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The effect of adding antimicrobial peptides to milk inoculated with Staphylococcus aureus and processed by high-intensity pulsed-electric field

A. Sobrino-Lopez^*, P. Viedma-Martínez, H. Abriouel, E. Valdivia, A. Gálvez and O. Martin-Belloso^*,1

^* Department of Food Technology, University of Lleida, Avda. Alcalde Rovira Roure 191, 25198 Lleida, Spain
Microbiology Division, Health Sciences Department, University of Jaen, Campus Las Lagunillas s/n, 23071 Jaen, Spain
Department of Microbiology, University of Granada, 18071 Granada, Spain

¹ Corresponding author: omartin{at}tecal.udl.es

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
The use of high-intensity pulsed-electric field (HIPEF) and antimicrobial substances of natural origin, such as enterocin AS-48 (AS-48), nisin, and lysozyme, are among the most important nonthermal preservation methods. Thus, the purpose of this study was to evaluate the combined effect on milk inoculated with Staphylococcus aureus of the addition of AS-48 with nisin or lysozyme, or both, together with the use of HIPEF. Synergy was observed in the reduction of Staph. aureus counts with the following combination methods: i) addition of AS-48 and nisin, ii) addition of AS-48 plus use of HIPEF, and iii) addition of AS-48 and nisin plus use of HIPEF. Specifically, when 28 arbitrary units/mL of AS-48 and 20 IU/mL of nisin were added to the milk, and it was treated with HIPEF for 800 µs, over 6 log reductions were observed in the microorganism. In general, Staph. aureus inactivation was dependent on HIPEF treatment time, antimicrobial doses, and medium pH. During storage of the treated milk, survivor population was related to peptide concentration and temperature. Final cell viability was influenced by the sequence in which the treatments were applied: the addition of AS-48 or AS-48 and nisin was more effective before than after HIPEF treatment. The results obtained indicate that the combination of HIPEF and antimicrobials could be of great interest to the dairy industry, although it is necessary to study further the way in which the combined treatments act.

Key Words: milk • high-intensity pulsed-electric field • enterocin AS-48 • nisin

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
The interest of the food industry in food preservation through nonthermal methods arises, in general, from the fact that they make it possible to guarantee the microbiological qualities of a product while, at the same time, minimizing the effect on its sensory properties. Of these methods, the use of high-intensity pulsed-electric field (HIPEF) and of antimicrobial compounds of natural origin are of special interest because of their great potential as preservation treatments in a wide range of food.

High-intensity pulsed-electric field is one of the most important nonthermal techniques for treating liquid foods for 2 reasons: first, because it is possible to use it industrially on continuous-flow processing lines, specifically before reaching the aseptic packaging equipment, and, second, because of its wide spectrum of microorganism inactivation. However, the resistance of different microorganisms to HIPEF treatment is dependent on many factors, such as food media (Bendicho et al., 2002), electrical variables (Sobrino-López et al., 2006) and type of microorganism, species, and even strains (MacGregor et al., 2000). On the basis of a hurdle approach, the combination of HIPEF with other preservation methods can improve the final effectiveness of the treatment. Thus, it has been observed that the simultaneous combination of HIPEF with certain added antimicrobial substances results in a synergistic effect in the cell death (Wu et al., 2005; Sobrino-López and Martín-Belloso, 2006).

Among antimicrobial compounds of natural origin, we should differentiate between those that form part of the intrinsic composition of the food, such as enzymes capable of cell-membrane damages, and those that are formed during its processing, such as antimicrobial substances of bacterial origin released during fermentation of the product. Lysozyme is a component usually present in milk whose muramidase activity focuses above all on gram-positive microorganisms, although certain gram-negative bacteria have also been identified as being sensitive to this substance (Masschalck and Michiels, 2003). As a preservative agent, lysozyme is generally recognized as safe by the Joint Food and Agriculture Organization-World Health Organization (WHO Food Additives Series 30) and as quantum satis by European directive 95/2/EC on food additives other than colors and sweeteners.

With regard to the natural compounds added during the food processing, bacteriocins are substances with antimicrobial activity produced by bacteria (Gálvez et al., 2007). Especially important are those bacteriocins produced by acid-lactic bacteria because they are often related with a large number of fermentations in the food industry. In particular, nisin is a peptide produced by Lactococcus lactis ssp. lactis. Its lethal activity covers a wide spectrum of gram-positive pathogenic and food-spoilage bacteria (Rodríguez et al., 2000) and it can be used in a wide variety of foods, including dairy products (Jung et al., 1992). Because of its history as a preservative agent, nisin was accepted as a food additive, E-234, in the European Union (EEC, 1983) and, later, was qualified as generally recognized as safe by the FDA (1988).

In contrast to the long-use history of nisin, other bacteriocins have been identified and characterized only recently, although some authors have already pointed out the great potential of some of them to be used in the food industry. Enterocin AS-48 (AS-48) is a circular peptide, which is stable over a wide pH range, heat-resistant, and displays a broad antimicrobial spectrum. It is produced by Enterococcus faecalis (Gálvez et al., 1986) and shows feasibly applications in food such as sausages (Ananou et al., 2005b), vegetable sauces (Molinos et al., 2005), fruit juices (Grande et al., 2005), and dairy products (Muñoz et al., 2007). However, there is still little or no information on the effectiveness of this bacteriocin and its degree of microorganism inactivation when combined with HIPEF, or even with other bacteriocins. Because Staphylococcus aureus is an important milk- and dairy product-related pathogen, the purpose of this study was to evaluate the effect of enterocin AS-48 added alone to milk inoculated with Staph. aureus or in combination with nisin or lysozyme, and processed simultaneously with HIPEF treatment. The influence of processing parameters, the storage conditions of the samples treated, and the sequence of application of each treatment were also studied.

	MATERIALS AND METHODS

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Staphylococcus aureus Culture
Staphylococcus aureus CECT 240 (Food Technology Department, University of Lleida, Spain) was the target microorganism. The strain was maintained routinely on slants of plate count agar (Biokar Diagnostics, Beauvais, France) at 4°C until it was used.

Strain growth was performed by incubating cultures on tryptone soy broth at 35°C for 6 h. Inoculum concentration was determined by optical measurement. A population density of approximately 10⁸ cfu/mL matches an absorbance value between 0.6 and 0.7 at 620 nm.

Skim Milk
Homogenized UHT skim milk was obtained from a dairy plant and stored at 4°C (Puleva, Mollerussa, Lleida, Spain). Natural pH of milk was 6.80 ± 0.02 measured by a pH meter (Crison 2001 pH-meter, Crison Instruments SA, Alella, Barcelona, Spain). The electrical conductivity of the skim milk was 5.55 ± 0.04 mS/cm. The measurement was performed at 25°C and determined with a conductivity meter (Testo 240 conductivimeter; Testo GmBh & Co, Lenzkirch, Germany).

Preparation of Enterocin AS-48 Solution
Enterococcus faecalis A-48-32 (Martínez-Bueno et al., 1990) was used as enterocin AS-48 producer. The bacteriocin solution was obtained by purification of cultured broths of the producer strain after concentration by cation-exchange chromatography in accordance with the method described by Abriouel et al. (2003). Bacteriocin concentrates were desalted through 2,000 Da cut-off dialysis tubing (Sigma-Aldrich, Steinheim, Germany), filtered through 0.22-µm pore size low-protein binding filters (Millex GV, Millipore Corp., Belford, MA) under sterile conditions. The AS-48 activity was determined by the agar well diffusion method (Gálvez et al., 1986). The enterocin AS-48 preparation resulted in a concentration of 3,500 arbitrary units per mL (AU/mL), which was 10-fold diluted with demineralized water to obtain the final solution.

Sample Preparation
Samples with Staph. aureus were prepared by inoculating the microorganism in skim milk to a final concentration of approximately 10⁷ cfu/mL. According to the experimental design, pH of milk was adjusted by adding lactic acid (L(+)-lactic acid, Panreac, Barcelona, Spain). When acidity was adjusted to 5.0 pH, electrical conductivity of milk was 6.99 ± 0.13 mS/cm.

HIPEF Equipment and Treatment
A continuous-flow HIPEF system was used to carry out this study. The treatment device was an OSU-4F HIPEF unit (Ohio State University, Columbus) that discharges square-shape pulses within 8 collinear chambers, in which gap distance was 0.29 cm and each treatment chamber volume was 0.012 cm³. Electrical parameters were set at 35 kV/cm of electric field strength, 6 µs of pulse width, and 75 Hz of pulse frequency in bipolar mode in accordance with previous studies. Treatment temperature was kept under 25°C using a cooled water bath to rule out thermal effects. The antimicrobial action of HIPEF treatment against Staph. aureus was determined by treating samples at their natural pH and pH 5.0 up to a final treatment time of 1,200 µs. Each trial was performed in triplicate.

Enterocin AS-48 in Combination with Nisin or Lysozyme
The single effect of enterocin AS-48, nisin (N 5764, 2.5% nisin, 1,000,000 IU/mg, Sigma-Aldrich) and lysozyme (L 2879, 43,560 IU of lysozyme/mg of solid, Sigma-Aldrich) against Staph. aureus was measured by exposing samples at their natural pH and pH 5.0 to a bacteriocin concentration up to 28 AU/mL, 20 IU/mL, and 5,000 IU/mL, respectively. The effectiveness of AS-48 was also evaluated when added together with nisin or lysozyme and holding samples for 1 h at room temperature. All assays were performed in triplicate.

Application of HIPEF Treatment in Combination with Added Enterocin AS-48
The susceptibility of Staph. aureus against the combination of added enterocin AS-48 and HIPEF was studied by adding the bacteriocin to milk samples and then applying the HIPEF treatment. A response surface methodology (RSM) was set to study the effect of the combined treatment (Table 1). Process variables were AS-48 concentration (3.5 to 28 AU/mL), pH of milk (5.0 to 6.8), and HIPEF treatment time (120 to 1,200 µs).

Storage Conditions of Treated Samples
Samples with single and combined additions of AS-48, nisin, and lysozyme or those treated with a combination of HIPEF and different mixtures of the antimicrobial compounds were stored at 4°C and 22°C. Population of Staph. aureus in the stored samples was evaluated after 24 and 48 h. Assays were performed in triplicate.

RSM
Response surface methodology was performed to study the simultaneous effect of HIPEF and added AS-48 and nisin or lysozyme (Tables 1 and 2). In each experimental model, a central composite and faced centered design was selected (Myers and Montgomery, 2002). Assays were replicated twice and the experimental order randomized within each block. Each trial was performed in triplicate. The effect of n independent variables was modeled by using a second-order polynomial equation:

where –log s is the microbial inactivation of Staph. aureus, β_i are the regression coefficients, and factor X_i represents the encoded values of the variables. Regression coefficients were estimated by model reduction, as such omitting nonsignificant terms (P > 0.05) from ANOVA. A 95% confidence interval was set for all procedures. Design Expert 6.0.1 software (Stat Ease Inc., Minneapolis, MN) was used in all RSM analyses and generated plots.

Microbial Inactivation of Staphylococcus aureus
The untreated and treated samples were serially diluted in peptone saline solution, plated on slants of plate count agar, and incubated for 48 h at 35°C. The number of viable cells of Staph. aureus after applying a treatment was expressed as survival fraction, s, which was calculated as N/N₀, where N₀ was the initial count in samples before any of the treatments and N was the count after each treatment. Microbial inactivation was calculated as –log s.

	RESULTS

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Effect of Adding AS-48, Nisin, or Lysozyme, or AS-48 with Lysozyme
The addition of 28 AU/mL of AS-48, 20 IU/mL of nisin, or 5,000 IU/mL of lysozyme alone, and the combination of enterocin AS-48 (28 AU/mL) with lysozyme (5,000 IU/mL) caused no variation in survival of Staph. aureus in milk at its natural pH and pH 5.0 after 1 h of exposure at room temperature, nor after 24 and 48 h of storage at 4 and 22°C. On the contrary, the Staph. aureus population fell when 28 AU/mL of AS-48 was combined with 20 IU/mL of nisin, although the level of inactivation depended on exposure time, milk pH, and temperature. Thus, after 1 h of exposure to the 2 bacteriocins, log reductions of 1.8 and 1.1 in Staph. aureus were observed at 6.8 and 5.0 pH, respectively (Table 3). During storage, the activity of the 2 bacteriocins, in general, led to an even greater reduction in Staph. aureus population, although the number of survivors was lower at 22°C or at natural milk pH than at 4°C or at 5.0 pH. Up to 6 log reductions of the microorganism were observed when the milk was kept at its natural pH with 28 AU/mL of AS-48 and 20 IU/mL of nisin for 24 h at 22°C. However, this tendency was blocked when storage was lengthened to 48 h. On the one hand, the recovery in Staph. aureus counts to the initial inoculated level at both of the 2 pHs under study revealed the ending of the bactericide effect of the 2 antimicrobials, whereas on the other hand, when the temperature was kept at 4°C, the reduction increased progressively up to practically 4 log cycles over the 48 h.

The effect of the addition of enterocin AS-48 to milk before HIPEF treatment was studied in accordance with the proposed RSM. The inactivation achieved for each combination of variables is shown in Table 1. The ANOVA revealed that the results fit a second-order model, with a coefficient of determination (R²), of 0.94 and a nonsignificant lack of fit (Table 4). The reduction in Staph. aureus population could be fitted by means of the following quadratic equation:

where –log s represents the number of log reductions, e the AS-48 concentration (AU/mL), t the treatment time (µs), and p the pH of milk. On the basis of this model, maximum destruction of Staph. aureus, of 4.5 log reductions, was achieved when 28 AU/mL of AS-48 was added to milk at its natural pH, which was then treated with HIPEF for 1,200 µs (Figure 1). This level of destruction represented 1.3 log reductions more than the cell death achieved by summing the inactivation obtained by each of the treatments applied separately. However, counts of Staph. aureus in the samples at pH 5.0 treated simultaneously with AS-48 and HIPEF were no different, in the best of cases than those obtained when using only HIPEF. The number of viable microorganisms in samples at pH 6.8 with 28 AU/mL of AS-48 processed with HIPEF for 1,200 µs remained constant during storage at 4°C for 48 h, whereas a growth in the population up to the initial inoculated level was observed in the same samples after 48 h if storage was held at 22°C.

View larger version (36K): [in this window] [in a new window]   Figure 1. Effect of increasing enterocin AS-48 concentration (arbitrary units/mL) and high-intensity pulsed-electric field (HIPEF) treatment time on the microbial inactivation of Staphylococcus aureus in milk at its natural pH (HIPEF set up at 35 kV/cm, 6 µs of pulse width, and 75 Hz at bipolar mode).

where n is the nisin concentration (IU/mL) and t is the HIPEF treatment time (µs). The greatest effect achieved by combined treatment with nisin, AS-48, and HIPEF was a log reduction of 6.3, which was obtained by combining 28 AU/mL of AS-48, 20 IU/mL of nisin, and HIPEF treatment for 800 µs at pH 6.8. This level of inactivation is, on the one hand, almost double that obtained when summing the maximum levels achieved by each of the treatments if applied separately and, on the other, shows an increase of approximately 1 log reduction compared with the level achieved by summing the maximum effect obtained by HIPEF and that achieved with the combined addition of AS-48 and nisin. Furthermore, after 48 h of storage at 4°C, viable microorganism counts in the samples treated with the previous combination of variables remained stable, although a growth in the Staph. aureus population was observed in the same samples stored at a temperature of 22°C.

Effect of the Sequence of Application of HIPEF and Bacteriocins AS-48 or Nisin, or Both
The addition of AS-48 to samples treated previously by HIPEF did not improve the lethality achieved by processing the samples with HIPEF alone or by processing them with HIPEF after the addition of AS-48. Likewise, after storage for 24 or 48 h at 4° or 22°C, no differences in the counts were observed among the samples treated with HIPEF before or after the addition of AS-48. Similarly, no differences were observed in the number of log reductions in samples with AS-48 (28 AU/mL) and nisin (20 IU/mL), whether treated previously with HIPEF or not, or even in the survivor counts during storage.

	DISCUSSION

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The addition to milk of each of the antimicrobials AS-48, nisin, or lysozyme on their own did not lead to any reduction in the Staph. aureus population within the range of concentrations and pH levels tested. Among the most important features of AS-48 is its stability under different pH and temperature conditions, as well as its wide antimicrobial spectrum (Diaz et al., 2003). In the particular case of Staph. aureus, Muñoz et al. (2007) found that the minimum dosage to succeed in inhibiting growth of the CECT 976 strain in milk was 50 µg/mL. That result explains the sublethal or nonexistent effect of the concentration of AS-48 used in this study (8 µg/mL, equivalent to 28 AU/mL) against the target strain of Staph. aureus. Similarly, other authors determined that the minimum lethal concentrations of nisin and lysozyme are higher than the maximums used in this study. Sobrino-López and Martín-Belloso (2006) observed that the concentration of nisin necessary to achieve a lethal effect on Staph. aureus in milk was 20 IU/mL, and Chun and Hancock (2000) detected a reduction in the population of the microorganism above 12,000 IU/mL of lysozyme. In the case of this latter antimicrobial, the modifications in the peptidoglycans of the Staph. aureus cell membrane, consisting of O-acetylation, may also explain the low activity of lysozyme against this microorganism (Clarke and Dupont, 1992).

Although the activity of each of the antimicrobials separately was not sufficient to inhibit the growth of Staph. aureus in the ranges of concentrations used, the combination of AS-48 (28 AU/mL) and nisin (20 IU/mL) did manage to reduce the population by 1.8 log cycles after an hour of exposure and almost up to detection level when the milk samples, at their natural pH, were exposed for 24 h at 22°C. These results suggest that the 2 peptides together act synergistically to destroy Staph. aureus even when their individual concentrations are at sublethal levels. However, the joint, simultaneous activity of the 2 peptides seems to be intrinsically dependent on pH, temperature, and exposure time to the 2 substances. Contrary to what might be expected, higher acidic levels in the milk reduced the combined activity of AS-48 and nisin, when their activity separately is higher precisely at low pH levels (Ananou et al., 2004). Abriouel et al. (2001) proposed that certain changes in the oligomerization of the AS-48 molecule, together with a change in the surface electrical charge of the target bacteria, could alter the activity of the bacteriocin at low pH conditions. This fact could probably explain the loss of synergy between the 2 molecules when the milk pH was 5.0 within the range of concentrations studied. Until now, no other study has highlighted the synergistic effect of the simultaneous addition of enterocin AS-48 and nisin to milk, although it has been observed that the bactericide effect of AS-48 increases when it is combined with organic acids, such as benzoic, sorbic, and lactic (Ananou et al., 2007), or chelating agents, such as sodium tripolyphosphate and EDTA (Ananou et al., 2005a).

Temperature and, above all, exposure time seem to indicate the level of involvement of each molecule in the inhibiting effect. On the one hand, it was observed that nisin acts immediately or in a very short period of time (Hyde et al., 2006), thus the reduction observed during the first hour is directed and controlled principally by this molecule. On the other hand, the action of AS-48 is mainly responsible for the inhibition achieved after 24 or 48 h of storage. However, temperature is the factor that acts as an accelerator or decelerator of the activity during that period of time. Thus, a temperature of 22°C during storage accelerated and intensified the joint activity of the peptides during the first 24 h, although that activity stopped during the following 24 h, with a consequent regrowth in the population of Staph. aureus. On the contrary, a temperature of 4°C first slowed the inhibiting effect of the bacteriocins, but prolonged the decrease in the population up to 48 h and, second, may have acted as a third treatment in itself with a bacteriostatic effect, given the thermophilic nature of the bacteria under study.

As can be seen in Figure 1, the number of log reductions achieved by HIPEF treatment of milk, at natural pH and with enterocin AS-48, increased as treatment time and AS-48 concentration reached their maximum values. The combination of HIPEF and AS-48 in inactivating Staph. aureus was synergistic. The loss of cell viability as a result of HIPEF treatment is basically due to cell damage that leads to permanent or transitory permeabilization of the membrane (Garcia et al., 2007). Thus, enterocin AS-48 may act with the induced electrical field in 3 possible ways: i) by maintaining and preventing the resealing of transient pores, ii) by enabling the formation of pores and disruption of the cell membrane by previous sensitization, and iii) by aiding in the extension of the permeabilization of the cell membrane above the critical level of cell viability. However, this pattern was not observed when milk pH was 5.0. Although the involvement of pH in the overall effectiveness of the treatment is not clear, a different conformation of the AS-48 molecule, together with a change in the sensitivity of the cell under these conditions (Abriouel et al., 2001; Ananou et al., 2007) or the ability of the bacteria to recover from possible sublethal damage inflicted by HIPEF treatment (Aronsson et al., 2001), could explain the results observed at acidic pH values.

From a hurdle concept, the more preservation methods that are combined, the greater the inactivation that may be achieved. Although the addition of lysozyme to milk with added AS-48 and then treated with HIPEF did not improve the level of reduction of Staph. aureus compared with the same treatment without lysozyme, the addition of nisin to milk with added AS-48 (28 AU/mL), at natural pH and then treated with HIPEF, did noticeably reduce the population of Staph. aureus. In this case, the number of log reductions depended on the concentration of nisin and the treatment time. Specifically, maximum destruction was observed for a HIPEF treatment time of 800 µs (Figure 2), and both lower and higher times were less effective in reducing the population. Similar behavior was described by Sobrino-López and Martín-Belloso (2006) when milk inoculated with Staph. aureus and with added nisin was treated with HIPEF. In this case, an increase in the treatment time decreased the effectiveness of the combined treatment. In spite of the high level of initial destruction, the microbial population in the samples treated remained constant during storage at 4°C up to 48 h. This result contrasts with the behavior observed at the same temperature when only the 2 bacteriocins were added, when decrease in the population was gradual (Table 3).

View larger version (32K): [in this window] [in a new window]   Figure 2. Microbial inactivation of Staphylococcus aureus in milk by combining nisin (1 to 20 IU/mL) and enterocin AS-48 (28 arbitrary units/mL) addition before applying high-intensity pulsed-electric field (HIPEF) treatment (35 kV/cm, 6 µs of pulse width, and 75 Hz of frequency at bipolar mode).

In conclusion, the treatment of milk with added AS-48 or nisin, or both, at sublethal doses with HIPEF acted in a synergistic way to destroy Staph. aureus. However, the possible application of the combined treatment should be studied on acidic dairy products, due to its loss of effectiveness under these pH conditions. It is also necessary to study in greater depth the joint mode of action of the 2 bacteriocins combined with HIPEF and to evaluate the effect of the application of HIPEF on the activity of the bacteriocins. However, the degree of destruction achieved with the combination of the above treatments, that is, the addition of AS-48 or nisin, or both, together with the application of HIPEF, and the prolongation of the inhibiting or bactericide effect during later storage could be of great interest for the preservation of both milk and other dairy products.

	ACKNOWLEDGMENTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
This work was supported by the Generalitat de Catalunya (research group 2005SGR-00058) and the University of Lleida (Spain). We also acknowledge the Research Programme of the University of Jaén (Spain), from which Pilar Martínez Viedma received a fellowship, and the Research Plan of the Junta de Andalucía (research group AGR230). We also thank Terence W. Roberts for revising the manuscript.

Received for publication December 23, 2008. Accepted for publication January 16, 2009.

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TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 


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