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Enhancing the Lethal Effect of High-Intensity Pulsed Electric Field in Milk by Antimicrobial Compounds as Combined Hurdles

Department of Food Technology, University of Lleida, 25198-Lleida, Spain

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

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
High-intensity pulsed electric field (HIPEF) is a nonthermal treatment studied for its wide antimicrobial spectrum on liquid food, including milk and dairy products. Moreover, the antimicrobial effect of HIPEF may be enhanced by combining HIPEF with other treatments as hurdles. Nisin and lysozyme are natural antimicrobial compounds that could be used in combination with HIPEF. Therefore, the purpose of this study was to determine the effect of combining HIPEF with the addition of nisin and lysozyme to milk inoculated with Staphylococcus aureus with regard to different process variables. The individual addition of nisin and lysozyme did not produce any reduction in cell population within the proposed range of concentrations, whereas their combination resulted in a pH-dependent microbial death of Staph. aureus. The addition of nisin and lysozyme to milk combined with HIPEF treatment resulted in a synergistic effect. Applying a 1,200-µs HIPEF treatment time to milk at pH 6.8 containing 1 IU/mL of nisin and 300 IU/mL of lysozyme resulted in a reduction of more than 6.2 log units of Staph. aureus. Final counts resulting from the addition of nisin and lysozyme and applying HIPEF strongly depended on both the sequence of application and the milk pH. Thus, more research is needed to elucidate the mode of action of synergism as well as the role of different process variables, although the use of HIPEF in combination with antimicrobial compounds such as nisin and lysozyme is shown to be potentially useful in processing milk and dairy products.

Key Words: nisin • lysozyme • high-intensity pulsed electric field • milk

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
Nonthermal treatments are being studied because of their antimicrobial effect with minimal alteration of sensory properties, as opposed to heat treatments. For that reason, nonthermal treatments such as high-intensity pulsed electric fields (HIPEF), together with the use of natural antimicrobial compounds, are gaining importance among novel technologies of food preservation. Because milk and dairy products are part of the human diet, the control of natural flora and bacterial growth is an important issue in milk processing. As liquid foods are suitable for treatment by HIPEF, the dairy industry is focusing its attention on the potential application of HIPEF as an acceptable alternative to classical heat treatments.

The antimicrobial spectrum of HIPEF includes a large number of gram-positive and gram-negative bacteria and, among these, the microbial death of pathogenic and spoilage bacteria such as Staphylococcus aureus (Sobrino-López et al., 2006), Escherichia coli (Martín et al., 1997), Listeria monocytogenes (Chen and Hoover, 2004), Pseudomonas fluorescens (Fernández-Molina et al., 2005), and Lactobacillus brevis (Grahl and Märkl, 1996) is particularly remarkable. However, the inactivation levels of viable cells recorded in milk as a result of HIPEF is noticeably lower than cell death of microorganisms inoculated in other media such as fruit juice. According to Bendicho et al. (2002a), these differences in the effectiveness of HIPEF in milk and other media may be due to the more complex composition of milk, its low resistivity, and the presence of proteins. Thus, inactivation of microorganisms is more difficult in complex food materials such as milk, than in simple solutions.

In recent decades, the use of naturally occurring antimicrobial agents to inhibit pathogen growth and prevent food spoilage has received special attention (Cleveland et al., 2001). Among these, nisin is considered especially relevant due to its international acceptance as a food additive by the Joint Food and Agriculture Organization/World Health Organization (FAO/WHO) Expert Committee on Food Additives (WHO, 1969). Moreover, its potential use in the food industry is based on its dairy origin and antimicrobial activity against pathogens (Chen and Hoover, 2003). On the other hand, lysozyme is a natural and broad-spectrum antimicrobial compound (Jollès, 1996) also present in milk and commonly classified as a milk enzyme (Chandan et al., 1965). Like nisin, lysozyme was granted a status of generally recognized as safe (GRAS) by WHO/FDA (WHO Food Additives Series 30) and, for matured cheese, received the "quantum satis" status through European directive 95/2/EC on food additives other than colors and sweeteners (European Union, 1995). Gram-positive bacteria are generally sensitive to lysozyme whereas gram-negative bacteria are commonly resistant (Masschalck and Michiels, 2003).

Although HIPEF treatment induces a loss of microorganism viability in general, the protective effect of milk composition implies a drop in HIPEF effectiveness (Martín et al., 1997), which may represent a drawback in its potential industrial implementation. Nevertheless, different studies have pointed out a synergistic effect when combining HIPEF and other nonthermal treatments, particularly antimicrobial compounds such as nisin and lysozyme. However, the combination of these treatments have rarely been studied in milk, and process variables, range of concentrations added, or effect of milk media have not been analyzed in depth. Sobrino-López and Martín-Belloso (2006) reported a synergistic activity in the reduction of Staph. aureus when milk samples were treated by adding 20 IU/mL of nisin before applying a 2,400-µs treatment time at 35 kV/cm field strength and 100 Hz of pulse frequency in bipolar mode. Smith et al. (2002) found that the addition of 4,250 IU/mL of lysozyme and a HIPEF treatment of 50 pulses at 80 kV/cm led to greater microbial inactivation of natural flora of milk than each treatment applied separately. Interestingly, the combination of 1,638 IU/mL of lysozyme and 38 IU/mL of nisin with a HIPEF treatment set at 50 pulses and 80 kV/cm produced an even more lethal effect on total microbial counts of raw milk than their effects when used alone (Smith et al., 2002). Hence, a hurdle approach by combining HIPEF and antimicrobial compounds seems to improve the microbial destruction in milk. However, there is a lack of information about the effect of the combined application of these treatments against milk pathogens as well as little information about the influence of treatment conditions on the final effect. Thus, the purpose of this study was to determine the combined effect of nisin, lysozyme, and HIPEF against Staph. aureus in milk affected by different treatment conditions, such as pH of milk, nisin and lysozyme concentrations, and the sequence of the combined treatments.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
Skim Milk
Homogenized UHT skim milk was obtained from a dairy plant (Puleva, Mollerussa, Lleida, Spain) and stored at 4°C. Natural pH of milk was 6.8 ± 0.02 and was measured by using 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 at pH 6.8 and 6.99 ± 0.13 mS/cm at pH 5.0. The measurement was performed at 25°C and determined with a conductivity meter (Testo 240 conductivimeter, Testo GmBh and Co, Lenzkirch, Germany).

Staph. aureus Culture
Staphylococcus aureus CECT 240 (Food Technology Department, University of Lleida, Spain) was used as the target microorganism. It was maintained on slants of plate count agar (PCA; Biokar diagnostics, Beauvais, France) at 4°C until used.

Strain growth was performed by incubating cultures on tryptone soy broth (TSB) at 35°C for 6 h. Inoculum concentration was determined by optical measurement. A population density of approximately 10⁹ cfu/mL is equivalent to an absorbance value between 0.60 and 0.70 at 620 nm.

Sample Preparation
Samples with Staph. aureus were prepared by inoculating the microorganism in skim milk to a final concentration of approximately 10⁷ cfu/mL. The milk pH was adjusted by adding lactic acid [l(+)-lactic acid, Panreac, Barcelona, Spain] to the desired value. Then, samples were treated by adding nisin or lysozyme or by combining the addition of these compounds before or after applying HIPEF.

HIPEF Equipment and Treatment Conditions
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, OH) that discharges square-shape pulses within 8 collinear chambers, in which the 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 according to previous studies (Sobrino-López et al., 2006). Treatment temperature was kept always under 25°C, using a cooled water bath to rule out thermal effects.

Effect of Individual and Simultaneous Addition of Nisin and Lysozyme
The antimicrobial effects of nisin (N5764; 2.5% nisin, 1,000,000 IU/mg, Sigma-Aldrich, Steinheim, Germany) and lysozyme (L2879; 43,560 IU lysozyme/mg of solid, Sigma-Aldrich) against Staph. aureus was measured by exposing milk samples to different concentrations of nisin (0 to 5 IU/mL) or lysozyme (0 to 3,000 IU/mL) at 2 different pH values (5.0 to 6.8). Exposure time was prolonged for 1 h in accordance with previous studies (Sobrino-López and Martín-Belloso, 2006).

A response surface design was set to study the effect of adding nisin and lysozyme (Table 1). On the central composite and faced centered design, nisin ranged from 1 to 5 IU/mL, lysozyme from 300 to 3,000 IU/mL, and milk pH from 6.8 to 5.0, while exposure time was set at 1 h. Assays were replicated twice and the experimental order was randomized within each block. The effect of the independent variables was modeled by using a second-order response function:

[1]

where factor X represents the encoded values of the variables and β are the constant coefficients. Confidence interval was set at 95% for all procedures.

Combined Effect of Nisin, Lysozyme, and HIPEF Treatment
The antimicrobial effect of HIPEF on Staph. aureus inoculated in skim milk at its natural pH or pH 5.0 was evaluated by setting a HIPEF treatment time of 1,200 µs. The assay was performed in triplicate. The response surface methodology was performed to observe the effect of the addition of nisin and lysozyme followed by HIPEF treatment on the microbial inactivation of Staph. aureus in skim milk. A central composite design with 4 factors and faced centered was the proposed experimental design. The independent variables were nisin concentration (1 to 5 IU/mL), lysozyme concentration (300 to 3,000 IU/mL), HIPEF treatment time (120 to 1,200 µs), and pH (5.0 to 6.8). The values for each variable and combination are shown in Table 2. The experimental design was performed in duplicate, providing 2 blocks of experiments and each assay in triplicate. The order of assays within each block was randomized.

The sequence of addition of the antimicrobial compounds was also evaluated by applying a HIPEF treatment before the addition of nisin and lysozyme. In that case, exposure time of the HIPEF-treated sample to nisin or lysozyme lasted 1 h. Each assay was performed in triplicate.

Microbial inactivation of Staphylococcus aureus
The untreated and treated samples were serially diluted in peptone solution, plated on slants of plate count agar and incubated for 72 h at 30°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, addition of nisin and lysozyme, application of HIPEF, or the proposed combinations, and N was the count after each treatment. Microbial inactivation was calculated as –log s.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
Effect of the Addition of Nisin, Lysozyme, or Both
The addition of nisin alone up to 5 IU/mL to milk caused no cell death of Staph. aureus within milk pH and exposure time ranges. These results are in agreement with different studies that have reported high doses of nisin to be lethal on Staph. aureus in milk and other media. Sobrino-López and Martín-Belloso (2006) found that concentrations lower than 20 IU/mL had no lethal effect on Staph. aureus population in milk, whereas Smith et al. (2002) observed a reduction of 0.8 log units on natural flora of milk at 100 IU/mL of nisin. Moreover, doses of lysozyme up to 3,000 IU/mL remained beneath the minimal bactericidal concentration within the conditions set up in this study. The susceptibility of other pathogenic and lactic acid bacteria to lysozyme has been shown to be significant (Branen and Davidson, 2004) at greater concentrations than the latter concentration. A lysozyme concentration of 4,250 IU/mL showed a decrease of only 0.2 log cycles on total microbial counts in milk (Smith et al., 2002), and Chung and Hancock (2000) reported no cell death of Staph. aureus at 12,000 IU/mL. The resistance of Staph. aureus to lysozyme has been explained through the cell envelope composition of the microorganism and the singular action mode of the compound. The inhibition mechanism of lysozyme is based on both lytic and non-lytic activity. While peptidoglycan hydrolysis of the bacterial cell wall is brought on by its muramidase activity, the conformation of the protein probably plays an important role in its nonlytic mechanism of inactivation (Masschalck and Michiels, 2003). Consequently, gram-negative bacteria are believed to be resistant because of their outer membrane (Hughey and Johnson, 1987), whereas inert activity against some gram-positive bacteria may be due to peptidoglycan modifications, such as O-acetylation (Clarke and Dupont, 1992). Thus, the resistance of Staph. aureus to lysozyme is thought to be based on the O-acetylation of its cell wall, which ranges from 35 to 90% depending on the strain and growth conditions (Clarke and Dupont, 1992).

The combined effect of nisin and lysozyme addition was studied by response surface modeling. Microbial inactivation achieved by combining the addition of both antimicrobial compounds is shown in Table 1. A maximal inactivation of 1.6 log units was observed when 5 IU/mL nisin and 300 IU/mL lysozyme were added to milk at pH 6.8, whereas almost no reduction on cell population was seen with the combination of 1 IU/mL and 300 IU/mL of nisin and lysozyme at pH 5.0, respectively. Microbial inactivation of Staph. aureus was satisfactorily fitted by a second-order equation (equation [1]) with a determination coefficient, R², of 0.995 and no significant lack of fit. Cell death of Staph. aureus was affected by nisin and lysozyme doses as well as by milk pH and their interaction (Table 3). Microbial inactivation was expressed by the following polynomial quadratic equation:

[2]

where –log s is the microbial inactivation, n is the nisin concentration (IU/mL), l is the lysozyme concentration (IU/mL), and p is the pH of milk.

Although the additions of nisin and lysozyme alone did not affect the viability loss of Staph. aureus within the studied ranges, the simultaneous combination of these antimicrobial compounds at sublethal doses acted synergistically in cell inactivation. However, the effect of the mixture clearly depended on the milk pH as well as the bacteriocin doses used at each pH (Figure 1). First, the resistance of Staph. aureus against the nisin and lysozyme combination sharply diminished at natural pH of milk. Second, the activity against Staph. aureus of adding nisin and lysozyme at acidic milk pH increased as the nisin and lysozyme concentration increased, whereas lower doses of lysozyme enhanced microbial inactivation when adding nisin at natural milk pH. Results reported by Smith et al. (2002) showed a decrease of 1.2 log units on total microbial counts when 38 and 1,638 IU/mL of nisin and lysozyme, respectively, were added to raw skim milk. The final counts of the latter study may diverge from our results in that the microbial target of the treatment was the natural flora of milk and a greater nisin concentration was added. Chung and Hancock (2000), who studied the effect of different mixtures of nisin and lysozyme in Staph. aureus population by fluorescence assay, observed that a combination of 12.5 µg/mL of nisin and 900 IU/mL of lysozyme had a greater effect compared with other mixtures with greater lysozyme concentrations. A similar behavior of an increase of cell death at any nisin dose if lysozyme concentration decreased was shown in our results at natural milk pH, whereas increasing the lysozyme dose acted efficiently only at acidic pH. Our results may differ from those of Chung and Hancock (2000) because of both the greater concentration of the antimicrobial compounds they used and the different media, whose pH was not determined. In this sense, Nattress et al. (2001) also found that the greater the lysozyme concentration in the mixture, the longer the antimicrobial efficacy was when evaluating the ability of lysozyme and nisin to control meat spoilage bacteria.

View larger version (20K): [in this window] [in a new window]   Figure 1. Microbial inactivation of Staphylococcus aureus in milk by combining nisin (IU/mL) and lysozyme (IU/mL) addition at different pH values: A) 5.0 and B) 6.8.

Combined Effect of HIPEF, Nisin, and Lysozyme
The individual effect of HIPEF treatment for 1,200 µs was performed in skim milk at its natural pH and at pH 5.0. A microbial reduction on counts of Staph. aureus of 3.8 ± 0.4 log units was induced in milk at its natural pH, whereas no difference on cell destruction was shown at pH 5.0. Few studies have considered pH as a variable process of HIPEF treatment. Some authors reported that pH had no effect on the inactivation achieved by HIPEF treatment (Smith et al., 2002; Sobrino-López and Martín-Belloso, 2006), whereas others claimed that a modification of milk conductivity (Wouters et al., 2001) and cell capacity for recovering from sublethal injuries caused by HIPEF treatment (Aronsson and Rönner, 2001; Liang et al., 2002) are directly affected by pH.

Microbial inactivation achieved by applying HIPEF after adding nisin and lysozyme in milk is shown in Table 2. The ANOVA for the second-order model fits well the data obtained, with a determination coefficient R² = 0.982 and an insignificant lack of fit (Table 4). The microbial inactivation was adjusted by the following quadratic polynomial equation:

[3]

where –log s is the microbial inactivation, n is the nisin concentration (IU/mL), l is the lysozyme concentration (IU/mL), t is the HIPEF treatment time (µs), and p is the pH of milk.

In general, the population of Staph. aureus decreased dramatically with a greater HIPEF treatment time. A decrease of over 2.6 log units in counts of Staph. aureus was observed when HIPEF treatment time was lowered from 1,200 to 120 µs in milk with 1 IU/mL of nisin and 3,000 IU/mL of lysozyme added at its natural pH. Interestingly, milk pH seems to have a strong influence on the inactivation effect of the combined treatment, because acidic milk pH reduced the susceptibility of the microorganism to the combined treatment compared with milk at its natural pH (Figure 2). The difference between the maximal inactivation at natural milk pH and pH 5.0 was over 1.2 log cycles when HIPEF treatment time was set at 1,200 µs. Thus, the combination of HIPEF with antimicrobial compounds may involve a pH-dependent and complex mode of action. Milk pH modified the final effect of the addition of nisin and lysozyme followed by the application of HIPEF. In this way, maximal inactivation at acidic pH was obtained by high doses of nisin and lysozyme, whereas low nisin and lysozyme concentration displayed better results on cell death at natural milk pH. Maximal inactivation of Staph. aureus was nearly 6.4 log cycles if 1 IU/mL of nisin and 300 IU/mL of lysozyme were added to milk at pH 6.8 before applying a 1,200-µs HIPEF treatment. Other authors obtained similar values on microbial death, although it has to be pointed out that different media, extreme HIPEF conditions, and greater antimicrobial compound doses make comparisons difficult and, in addition, the process temperature set in these studies may have had a relevant effect. A greater than 7.0-log reduction on natural flora of milk was registered when skim milk was added to 38 IU/mL of nisin and 1,638 IU/mL of lysozyme and submitted to HIPEF treatment at 80 kV/cm and 50 pulses at 52°C (Smith et al., 2002). Wu et al. (2005) observed a microbial reduction of 5.9 log units on natural spoilage flora of grape juice when 20 IU/mL of nisin and 6,550 IU/mL of lysozyme were added and mixed for 2 h before applying 20 pulses of 65 kV/cm at 50°C. Similarly, a microbial reduction of over 6.5 log on population of Salmonella typhimurium was produced by a mixture of 27.5 IU/mL of nisin and 690 IU/mL of lysozyme combined with 30 pulses of 90 kV/cm at 45°C (Liang et al., 2002).

View larger version (20K): [in this window] [in a new window]   Figure 2. Microbial inactivation of Staphylococcus aureus in milk by combining nisin (IU/mL) and lysozyme (IU/mL) addition before applying high-intensity pulsed electric field (HIPEF) treatment (35 kV/cm, 6-µs pulse width, 75 Hz frequency, and bipolar mode) at different milk pH: A) pH 6.8 and 120-µs HIPEF treatment time; B) pH 6.8 and 1,200-µs HIPEF treatment time; C) pH 5.0 and 1,200-µs HIPEF treatment time.

The mechanism of the nisin-lysozyme-HIPEF synergism is not fully understood. It is believed that HIPEF and nisin have a mutual influence in inactivating micro-organisms, and the inclusion of a third preservation hurdle, lysozyme in this case, may intensify their reciprocal action. The HIPEF treatment may facilitate the binding of nisin to their target on the cell membrane causing sublethal injuries to the cell envelope (Calderon-Miranda et al., 1999; Terebiznick et al., 2000), and, simultaneously, nisin may diminish the resistance of the target cell lowering its critical field strength (Ho et al., 1995), whereas lysozyme might also facilitate and accelerate nisin binding (Chung and Hancock, 2000).

Surprisingly, increasing lysozyme concentration failed to achieve lower counts of Staph. aureus in milk at its natural pH followed by HIPEF treatment. In contrast, acidic milk pH led to a greater antimicrobial effect as nisin and lysozyme concentration increased, although the final inactivation goal reached lower values than those at natural pH. Two hypotheses may explain these phenomena. In this respect, it has been suggested that this behavior is related to the loss of activity caused by exposure to HIPEF. Furthermore, HIPEF is shown to inactivate bacteria but also enzymes (Bendicho et al., 2002a), to cause feasible structural changes on proteins (Bendicho et al., 2002b), and to alter some soluble components of the media (Gallo et al., 2007). In a previous work, the variation of nisin solubility at different pH, and leakage of intracellular content due to pore formation were proposed as possible causes of the loss of nisin activity depending on milk pH (Sobrino-López and Martín-Belloso, 2006). Nevertheless, because lysozyme activity is stable within acidic and neutral pH, more evidence is needed to clarify the susceptibility of nisin to HIPEF and, above all, the influence and resistance of lysozyme to HIPEF. As a second hypothesis, nisin pore formation and its functionality may explain differences in nisin activity at acidic and neutral pH. Nisin molecules may compete for the same target site on the cell membrane at high nisin concentration, which may lead to activity decay (Moll et al., 1997). In our case, lysozyme and nisin bind to the phospholipids of the cell membrane and may compete for them. On the other hand, the low efficiency of the nisin pore in dissipating the transmembrane electrical potential may explain the loss of nisin activity and a lower final microbial inactivation at acidic pH (Moll et al., 1997), even when combined with lysozyme.

Different concentrations of nisin and lysozyme were added to HIPEF-treated milk inoculated with Staph. aureus to better understand the behavior and mode of action of the synergism exerted by nisinlysozyme-HIPEF treatment. As seen in Table 5, microbial death as a result of the combined treatment did not depend on the sequence of application at acidic pH of milk. This result disagrees with those of Gallo et al. (2007), who observed that nisin addition after HIPEF treatment did not modify the final effect. In contrast, García et al. (2007) concluded that damage to the cell envelope by HIPEF depended on the bacterial species and, particularly, on the treatment medium pH. In this sense, the degree of permanent membrane permeabilization or the inability to reseal due to HIPEF may collaborate in the final microbial death achieved once the mixture of nisin and lysozyme is added to milk at acidic pH.

	CONCLUSIONS

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In conclusion, no cell death was observed when nisin and lysozyme were added individually to milk up to 5 and 3,000 IU/mL, respectively. However, the simultaneous addition of nisin and lysozyme to milk at sublethal doses acted synergistically in inactivating Staph. aureus. In general, acidic milk pH acted to strengthen the resistance of Staph. aureus against the mixture of nisin and lysozyme and, consequently, to diminish the level of inactivation. Nevertheless, the optimal concentration of each compound was also pH-dependent, in such a way that a lower lysozyme concentration enhanced the nisin activity to a greater degree against Staph. aureus at natural milk pH than at acidic pH. The use of a third preservation method such as HIPEF in combination with the addition of nisin and lysozyme to milk at different pH effectively inactivated Staph. aureus. The effect of the combined treatment was clearly synergistic at natural milk pH when low doses of nisin (1 IU/mL) and lysozyme (300 IU/mL) were added and a 1,200-µs HIPEF treatment time was applied. However, a greater concentration of both antimicrobial compounds was needed at acidic milk pH to observe synergism, which was less efficient in terms of microbial inactivation than that at natural pH. Moreover, the combination of nisin and lysozyme with HI-PEF treatment made it possible to obtain similar cell death compared with nisin-HIPEF treatment but with a reduced nisin concentration, which may represent an economic advantage.

The mode of action of combining added nisin and lysozyme with HIPEF has been little studied. However, the results derived from applying HIPEF treatment after or before the addition of the antimicrobial compounds suggest that the loss of nisin activity may occur during HIPEF because of a possible inactivation of the bacteriocin. Despite that, the mechanism of synergism seems to be influenced in a complex manner by process variables. Moreover, more evidence is needed to explain the role of lysozyme in the synergism as well as the possible effect of HIPEF on its activity. Hence, the application of the hurdle concept through the application of nonthermal technologies, such as the combination of HIPEF treatment with the addition of nisin and lysozyme, opens up a feasible possibility of preserving milk and dairy products with minimal loss of their sensory properties.

	ACKNOWLEDGEMENTS

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This work was supported by the Spanish Ministry of Education (research project AGL 2005-07665-C02-02/ ALI) and the Generalitat de Catalunya (research group 2005SGR-00058) and the University of Lleida. Our thanks to Terry W. Roberts for revising the translation into English.

Received for publication December 29, 2007. Accepted for publication January 23, 2008.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 


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