: +34 953 212160; fax: +34 953 212943. E-mail address: [email protected] (A. Ga´lvez). 0740-0020/$ - see front matter & 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.fm.2008.05.001 products, vegetables, meat, cakes and other desserts (Granum, 2001). B. cereus produces several toxins (Schoeni and Wong, 2005), including emetic toxin (Agata et al., 1995) and at least four other enterotoxins: hemolysin BL or Hbl (Beecher and Wong, 1997), nonhemolytic enterotoxin or Nhe (Lindba¨ck et al., 2004) and cytotoxin K or CytK (Lund et al., 2000). Bacillus weihenstephanensis is a psychrotolerant bacterium belonging to the B. cereus group (Lechner et al., 1998). Some strains may be highly cytotoxic, and have been shown to carry at least some of the genetic determinants of the enterotoxins as well as the emetic toxin described in B. cereus (Pru¨ß et al., 1999; Stenfors et al., 2002; Thorsen et al., 2006; Baron et al., 2007). B. cereus has been isolated from sprouts and sprouting . cereus group alone and in combination with other antimicrobials in search for synergistic effects against the bacilli.. 2006. potassium sorbate. 2006)..ARTICLE IN PRESS A. monocytogenes (Cobo Molinos et al. 2005). 2005a.. D. I).. Washing solutions consisting of LAB bacteriocins produced on a lettuce extract (nisin Z. 2007b). cereus (Grande et al. Enterocin AS-48 was tested previously in washing treatments for decontamination of seed sprouts against L. F. 1. 2005). 2007. Enterocin AS-48 may be an interesting bacteriocin for decontamination of raw vegetables. 2004. Ananou et al. 2003).. cereus levels in fruits and vegetables has been proposed as a safety measure (Beuchat et al. 2005. cereus LWL1 inoculated on soybean sprouts (A–C). coagulin and a nisin:coagulin cocktail) reduced viable cell counts of L. The use of sanitizers to reduce the B. b. 2005). G). and is now being ˜ oz tested as a natural preservative in different food systems (Mun 4 6 8 15°C 2 0 4 2 1 3 5 1 3 5 7 0 22°C 4 2 0 7 1 3 5 Time (days) 0 3 5 Time (days) 22°C Log CFU/ml Log CFU/ml 7 7 8 4 2 0 0 Time (days) 1 15°C 2 5 2 7 6 6°C 3 4 0 0 4 7 6 Log CFU/ml Log CFU/ml 0 1 5 15°C 2 3 5 Time (days) 3 Time (days) 6 6°C 0 1 Time (days) 4 1 4 0 0 7 Time (days) 0 6 2 0 0 Log CFU/ml 22°C Log CFU/ml Log CFU/ml Log CFU/ml 6°C Log CFU/ml 763 6 4 2 0 0 1 3 5 Time (days) 7 0 1 3 5 7 Time (days) Fig. Effect of immersion treatments with solutions containing enterocin AS-48 on survival and proliferation of B.. 2002).. After immersion for 5 min at working temperatures in solutions containing final bacteriocin concentrations of 0 (O) and 25 mg/ml (m). cereus has also been isolated from raw vegetables used to prepare refrigerated minimally processed foods (Valero et al. Bacteriocins of lactic acid bacteria (LAB) have seldom been used for decontamination of fresh produce (reviewed by Ga´lvez et al.. added enterocin AS-48 caused a strong inhibition of Staphylococcus aureus and completely inactivated L. cereus in rice-based foods (Grande et al. reducing the population of viable listeria below detection limits in the treated sprouts after treatment and preventing regrowth of the listeria during storage of the treated samples (Cobo Molinos et al. and 53 out of 55 B. Cobo Molinos et al. 2004. alfalfa sprouts (D–F) and green asparagus (G–I). endospore-forming bacteria were found in the order of 2 logunits/g. 1989. This bacteriocin has been purified on a semi-preparative scale (Abriouel et al. Maqueda et al. 2004). In soybean sprouts. but did not prevent further growth of survivors during refrigeration storage (Allende et al. Pao et al. The purpose of the present study was to determine the effectiveness of AS-48 applied in washing treatments against two toxicogenic species of the B. limiting the protection of samples during storage. cereus isolates were found to produce diarrheic enterotoxins (Kim et al. Grande et al. phytic acid and citric acid) were tested as possible sanitizer treatments for reducing the population of Listeria monocytogenes on cabbage... H) or 22 1C (C. E. 1986. 2007).. broccoli and mung bean sprouts (Bari et al. 2004). Lucas et al. samples were incubated at 6 1C (A. While added bacteriocin completely inactivated B.. . 2005). Results from our previous studies indicate that the effectiveness of AS-48 treatments greatly depends on the target bacteria and the food system... The antilisterial activity of AS-48 was enhanced greatly by several antimicrobial compounds. 2004). 2005). B. In lettuce juice. Data represent the average of two independent experiments plus standard deviation (error bars).. monocytogenes and B. 2006). Nisin and pediocin individually or in combination with antimicrobial agents (sodium lactate. 2008). / Food Microbiology 25 (2008) 762–770 et al. Enterocin AS-48 is a cyclic peptide with a broad antibacterial spectrum (Ga´lvez et al... 15 1C (B. In washing treatments. the bacteriocin concentration in samples will decrease after treatment. seeds (Kim et al. monocytogenes on fresh-cut iceberg lettuce stored in microperforated plastic bags.. 2007a.. b. higher bacteriocin concentrations were required to control mixed populations of bacilli in pure´es (Grande et al. carvacrol and peracetic acid were from Fluka (Madrid).2. Bacterial strains and cultivation conditions Enterococcus faecalis A-48-32 (Martinez Bueno et al. UK) and dipped for 5 min in 5 ml sterile distilled water (negative controls) or in 5 ml of Bacillus cell suspension at 6. Barcelona. 2003). 15 and 22 1C. hexadecylpyridinium chloride. This Bacillus cell suspension was used to artificially contaminate the vegetables being tested. 15 and 22 1C. Spain) broth at 37 1C. Assay of enterocin AS-48 in vegetables artificially contaminated with bacilli 22°C 6 Log CFU/ml The effect of immersion in solutions containing enterocin AS48 (25 mg/ml. Belford. 15 1C (B) or 22 1C (C). Data represent the average of two independent experiments plus standard deviation (error bars).M. they were deposited on a sterile filter paper to drain excess water.. Madrid) and alfalfa sprouts (Productos Fanya. Henkel Ibe´rica. Effect of immersion treatments with solutions containing enterocin AS-48 on survival and proliferation of B. van Leusden (Microbiological Laboratory for Health Protection. All solutions were prepared fresh before use. dipped for 5 min... 12.1.. 2. Then. soybean sprouts (Alleuras Industries. 5–10 mm diameter). Spain). Cobo Molinos et al. Stone. tri-sodium trimetaphosphate. B. weihenstephanensis CECT 5894 was obtained from the Spanish type culture collection (CECT). Fresh green asparagus (Mary Washington variety.ARTICLE IN PRESS A. and p-hydoxybenzoic acid methyl esther were purchased from Sigma (Madrid. Preparation of bacteriocin and chemical preservatives 0 0 1 3 Time (days) 5 7 8 15°C 6 Log CFU/ml Enterocin AS-48 was recovered from cultured broths of the producer strain E.5 or 25 mg/ml). All strains were cultivated routinely on brain–heart infusion (BHI. Immersion solutions were prepared by diluting bacteriocin concentrates (500 mg/ml) in sterile distilled water or in aqueous solutions of the chemical preservatives to be tested in the case of combined treatments. faecalis S-47 by the agar well diffusion method (Abriouel et al. kindly supplied by Dr. n-propyl p-hydroxybenzoate.. 4 2 0 0 1 3 5 7 Time (days) 8 2. sodium lactate. weihenstephanensis CECT 5894 inoculated on soybean sprouts. The commercial solutions or concentrated stock solutions prepared by dissolving the solid compounds in sterile distilled water or in ethanol were diluted at least 20-fold in sterile distilled water in order to prepare the immersion solutions to be used for the washing treatments. Millipore Corporation. Lactic acid. Madrid) were purchased from local supermarkets. polyphosphoric acid. Inst. MA. Health and Environ. . samples were incubated at 6 1C (A). USA) under sterile conditions and tested for bacteriocin activity against the indicator strain E. B. hydrocinnamic acid. 1995). F. and stored at 4 1C on BHI-agar slants.5 log CFU/ml. Bacteriocin concentrates were filtered through 0. 1990) was used for the production of enterocin AS-48. cereus LWL1 is a psychrotrophic and enterotoxigenic strain (Dufrenne et al. Samples of the vegetable being tested (2. 4 6°C Log CFU/ml 764 2 2. Natl. at room temperature in 5 ml of sterile distilled water (controls) or distilled water containing enterocin AS-48 (at final concentrations of 5. Publ. final concentration) on survival and growth of B. The Netherlands). Asparagus were cut onto 2 cm pieces before treatment application. After immersion for 5 min at working temperatures in solutions containing final bacteriocin concentrations of 0 (O) and 25 mg/ml (m). 2003). weihenstephanensis CECT 5894 on soybean sprouts was investigated at storage temperatures of 6. Scharlab. Cinnamic acid. Barcelona. Sodium hypochlorite was a commercial concentrated bleach (ConejoTM. faecalis A-48–32 in CMG-medium by cation exchange chromatography as described elsewhere (Abriouel et al. Cultures of bacilli grown overnight in BHI broth at 37 1C were diluted 1:100 in sterile saline solution to a final cell density of approx. Spain). Immersion solutions were held at room temperature for at least 1 h before use. / Food Microbiology 25 (2008) 762–770 2. The artificially contaminated samples were 4 2 0 0 1 3 5 7 Time (days) Fig. soybean sprouts and green asparagus as well as B. Materials and methods 2. and E. 5.5 g each) were deposited inside sterile capped 50 ml polypropylene tubes (Sterilin.3. cereus LWL1 inoculated onto fresh alfalfa. faecalis S-47 from our collection was used for standard determination of bacteriocin activity.22 mm pore size low protein-binding filters (Millex GV. 1%) + Polyphosphoric acid + AS-48 + * + * Polyphosphoric acid(0.5%) + + p-Hydroxibenzoic acid methyl esther + AS-48 0 1 2 + 3 4 5 6 Log CFU/g Fig. statistically significant reduction (Po0.85% NaCl) and pummelled for 3 min in a Stomacher 80 (Biomaster) before they were serially diluted in sterile saline solution and spread in triplicate on plates of B. Plates were incubated at 37 1C for 48 h. At each step. Schwabach. *. cereus agar (Scharlab.05) of the combined treatment compared with treatment with the chemical agent alone.5%) Trisodium trimetaphosphate + AS-48 Hexadecylpyridinium + AS-48 Peracetic acid (40 ppm) Treatment + * + + Hexadecylpyridinium (0. 15.5%) + + * + Peracetic acid + AS-48 + * + * + Polyphosphoric acid (0. Viable cell counts were determined following application of treatment (open bars) and also after 24 h (closed bars) of storage at 15 1C. Barcelona).5%) + + n-Propyl-p-hydroxybenzoate + AS-48 + p-Hydroxybenzoic acid methyl esther (0. Control AS-48 Lactic acid (0.3%) + + * + * + + Cinnamic acid + AS-48 Hydrocinnamic acid (0.5%) + Lactic acid + AS-48 + Sodium lactate (0. Cobo Molinos et al. cereus were determined in order to calculate viable cell counts.05) compared with the untreated control. Data represent the average of two independent experiments plus standard deviation (error bars). samples (2. and samples were stored in sterile capped 50 ml polypropylene test tubes placed in refrigerated storage or incubation chambers (Memmert. Viable cell counts of bacilli were determined as described above following immersion treatment (time zero) and after 24 h incubation at 22 1C. Confirmation of B. (2004). followed by detection of the 71-bp amplicon by agarose gel electrophoresis. statistically significant reduction (Po0. cereus was done by PCR amplification of the sspE gene with specific primers sspE1-F (50 -GAGAAAGATGAGTAAAAAA CAACAA-30 ) y sspE1-R (50 -CATTTGTGCTTTGAATGCTAG) as described by Kim et al. cereus LWL1 in alfalfa sprouts. Following immersion treatments. 22 1C) for different periods of time.5%) + * + * Hydrocinnamic acid + AS-48 n-Propyl-p-hydroxybenzoate (0.3%) + + * + * Carvacrol + AS-48 Cinnamic acid (0. 3.5%) + Sodiumlactate + AS-48 + Sodium hypochlorite (100 ppm) + Sodium hypochlorite + AS-48 + * + * Trisodium trimetaphosphate (0. +. .5%) + + * + * Polyphosphoric acid + AS-48 Carvacrol (0.ARTICLE IN PRESS A.5 g) were mixed with 5 ml of sterile saline solution (0. excess immersion solution was removed as above. by using immersion solutions containing enterocin AS-48 (25 mg/ml final concentration) and/or the corresponding chemical compounds (at the final concentrations indicated in previous heading). and the number of colonies showing features typical of B. Germany) at desired incubation temperatures (6. Barcelona) supplemented with egg yolk and polymixin B (Panreac. Effect of enterocin AS-48 (25 mg/ml) in combination with chemical antimicrobial compounds on the viability of B. Standard deviation is shown by error bars. / Food Microbiology 25 (2008) 762–770 765 Combined treatments of enterocin AS-48 and chemical preservatives were carried out on Bacillus artificially contaminated food samples at room temperature essentially as described above. During prolonged storage. For the bacilli. Bacilli were confirmed by PCR amplification as described above. Best results were obtained for samples refrigerated at 6 1C.1 (Statistical Graphics Corp. In order to determine the statistical significance of the data.. 2.ARTICLE IN PRESS A. Statistical analyses The average data from duplicate trials7standard deviations were determined with Excel programme (Microsoft Corp.3 and 1. Effect of enterocin AS-48 (25 mg/ml) in combination with the phenolic compounds carvacrol (A). alfalfa sprouts and green asparagus (Fig. followed by 48 h incubation at 37 1C. cinnamic acid (B) and hydrocinnamic acid (C) on the viability of B. or AS-48+phenolic compound (K). no viable bacilli were detected after day 1 of storage at 6 1C (Fig. Vegetable samples (2. 2). However.59 log units were obtained after washing treatments.1. D and G). B. The significance of combined treatments was determined by comparison of data from the same incubation time. cereus agar was supplemented with ampicillin (80 mg/ml. Co-cultivation experiments 4 2 0 0 1 3 Time (days) 5 7 0 1 3 Time (days) 5 7 0 1 3 Time (days) 5 7 6 Log CFU/ml 766 4 2 0 3. By contrast. samples were plated for viable cell counts of bacilli and enterococci. 4.4. counts by 1. the surviving 6 Log CFU/ml 2. At desired intervals of incubation at the test temperature (15 or 22 1C).06. statistically significant (Po0. weihenstephanensis CECT 5894 inoculated on soybean sprouts (Fig. In all cases. . viable cell counts of treated samples were significantly lower than the untreated controls only for alfalfa sprouts and asparagus at day 3 and for soybean sprouts after day 3 of storage. This suspension was added to the bacilli contamination suspensions to obtain a co-culture suspension with an approximate enterococci-to-bacilli ratio of 100:1.5 g each) were immersed in the co-culture suspension for 5 min at room temperature as above and excess suspension was removed as above. USA). Effect of washing treatments containing enterocin AS-48 alone 4 2 0 Fig. Cobo Molinos et al. 1. reductions of viable cell counts obtained after treatments at 15 or 22 1C were not statistically significant for the three types of food tested. In the bacteriocin-treated samples. faecalis S-47 as the sensitive indicator strain and A-48-32 strain as the AS-48resistant indicator strain. USA). Controls (J). Results 6 The effect of washing treatments with solutions containing 25 mg/ml AS-48 was studied on B. Bacteriocin activity in samples was determined by testing 100 ml of the pummelled vegetable suspension by the agar-well diffusion method. faecalis A-48-32 were recovered by centrifugation (5000g for 10 min at room temperature) and resuspended in sterile saline solution to the initial culture volume.05) reductions of viable cell counts of 1. cereus LWL1 in alfalfa sprouts stored at 15 1C for 1 week. 1A. Colonies that showed inhibition against strain S-47 but failed to inhibit strain A-48-32 were considered to be AS-48 producers. and the remaining viable population was reduced below detection levels after days 1–3 of storage (Fig. phenolic compound (’).5. 1) stored at 6. by spreading samples in triplicate on kanamicin esculin azide agar (KAA. final concentration) in order to inhibit growth of enterococci. and the remaining viable cells multiplied as the untreated controls at least for the first day of storage. cereus LWL1 in soybean.38 log units at all temperatures tested. The bacteriocin treatment significantly reduced viable cell Log CFU/ml 3. / Food Microbiology 25 (2008) 762–770 Cells from an overnight culture (grown at 37 1C in BHI broth) of the enterocin AS-48 producer strain E. a t-test was performed at the 95% confidence interval with Statgraphics Plus version 5.. 2A). Data represent the average of two independent experiments plus standard deviation (error bars). Viable cell counts of enterococci were also carried out. samples treated with AS-48 (m). Scharlab).5–2. Confirmation of the AS-48 producer strains from KAA plates was done by testing colonies isolated at random for bacteriocin production by the spot on a lawn method with E. 15 and 22 1C. Washing treatments with AS-48 (25 mg/ml) were also applied to B. Effect of washing treatments containing enterocin AS-48 in combination with other antimicrobial compounds Enterocin AS-48 (25 mg/ml) was tested on B. either at time 0 or after 24 h storage or both (Fig. samples treated with AS-48 (m). 4C). 6). 767 alfalfa sprout samples stored at 15 1C for 1 week. Effect of enterocin AS-48 (25 mg/ml) in combination with the chemical compounds sodium hypochlorite (A). . hexadecylpyridinium chloride (B). none of the compounds was able to completely eliminate B. polyphosphoric acid and hydrocinnamic acid) reduced the viable cell counts of B. faecalis A-48-32 as a 6 Log CFU/ml Log CFU/ml 6 4 2 0 4 2 0 0 1 3 5 7 0 1 Time (days) 5 7 6 Log CFU/ml 6 Log CFU/ml 3 Time (days) 4 2 0 4 2 0 0 1 3 5 Time (days) 7 0 1 3 5 Time (days) 7 Fig.2. and also for trisodium trimetaphosphate at 24 h. cereus below detection levels.ARTICLE IN PRESS A. cinnamic and hydrocinnamic acids in combination with AS-48.5%). cereus LWL1 remained significantly lower compared with each individual treatment (Po0. chemical compound (’). 3). However. peracetic acid. some of the combined treatments (with sodium hypochlorite.1% and 0. In the case of polyphosphoric acid. At this concentration. 3. polyphosphoric acid (at 0. Addition of the antimicrobial compounds alone had much more limited effects in most cases. 2B and C).4. carvacrol. By contrast. the bacteriocin alone did not reduce significantly the number of viable B. 4). for which no viable cells were detected except at day 2 (Fig.05) compared with the untreated controls. or AS-48+chemical compound (K). viable cell counts of B. peracetic acid and polyphosphoric acid reduced the population of B. cereus LWL1 in alfalfa sprouts stored at 15 1C for 1 week.3. Data represent the average of two independent experiments plus standard deviation (error bars). Inhibition of B. 6E and F). Cobo Molinos et al. Best results were obtained for the combined treatment with hydrocinnamic acid.05) for cinnamic and hydrocinnamic acids. 3. Treatment with bacteriocin alone reduced viable cell counts below detection levels from the beginning. hexadecylpyridinium chloride and sodium hypochlorite. cereus when tested without bacteriocin. Preservation of sprouts after treatment 3. 3). Comparison of viable cell counts obtained for each chemical preservative and the combination bacteriocin–chemical preservative indicated that the bactericidal effect of treatments was enhanced significantly by addition of AS48 (Po0. 6). the combinations of 25 mg/ml AS-48 and hydrocinnamic acid. 5. peracetic acid. cereus below detection levels for the whole or at least most of the storage period when tested in combination with AS-48 (Fig. Antimicrobial compounds were also tested against B. a higher concentration of 0. cereus in sprouts by cocultivation with a bacteriocin-producing strain The efficacy of combinations of selected preservatives and enterocin AS-48 for prolonged inhibition of bacilli were tested in B. weihenstephanensis below detection levels for the whole or at least most of the storage period (Fig.05) during most part or the whole storage period (Fig. hexadecylpyridinium chloride. peracetic acid. Interestingly. Controls (J).5% was required to achieve such a degree of inactivation (Fig. The chemical compounds sodium hypochlorite. especially at 15 1C and to a less extent also at 22 1C (Fig. both at 0 and 24 h of storage. In samples treated with carvacrol. cereus in combination with several other antimicrobial compounds in alfalfa sprouts stored at 15 1C. cereus LWL1 was inoculated on soybean sprouts in combination with the bacteriocin-producing strain E. cereus after treatment or during the following 24 h of storage (Fig. weihenstephanensis CECT 5894 inoculated on alfalfa sprouts stored a 15 1C. 5). / Food Microbiology 25 (2008) 762–770 population multiplied during storage at higher temperatures. but did not prevent further proliferation of the surviving bacteria (Fig. sodium hypochlorite as well as hexadecylpyridinium chloride reduced the population of B. Many of the antimicrobial compounds tested individually reduced the numbers of viable cells significantly (Po0. peracetic acid (C) and polyphosphoric acid (D) on the viability of B. that can germinate and reach high population densities greater than risk levels. samples treated with AS-48 (m). weihenstephanensis in sprouts had still not been investigated. Controls (J). cereus was completely inhibited for the whole storage period. Effect of enterocin AS-48 (25 mg/ml) in combination with hydrocinnamic acid (A)..5% polyphosphoric acid (F) on the viability of B. 7B). In both cases. during the first 3 days of storage (Fig. 2004.2–5.1% polyphosphoric acid (E) and 0. 2006. cereus. Previous studies have demonstrated that the addition of enterocin AS-48 presents a good inhibitory activity against this bacteria in foods of milk origin. earth. peracetic acid (B). Seed sprouts can contain spores of B. 2005).4 min and. Enterococci multiplied rapidly on sprouts both at 15 and 22 1C. 4. 7). cereus coming from diverse sources (vegetal substrate. / Food Microbiology 25 (2008) 762–770 4 Log CFU/ml Log CFU/ml 4 2 0 0 0 1 3 5 Time (days) 7 0 1 0 1 Log CFU/ml 2 7 2 0 0 0 1 3 5 7 3 5 7 Time (days) Time (days) 4 Log CFU/ml 4 Log CFU/ml 3 5 Time (days) 4 4 Log CFU/ml 2 2 2 0 0 0 1 3 5 Time (days) 7 0 1 3 5 Time (days) 7 Fig. in addition. weihenstephanensis CECT 5894 in alfalfa sprouts stored at 15 1C for 1 week. Choma et al. cereus survive pasteurization processes of 100 1C during 2. cereus in the food chain could be solved by using natural antibacterial substances like bacteriocins. By contrast. The spores of B. 1995. hexadecylpyridinium chloride (D). 6. with viable cell counts being significantly lower than monocultures for the first 5 days of storage (Fig. sodium hypochlorite (C). or AS-48+chemical compound (K). Data represent the average of two independent experiments plus standard deviation (error bars). the (Mun potential of this bacteriocin in the control of B. growth of B. Nevertheless.. Cobo Molinos et al. of 4–5 1C (Dufrenne et al.ARTICLE IN PRESS 768 A. 0. The results obtained in the present study are clearly promising for the decontamination of these two species. but not at other points of storage. boiled rice and vegetable purees ˜ oz et al. chemical compound (’). bacteriocin activity could be recovered from the sprouts during days 1–3.. cereus as well as B. weihenstephanensis is now being investigated as it may also produce food-poisoning toxins. The closely related psychrotolerant species B. cereus is the main aerobic mesophilic endospore former of concern in the food industry (Schoeni and Wong. vegetative cells from some strains can grow at low temperatures. 7A). fertilizers). Grande et al. cocultivation with the producing strain at 22 1C only produced some growth inhibition of B. protective culture at 15 and 22 1C (Fig. mainly for samples stored . Discussion B. In coculture samples stored at 15 1C. The need to include additional hurdles for the control of B.. 2000). 2007b). under refrigeration. In the present study. 2003. Mori... For this reason. cereus LWL1 and the enterocin AS-48 producer strain E. Acknowledgments This work was supported by the Spanish Ministry of Education (Research Project AGL2005-07665-C02-02/ALI). Hugas... Valdivia. 24. These results suggest that such combinations could be used as highly effective decontamination treatments against Bacillus in germinated seed sprouts. 129.. M. cereus and B. Valdivia. 2007). cereulide. reaching elevated cell densities within the three first days of storage of the samples at 15 and 22 1C. the strains used for food applications should be devoid of such traits. A. For most of them. Ananou. in order to increase the temperature safety margins it would be necessary to apply combined treatments with other antimicrobial agents. and are also found in many different types of foods (revised by Foulquie´ Moreno et al. Maqueda. 549–576. Results from the preliminary screening carried out in the present study were highly satisfactory for the combined treatments of AS-48 with the cinnamic and hydrocinnamic acids.. In other cases (lactic acid. this is the first example of application of washing treatments against endospore-forming bacteria and also for application of a bacteriocin against B. cereus and B. A novel dodecadepsipeptide. Maqueda. viable cell counts in the bacteriocintreated samples were significantly lower compared to the nontreated controls.. weihenstephanensis. Ga´lvez. A. the treatment with bacteriocin did not provide any protection under abuse temperature conditions of 15 or 22 1C. Since there is now a concern that enterococci may be involved in the spread of antibiotic resistance and virulence traits in foods. reducing the concentration of viable cells below the detection limits for most of the samples. cereus under suitable temperature conditions that allow production of sufficient amounts of bacteriocin without favouring an excessive growth of B. Control of Staphylococcus aureus in sausages by enterocin AS-48. Enterococci are part of the normal microbiota of the human gastrointestinal tract. Food Microbiol. We also acknowledge the Research Programme of the University of Jae´n.. 2007. cereus were reduced below the detection limits. V. FEMS Microbiol.. M.. 17–20. E.. M. faecalis A-48-32 on soybean sprouts stored at 15 1C (A) and 22 1C (B). cereus in soybean sprouts also indicate that the AS-48 producing strains could serve to control the growth of B. J. 7. hexadecylpyridinium chloride and sodium hypochlorite. is an emetic toxin of Bacillus cereus. liquefaciens A-4832.... viable cell counts of B. Valdivia. M. in which the bacteriocin-producing strains are used to inhibit the growth of pathogenic or toxicogenic bacteria.. weihenstephanensis during 1 week storage of sprouts at 15 1C. Martı´nez-Bueno. Ananou et al. Ananou. Sua´rez.. M. Therefore.. It is noteworthy the good implantation shown by the bacteriocinogenic strain A-48-32 in soybean sprouts. Meat Sci. Microbiol. 179–190. Gil. M. Lett. the use of producing strains of enterocin AS-48 has shown good results in the control of B. Martı´nez-Bueno. those combinations of AS-48 that were more effective in reducing viable cell counts during treatment also provided a very good protection against B... aureus in cheese as ˜ oz well as of S. peracetic acid. Data represent the average of two independent experiments plus standard deviation (error bars). Cocultivation of B. but not with the chemical preservatives alone. 2004..E. 2007. b). Isobe. The results obtained in the present study against B. M. faecalis A-48-32 in cocultures (m) and bacteriocin production (bars).. However. A. Selma.. Ga´lvez. polyphosphoric acid. Growth of strain E. Maqueda. M. Ohta.. However. aureus and L. One of the main problems of decontamination treatments is that the reductions of viable cell counts obtained may not be 769 sufficient to avoid growth of survivors and sublethally injured cells during storage.ARTICLE IN PRESS A. B.. Martı´nez. 1995.. cereus LWL in control monocultures (J) and in cocultures (K) are shown. Growth and bacteriocin production by lactic acid bacteria in vegetable broth and their effectiveness at reducing Listeria monocytogenes in vitro and in fresh-cut lettuce. weihenstephanensis on sprouts.. A. References Abriouel. 103. H. In this respect. Cobo Molinos et al. 759–766. In this sense. / Food Microbiology 25 (2008) 762–770 15 12 8 6 10 4 AS-48 (mm) Log CFU/ml 10 2 0 5 0 1 2 3 5 Time (days) 7 12 15 8 6 10 4 AS-48 (mm) Log CFU/ml 10 2 0 5 0 1 2 3 5 Time (days) 7 Fig. 599–605. A simple method for semi-preparative-scale production and recovery of enterocin AS-48 derived from Enterococcus faecalis subsp.. n-propyl p-hydroxybenzoate and p-hydoxybenzoic acid methyl ester). While previous reports have studied the synergistic effects of antimicrobial compounds with bacteriocins (reviewed by Ga´lvez et al. cereus. Garriga. and the Research Plan of the Junta de Andalucı´a (Research Group AGR230). Methods 55. lactate sodium.. Allende. the application of this strain as a protective culture would be limited by the low bacteriocin production under refrigeration and the lack of effectiveness shown at 22 1C. M. M. Int. Another possible alternative to control undesired microorganisms in foods is based on the application of competitive exclusion techniques. 2006). 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