Evaluation of an enterocin AS-48 enriched bioactive powder obtained by spray drying

Food Microbiology 27 (2010) 58–63

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Evaluation of an enterocin AS-48 enriched bioactive powder obtained by spray drying ˜ oz a, M. Martı´nez-Bueno a, P. Gonza´lez-Tello b, A. Ga´lvez c, S. Ananou a, A. Mun M. Maqueda a, E. Valdivia a, b, * a b c

Departamento de Microbiologı´a, Facultad de Ciencias, Universidad de Granada, Fuentenueva s/n, 19071 Granada, Spain Departamento de Ingenierı´a Quı´mica, Facultad de Ciencias, Universidad de Granada Fuentenueva s/n 19071 Granada, Spain ´ rea de Microbiologı´a, Facultad de Ciencias Experimentales, Universidad de Jae´n, Paraje Las Lagunillas, 23071 Jae´n, Spain A

a r t i c l e i n f o

a b s t r a c t

Article history: Received 31 March 2009 Received in revised form 31 July 2009 Accepted 1 August 2009 Available online 5 August 2009

Enterocin AS-48 is a cationic cyclic bacteriocin produced by Enterococcus faecalis with broad bactericidal activity. Currently we are assaying the efficacy of AS-48 as biopreservative in foods. In this work we have applied the spray drying process to different AS-48 liquid samples to obtain active dried preparations. We have also assayed different methods, heat, UV irradiation and filtration, to inactivate/remove the AS-48 producer cells from the samples. Best results were obtained for the sample from CM-25 cation exchange, for which it was also possible to completely eliminate/inactivate the producer cells by heat or UV irradiation without loss of activity. When added at 0.016% or 5% to Brain Heart Infusion broth or to skim milk, respectively, the AS-48 powder caused early and complete inactivation of Listeria monocytogenes. A partial inhibition of Staphylococcus aureus was achieved in broth and in skim milk supplemented with 2.5% and 10% AS-48 powder, respectively. Ó 2009 Elsevier Ltd. All rights reserved.

Keywords: Enterocin AS-48 Spray drying Bioactive powder Listeria monocytogenes Staphylococcus aureus

1. Introduction Many of the chemicals licensed for use as food preservatives are increasingly being questioned with regard to their effects on humans, creating pressure on food suppliers to consider the use of natural alternatives. This circumstance has increased interest in research on lactic acid bacteria (LAB) and LAB bacteriocins, since they have been in foods from time immemorial and can more easily be recognised as safe compounds (GRAS). Consequently, several LAB bacteriocins are currently being assayed for food-manufacturing processes in attempts to improve the safety and shelf life of foods (Stiles, 1996; Chen and Hoover, 2003; Deegan et al., 2006; Ga´lvez et al., 2008). Bacteriocins can be applied to food in two basic ways: inoculation of the bacteriocinogenic strain as a bioprotective culture or by adding bacteriocin previously produced by conventional fermentation. In the latter case, for effective commercial use the production processes must be optimized to make the product economically feasible. However, for the addition of preformed bacterocins to food, it is mandatory that they be produced in a food-grade substrate and that * Corresponding author at: Departamento de Microbiologı´a, Facultad de Ciencias, Universidad de Granada, Fuentenueva s/n, 19071 Granada, Spain. Tel.: þ34 958 243244; fax: þ34 958 249486. E-mail address: [email protected] (E. Valdivia). 0740-0020/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.fm.2009.08.002

the preparation of bacteriocin be stable and free of producer cells. In this respect spray drying is a potentially useful process for the largescale production of dried powders containing bioactive molecules that can be transported at low cost and stored in a stable form for prolonged periods (Gardiner et al., 2000). In addition, this technology is less expensive and time-consuming than other processes such as freeze-drying. The most widely researched example of bacteriocin is nisin, which has been approved globally for use in many foods to extend product shelf life; it is commercially available as a powder known as Nisaplin. The product is extremely stable over time (two years from the date of manufacture provided it is stored dry, away from direct sunlight and between 4  C and 25  C) (Morgan et al., 1999). However, application of nisin on food systems such as meat products is clearly limited, especially if the pH is above 5.0 (ElKhateib et al., 1993; Fang and Lin, 1994). In fact, it is currently applied only on a few meat products, and a high concentration (250 mg/g) is recommended in these cases (Thomas et al., 2000). Nisaplin is ineffective against Gram negative bacteria and, as in the case of most antimicrobials, nisin-resistant organisms develop (Mazzotta and Montville, 1997; Ming and Daeschel, 1993). Therefore, other bacteriocin-producing lactic acid bacteria are being examined for antimicrobial substances as alternatives to nisin. Enterocin AS-48 is a cationic circular bacteriocin produced by Enterococcus faecalis S-48 with broad bactericidal activity against

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most of the Gram positive bacteria, including several pathogens like Listeria monocytogenes, Staphylococcus aureus, Mycobacterium spp., Bacillus cereus (Ga´lvez et al., 1989a; Abriouel et al., 1998, 2002; Mendoza et al., 1999). Even some Gram negative bacteria, such as some Escherichia coli and Myxococcus strain, are susceptible to the enterocin (Ga´lvez et al., 1989a). In addition is it possible to sensitise resistant Gram negative bacteria such as Salmonella choleraesuis or E. coli O157:H7, by combining AS-48 with agents that destabilise the outer membrane (Abriouel et al., 1998; Ananou et al., 2005c). Various characteristics of AS-48 (broad spectrum of antimicrobial activity, stability over a wide range of temperature and pH, and sensitivity to digestive proteases (Ga´lvez et al., 1986; Samyn et al., 1994) indicate it is a promising alternative to chemical preservatives for use as a biopreservative in foods. In fact, AS-48 has demonstrated itself to be effective in the control of various food-borne pathogens in dairy, ˜ oz et al., 2004, 2007; Ananou, meat, and vegetable products (Mun 2005a,b,c; Cobo-Molinos et al., 2005; Grande et al., 2006). Currently we are assaying the efficacy of enterocin AS-48 as a biopreservative in different food systems; this application raises the need to obtain sufficient bacteriocin amounts from a food-grade substrate and in an applicable, dried form. Along these lines, we have recently developed an easy and economical process to obtain large amounts of bacteriocin by culturing the producer strain in a whey-derived substrate, Esprion-300 (Ananou et al., 2008). In the present study, we discuss the results of the application of spray drying AS-48 samples with the aims of obtaining the enterocin in a cell-free active powder suitable for food application and also the evaluation of its inhibitory ability in broth and in skim milk against L. monocytogenes and S. aureus. 2. Materials and methods 2.1. Bacterial strains and culture conditions E. faecalis A-48-32 was used as a AS-48 producer. E. faecalis S-47, from our laboratory collection, was used as the standard indicator strain for bacteriocin activity assays. To prove the identity of the antimicrobial substance present in powders with AS-48 we have used as indicator strain E. faecalis JH2-2 (Yagi and Clewell, 1980), susceptible to the enterocin and E. faecalis JH2-2 (pAM401-81), an isogenic transformed strain harbouring the plasmid pAM 401-81 which encoded AS-48 production and immunity (Dı´az et al., 2003). L. monocytogenes CECT 4032 and S. aureus CECT 976 were used to test the inhibitory activity of AS-48 dried preparations. All strains were routinely grown in brain heart infusion broth (BHI) (Scharlau, Barcelona, Spain) at 28  C. Solid media were prepared by adding 1.5% agar (Scharlau) to the broth (BHA). Bacteria cultures were maintained at 5  C in BHA slants. 2.2. Bacteriocin assays To quantify enterocin AS-48, samples were tested for inhibitory activity against the indicator strain S-47 by the agar well diffusion method (Ga´lvez et al., 1986). Briefly, stainless steel cylinders (Oxford towers, 8 mm diameter by 10 mm height; Scharlau) were deposited on a Petri dish containing 10 ml of solidified buffered BHA (in 0.1 mol/l sodium phosphate buffer, pH 7.2). Then, 6 ml of molten soft BHA (1.5% BHI plus 0.8% agar) buffered as above was tempered to 45  C and inoculated (3  108 CFU/ml) with a BHI culture of the standard indicator strain E. faecalis S-47 and then poured onto the plate. After solidification, the Oxford towers were removed and 100 ml of the samples to be tested were deposited in the resulting agar wells. Plates were incubated at 28  C for 18–24 h, and the diameters (in mm) of the inhibition zones around the wells were measured. The activity in arbitrary units per ml (AU/ml) was calculated from a titration curve obtained previously in our laboratory

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relating the diameter of inhibition zones (in mm) produced by a series of two-fold diluted enterocin samples with the reciprocal of the highest dilution showing inhibition of the indicator lawn. 2.3. Enterocin AS-48 production and recovery AS-48 was produced by culturing the strain E. faecalis A-48-32 in a food-grade whey-derived substrate, Esprion-300 (E-300) (DMV Int., Veghel, Netherland) supplemented with 1% glucose according to Ananou et al. (2008). The substrate was reconstituted at 5% total solids in sterile distilled water and inoculated (8%, vol/vol) with an 18-h E. faecalis A-48-32 culture in skim milk. Cultivation lasted 18–20 h at 28  C at a controlled pH of 6.5 by the addition of 5 M NaOH on demand via a 719 S Titrino (Metrohm, Herisau, Suiza) with shaking at 400 rpm with a magnetic stirrer to homogeneously distribute the added NaOH. AS-48 was recovered from E-300 cultures by cation exchange chromatography on carboxymethyl Sephadex CM-25 (as described in Abriouel et al., 2003). When indicated, eluted fractions were dialysed at 4  C against distilled water through a 2000-Da cut-off membrane to eliminate NaCl. 2.4. Elimination/inactivation of AS-48 producer cells One of the requirements for food-grade bacteriocin preparations is that they be free of viable producer cells. So, before use, attempts were made to remove/inactivate the bacteriocinogenic cells by different procedures: membrane filtration, heat, and UV light irradiation. Cell removal from samples (10–20 ml) by transverse flow filtration was performed with 0.22-mm pore filters of different composition (all from Millipore, Belford, MA, USA): cellulose acetate (CA), polyvinylidene difluoride (PVDF), and polyethersulfone (PES). Heat inactivation of cells was accomplished by heating samples (45 ml)in 50 ml polypropylene tubes at different temperatures for different times. Temperature was measured in the centre of tubes with a thermometer. Inactivation of cells by UV light was carried out by irradiation with a 253 nm 30 W lamp (Telstar, Terrassa, Spain) sited 30 cm above the samples (60 ml volume, 6 mm thick) for different times. The efficacy of each treatment was determined by measuring the inhibitory activity of samples by the agar well diffusion method and the remaining viable cell numbers by decimal dilution in sterile saline solution and direct plating in triplicate in a BHA medium. For treatment in which no colonies were obtained by direct plating, 1 ml of the treated sample was mixed with 1 ml of double concentrated BHI broth, incubated at 28  C for 48 h and then examined for the presence of viable cells by measuring optical density at 620 nm and also by plating on BHA plates. In samples in which growth was observed after 48 h, an enrichment in BHI broth of decimal dilutions of the samples was also carried out in order to determine the approx. number of viable cells in the undiluted origin sample. Two independent replicates of each experiment were made and in each experiment two batches of each treatment were stated. In each batch values of cfu/ml and enterocin titer were assayed in triplicate. The results are expressed as the average of the values obtained. Error bars in figures represent standard deviations. 2.5. Spray drying conditions Different preparations of AS-48 (500 ml each) were directly subjected to spray drying: A. Raw AS-48 fermentate produced in an E300 medium. B. AS-48 preparation recovered from an E300 medium by cation exchange on carboxymethyl Sephadex CM-25.

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Samples were spray-dried in a laboratory-scale Bu¨chi 190 minispray dryer (Laboratoriums-Technik AG, Flawil, Switzerland). Spray droplets (produced by the atomization of the feed liquid by a twin-fluid atomizers vaned wheel rotating at high speed) were evaporated in a vertical co-current drying chamber. Powder was collected in a single cyclone separator. The spray-drier conditions were: inlet air-drying temperature of 156  C, outlet air-drying temperature of 87  C, and atomizing with a compressed-air flow (6 Bar) in the twin-flow nozzle. After drying, the powder was distributed in impermeable plastic bags and stored at 5  C. In each experiment, the cell counts and inhibitory activity of the samples were determined before and after drying. 2.6. Stability of AS-48 powders The AS-48 bioactive powders (obtained by drying the preparations recovered from CM-25) were assessed for stability over 8 months of storage at 20  C, 5  C, or at room temperature. Periodically, the powders were reconstituted in sterile distilled water to the original solid concentration, and the activity of AS-48 was determined by the agar well diffusion assay as described above. All experiments were carried out in duplicate and the results are expressed as the average of the values obtained. Error bars in figures represent standard deviations. 2.7. Effect of AS-48 bioactive powder against L. monocytogenes and S. aureus in liquid media, BHI and skim milk Experiments were performed in BHI broth or skim milk as follows: an overnight culture of listeria was diluted in fresh medium (BHI or skim milk) to reach a cell concentration of 103 CFU/ml, aliquoted in 4 ml batches (in duplicate for each AS-48 concentration) and then added with 0.016% or 0.008% (weight/vol) of the AS-48 powder (obtained from CM-25 preparations), in experiments in BHI broth or 5% in experiments in skim milk, and then incubated at 28  C. Given its greater resistance to AS-48, staphylococci cultures, prepared as for listeria experiments, were added with 2.5% or 10% of the powder in BHI or in skim milk respectively. At desired intervals, samples were removed and serially diluted into sterile saline solution (0.85% NaCl). The appropriate dilutions were plated in triplicate on BHA plates, and the average number of colonies (CFU/ml) obtained after 24 h incubation at 28  C was used to establish the growth and survival curves. Control batches had no AS-48 powder but they were supplemented with 1.9%, 3.7% and 7.5% NaCl in order to achieve equivalent salt concentrations as the batches challenged with 2.5%, 5% and 10% of active powder respectively. Two independent replicates of each experiment were made and in each experiment two batches of each treatment were stated. In each batch values of CFU/ml were determined in triplicate. The results are expressed as the average of the values obtained. Error bars in figures represent standard deviations. 3. Results and discussion

AS-48 activity was reduced in 80% whereas the Enterococcus counts remained at 2.8  104 CFU/ml). Neither it was possible to inactivate the producer cells after the drying process in the resuspended powder. Morgan et al. (2001) eliminated producer lactococci in lacticin 3147 fermentates by pasteurization prior to spray drying, but the greater thermal resistance of enterococci made this approach unviable in the present case. In contrast, heat revealed itself to be an effective approach to inactivate cells in samples deriving from CM-25, with a treatment of 80  C for 20 min being sufficient to inactivate approx. 107 CFU/ml (Fig. 1) without further cell recovery after 48 h enrichment in BHI broth. UV light irradiation was also tested to inactivate producer cells. Due to the high turbidity of the raw fermentate, this approach was not applicable to this type of sample. However, UV light was very effective at inactivating enterococci in undialysed CM-25 samples and 5 min irradiation under the prescribed conditions was enough to kill all viable cells in the samples as measured right after treatment and also after 48 h enrichment in BHI broth (Fig. 2). In this regard, it is worth taking particular note of the effective, cheap, and easy application of UV light. Filtration using transverse flow through membranes has previously been used as the preferred means of removing the producer cells from AS-48 samples deriving from CM-25 (Abriouel et al., 2003). This approach was not applicable to raw fermentate due to the rapid formation of a filter cake that clotted the membranes. Even in the case of CM-25 samples, problems were found related with the retention of part of the activity and also with the passage through membranes of a low number of cells (approx. 10 CFU/ml) when moderately large volumes of samples were processed (up to 15–20 ml). In order to solve these problems, membranes of different composition were tested. None of the membranes used (CA, PVDF, and PES), however, were effective in preventing the passage of a few enterococcal cells through the filters. The PES membrane was the only one that did not retain any inhibitory activity. The other types of membranes (CA and PVDF) retained 87.5% and 50% of inhibitory activity respectively. It is worth noting that after dialysis against distilled water through a 2000-Da cut-off membrane of the samples eluted from CM-25 no growth was obtained by direct plating of the dialyzed samples (data not shown). This result can be probably due to the combination of osmotic stress and low temperature during dialysis (4  C). However, after enrichment of dialyzed samples in BHI broth for 48 h, cell recovery was observed by the evident growth even in 7

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the 106 dilution. So we can conclude that sublethal stressed cells recovered viability after culturing in a favourable liquid medium. According to the results obtained, UV irradiation for 5 min was the method of choice to inactivate the cells in samples recovered from CM-25, previously to drying process.

Spray drying of sample B (recovered from CM-25 cation exchange) resulted in a fine, amorphous material that was easily redissolved at the original concentration (7.4% solids) and showing the same inhibitory activity as the sample before drying (600 AU/ ml). In contrast to sample A, in this case it was possible to eliminate/ inactivate the bacteriocin-producer cells by heat or UV irradiation or after spraying. In addition to concentrating the enterocin, reducing the bacterial load by approx. 3 log CFU/ml, and eliminating most of the Esprion-300 components, CM-25 chromatography clarified the preparations and allowed the removal/inactivation the producer cells by either heat, UV irradiation, or filtration. Sample C also yielded an active powder when spray-dried. Nevertheless, taking into account the low solid concentration (1.1%) in this sample and the fact that most of the material remained adhered to the walls of the spray-drier, the weight of active substance recovered in the container for collecting the finished product was quite insignificant. It will be necessary to further evaluate the feasibility of this process since the absence of NaCl in this type of preparation would allow the inclusion of other carriers (such as those of a dairy origin) in order to develop bacteriocin preparations suitable for food systems that do not require added salt. In all cases, the inhibitory activity of the powders can be attributed to AS-48 rather than other metabolites such as organic acids or other bacteriocins since it was active against the E. faecalis JH2-2 strains but did not show any inhibitory effect against the transformed isogenic strain E. faecalis JH2-2 (pAM 401-81), which produces and resists AS-48 (data not shown).

3.2. Production of a spray-dried AS-48 powder

3.3. Stability of AS-48 powders

Spray drying technology has been applied to obtain powders in many areas, including foods (e.g. dairy industry). It has been used to obtain active powder of lacticin 3147 (Morgan et al., 1999, 2001), mixtures of several lactococci and lactobacilli strains (Mauriello et al., 1999), or bacteriocinogenic probiotic cultures of Lactobacillus paracasei and Lactobacillus salivarius (Gardiner et al., 2000) without loss of viability nor of the bacteriocin production capacity of strains, as well as preparations containing mixtures of bacteriocinogenic strains: Lactobacillus sakei, Lb. salivarius, and Carnobacterium divergens (Silva et al., 2002). In this work we have tried to produce a dry bioactive AS-48 powder suitable for food application, cell-free and stable during storage by applying the spray drying process. Spray drying of the raw fermentate (sample A) resulted in the production of an active powder that, when resuspended at the original concentration (5% solids), contained the same inhibitory activity as the initial sample (360 AU/ml). Nevertheless, it was not possible to recover most of the powder that remained deposited on the inner walls of the device, as it had a sticky, gummy consistency. In addition, as described above, the main concern with this powder was the failure to completely eliminate/inactivate the enterococci in this type of sample without affecting AS-48 activity. In this regard, the drying process only decreased viable cell numbers by one log unit at most (e.g. from 10 log CFU/ml to 9 log CFU/ml in the case of raw fermentate), and the application of the different methods (filtration, heat, or irradiation) before or after drying was unpractical or ineffective due to the physical properties of the growth medium (a suspension rather than a solution). Another difficulty related with this type of powder is the low concentration of the active substance in the crude fermentate. Even after concentration of the enterocin by drying, it would still be necessary to add a large amount of powder to achieve an inhibitory effect because of the high proportion of solids in the growth substrate in relation with the active substance. So, although this is the simplest and cheapest approach to obtaining an AS-48 active powder, we are not considering this alternative at the moment.

AS-48 bioactive powders obtained from undialysed CM-25 samples were aliquoted in hermetically closed bottles and stored at 20  C, 5  C at room temperature for 8 months. At 20  C, the bioactive powders remained active for at least 8 months. At 5  C, the powders retained full activity for at least 4 months, but sampling at 8 months indicated that the product had lost 50% activity. At room temperature, powder activity decayed to 50% by month 4 and to 25% by month 8 (Fig. 3). These results show that AS-48 powder is, on the whole, as stable as the lacticin 3147 powder, and is even more stable under freezing conditions than lacticin (Morgan et al., 2001). Nevertheless, the stability of AS-48 powder was clearly lower than the commercial powdered form of nisin, Nisaplin, which is highly

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stable between 4  C and 25  C over two years from the date of manufacture (Morgan et al., 2001). 3.4. Antimicrobial effect of bioactive powder against L. monocytogenes and S. aureus The bioactive powder was tested in liquid media against the pathogens L. monocytogenes and S. aureus. Since it was impossible to eliminate producer enterococci in raw fermentate, all experiments were carried out with the powder obtained by drying undialysed CM25 samples. In cultures in BHI broth with added bioactive powder (0.016%, weight/vol), Listeria counts fell drastically below detection levels (10 CFU/ml) after 2 h of incubation and remained undetectable throughout the experiment. According to the relationship established in previous works for AS-48 between AU/ml and mg/ml (Ga´lvez et al., 1989b), this concentration of powder correspond with approx. 0.1 mg/ml. Earlier results on the activity against L. monocytogenes of AS-48 liquid samples deriving from CM-25 in BHI broth (Mendoza

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et al., 1999) revealed a Minimal Bactericidal Concentration (MBC) of 0.1 mg/ml, that was similar to that obtain with bioactive powder. In cultures treated with 0.008% bioactive powder a drastic reduction of viable counts was also achieved within the first 2–8 h of incubation but, in this case, the cultures partially recovered growth after 8–10 h of incubation (Fig. 4A). The experiment was reproduced in skim milk but, based on previous experience, the greater complexity of this food matrix advised the use of a higher concentration (5%, 400 AU/ml) of AS-48 powder (Fig. 4B). The bacteriocin-treated samples remained free of detectable listeria from 2 h after treatment to at least 168 h. As expected, AS-48 powder was less active against S. aureus, one of the most resistant Gram positive bacteria to AS-48. Yet a drastic reduction of viable counts was achieved in BHI cultures treated with 2.5% powder (200 AU/ml, approx. 15 mg AS-48/ml) within the first 2 h. Nevertheless, S. aureus resumed growth rapidly, reaching viable counts (approx. 9 log units) similar to the control after 24 h of incubation (Fig. 5A). In skim milk, Staphylococcus cultures with added AS-48 powder (10%, (800 AU/ml) underwent a drop of 0.8 log units in the first 2 h (which represents a difference of 2.6 log units with respect to the control at the same elapsed time) and afterwards a progressive reduction in viable counts occurred up to 96 h incubation (Fig. 5B). This reduction represented differences of 4.0, 3.8, and 2.3 log units with respect to control cultures at 24, 96, and 168 h, respectively. Previous experiments treating S. aureus with

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CM-25 AS-48 liquid samples yielded MBCs of 15 mg/ml in BHI (Ananou et al., 2004), lower than in this experiment. In any case, the efficacy of this powdered preparation will still need to be tested in each specific food system since it is well known that bacteriocin activity can be influenced by the chemical composition and the physical conditions of food (Cleveland et al., 2001). Other authors have reported on the applications of powdered bacteriocins as biopreservatives. Morgan et al. (2001), for instance, demonstrated the potential of a lacticin 3147 powder as a biopreservative in natural yogurt, cottage cheese, and soup against L. monocytogenes and B. cereus, suggesting that lacticin 3147 may have widespread applications in food systems. Nevertheless, our results cannot be compared with those found by these authors since the lacticin 3147 powder was prepared directly from fermentates without any separation step. In fact, a high concentration of lacticin active powder (10%) was necessary to achieve a complete inhibition of L. monocytogenes in yogurt and in cottage cheese, thereby indicating the desirability of obtaining a more concentrated preparation like the one developed in the present study for enterocin AS-48. The results presented here are the first evidence for the production of an active dried AS-48 powder, free of producer cells and stable under freezing or refrigeration that might have widespread applications in food systems to control L. monocytogenes. Complete AS-48 activity is preserved after spray drying independently of the dispersion medium used. Nevertheless, this is a preliminary study and numerous technological limitations need to be considered before a commercial preparation could be made available, such as the generation of a product with greater activity in order to reduce the amounts of added powder required for inhibition. The process would also have to be scaled-up in order to minimize loss during spray drying. Acknowledgements This work was supported by a grant (AGL2005-07665-C02-01) from the Spanish Ministry of Science and Technology. Samir Ananou received several grants from the Junta de Andalucı´a (PAI ˜ oz has been a recipient Research Group CVI 160) and Arantxa Mun of fellowships from the Spanish Ministry of Education and Culture. We thank Antonio Carmona for assistance with the Spray-Drier. We would also like to acknowledge the work of Christine Laurin in editing the text. References Abriouel, H., Valdivia, E., Ga´lvez, A., Maqueda, M., 1998. Response of Salmonella choleraesuis LT2 spheroplasts and permeabilized cells to the bacteriocin AS-48. Appl. Environ. Microbiol. 64, 4623–4626. Abriouel, H., Maqueda, M., Ga´lvez, A., Martı´nez-Bueno, M., Valdivia, E., 2002. Inhibition of bacterial growth, enterotoxin production, and spore outgrowth in strains of Bacillus cereus by bacteriocin AS-48. Appl. Environ. Microbiol. 68, 1473–1477. Abriouel, H., Valdivia, E., Martı´nez-Bueno, M., Maqueda, M., Ga´lvez, A., 2003. A simple method for semi-preparative-scale production and recovery of enterocin AS-48 derived from Enterococcus faecalis subsp. liquefaciens A-48-32. J. Microbiol. Methods 55, 599–605. Ananou, S., Valdivia, E., Martı´nez-Bueno, M., Ga´lvez, A., Maqueda, M., 2004. Effect of combined physico-chemical preservatives on enterocin AS-48 activity against the enterotoxigenic Staphylococcus aureus CECT 976 strain. J. Appl. Microbiol. 97, 48–56. Ananou, S., Garriga, M., Hugas, M., Maqueda, M., Martı´nez-Bueno, M., Ga´lvez, A., Valdivia, E., 2005a. Control of Listeria monocytogenes in model sausages by enterocin AS-48. Int. J. Food Microbiol. 103, 179–190. Ananou, S., Maqueda, M., Martı´nez-Bueno, M., Ga´lvez, A., Valdivia, E., 2005b. Synergistic effect of enterocin AS-48 in combination with outer membrane permeabilizing treatments against Escherichia coli O157:H7. J. Appl. Microbiol. 99, 1364–1372. Ananou, S., Maqueda, M., Martı´nez-Bueno, M., Ga´lvez, A., Valdivia, E., 2005c. Control of Staphylococcus aureus in sausages by enterocin AS-48. Meat Sci. 71, 549–556.

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