Inhibition of Listeria and Staphylococcus aureus by Bovicin HC 5 and Nisin Combination in Milk

The aim of this work was to evaluate the effect of the bacteriocins bovicin HC5 and nisin against Listeria and Staphylococcus aureus in synthetic media and in milk. Growth of Listeria monocytogenes, Listeria innocua and S. aureus was carried out at 37oC in tryptic soy broth (TSB) and in ultra-high temperature whole milk containing bovicin HC5 and nisin added either individually or in combination. Concentrations above 100 AU ml of bovicin HC5 or 50 AU ml of nisin inhibited the growth of Listeria species in TSB. Bacteriocins at concentrations of at least 50 AU ml clearly increased the lag phase, but did not prevent the growth of S. aureus. The combination of both bovicin HC5 and nisin in TSB inhibited the growth of Listeria and S. aureus Embrapa 4018 at lower concentrations than the bacteriocins added separately. Bactericidal effect against L. monocytogenes and S. aureus cells was observed when both bacteriocins were added together in milk in concentrations larger than 400 AU ml of each one. The present results demonstrate that bovicin HC5 and nisin were effective against Listeria and S. aureus assessed in milk, especially when used in combination.


Introduction
Outbreaks of listeriosis resulting from consumption of dairy products contaminated with L. monocytogenes have prompted concern about the behavior of this microorganism during processing and subsequent storage of various dairy products (Silva, Almeida, Alves, & Almeida, 2003).L. monocytogenes, a ubiquitous foodborne pathogen, can be potentially introduced in raw milk in a dairy industry environment.Growth of Listeria in dairy products is often favored by its psychrotrophic nature and tolerance to high salt concentration and relatively low pH values (Farber & Peterkin, 1991).L. monocytogenes causes disease in high-risk groups, including pregnant women, neonates, and immunocompromised adults, and has a high mortality rate (Arques, Rodriguez, Nunez, & Medina, 2008).
Food from animal origins, such as milk, is naturally susceptible to contamination by Staphylococcus aureus, an important pathogen able to grow in a wide range of temperatures, pH and sodium chloride concentration up 15%, and then able to produce enterotoxins.These toxins are thermostable and maintain their stability even after thermal treatments (Dinges, Orwin, & Schlievert, 2000;Le Loir, Baron, & Gautier, 2003).
Although Listeria and S. aureus are inactivated under normal conditions of pasteurization, problems can arise from post-pasteurization contamination, representing a risk to consumers, making necessary an effective control during the steps of food production.
Bacteriocins from lactic acid bacteria are widely studied and have been suggested as a potential biological alternative to improve food safety (Cleveland, Montville, Nes, & Chikindas, 2001).Nisin is a well known broad spectrum bacteriocin that can inhibit gram-positive bacteria and prevent the outgrowth of spores of Bacilli and Clostridia associated with food (Arques et al., 2004;de Arauz, Jozala, Mazzola, & Penna, 2009).Although nisin has been widely used in food industries to increase the shelf life of food products, previous studies indicated that many sensitive gram-positive bacteria have developed resistance to nisin (Arques et al., 2008;Zapico, Medina, Gaya, & Nunez, 1998).
A combination of preservative methods may work synergistically or at least provide greater protection than a single method alone, thus improving the safety and quality of food (Deegan, Cotter, Hill, & Ross, 2006).Based on this assumption, a combination of bacteriocins has been tested in order to increase antimicrobial activities and improve food safety (Galvez, Abriouel, Lopez, & Ben Omar, 2007).
The objective of the present work was to study the combined effect of bovicin HC5 and nisin on L. monocytogenes, L. innocua and S. aureus in whole milk.Although the effect of nisin combined with other antimicrobial agents has already been extensively studied, this is the first study evaluating the effect of nisin combined with bovicin HC5 on foodborne pathogens.

Preparation and Activity of Bovicin HC5 and Nisin
Extracts of bovicin HC5 were prepared as described by Mantovani et al. (2002).Bovicin HC5 concentration was estimated by serial two-fold dilutions of extract followed by spotting 25 µl on MRS agar using L. lactis ATCC 19435 as the indicator organism.Plates were incubated at 37°C for 24 h.One arbitrary unity (AU) was defined as the reciprocal of the highest dilution that showed a zone of inhibition with at least 5 mm diameter.
Nisin solution (Nisaplin ® , Danisco, Copenhagen, Denmark) was prepared in phosphate buffer (pH 2.0) and the bacteriocin activity was determined as described earlier.

Effect of Bovicin HC5 and, or Nisin on the Growth of Listeria and S. aureus in TSB
To determine the concentration of bovicin HC5 and nisin (individually or combined) that could inhibit Listeria and S. aureus we performed in vitro studies in 96-well microtiter plates.Strains of L. monocytogenes, L. innocua and S. aureus were activated in TSB and incubated at 37°C for 18 h.The cells were harvested by 3000 g centrifugation, washed with 0.1% salt peptone water, and then resuspended in TSB.Listeria and S. aureus cultures (10 6 CFU ml -1 ) were treated with bovicin HC5 and nisin at concentration of 10, 50, 100 and 150 AU ml -1 for the tests with isolated bacteriocins.In the assays using bovicin HC5 and nisin combined, the concentrations varied from 10 to 50 AU ml -1 .
The bacteria were incubated at 37°C and the growth was monitored via changes in optical density at 630 nm in an ELISA reader (Thermo Plate, model TP-Reader) for up to 10 h of incubation.Control treatments were performed in TSB inoculated with bacterial cultures without bacteriocins.

Effect of Bovicin HC5 and, or Nisin on the Growth of Listeria and S. aureus in Whole Milk
Cultures of L. monocytogenes Scott A, L. innocua LMA83, and S. aureus ATCC 6538 were selected because they presented more resistant after being treated with bacteriocins in TSB.Cells were activated in 5 ml TSB and incubated at 37°C for 18 h, harvested by centrifugation at 3000 g for 15 min, washed with 0.1% salt peptone water and then, resuspended in 1 ml of ultra-high temperature (UHT) whole milk to reach 10 7 CFU ml -1 .Bovicin HC5 and nisin were added individually (400, 800, and 1200 AU ml -1 ) or in combination (400, 600, and 800 AU ml -1 of each bacteriocin).
The tubes were incubated at 37°C and samples were taken at 0, 3, 6, 9, and 12 h for determination of viable cell number by microdrops (Herbert, 1990) plating aliquots of 25 μl in tryptic soy agar (Oxoid, England).Plates were incubated at 37°C for 12 or 24 h.Control treatments were performed in milk inoculated with bacterial cultures without bacteriocins.

Statistics
Each experiment was performed at least two times in duplicate.The log of the absorbance and colony forming units was plotted versus time (SigmaPlot version 11.0, USA) and the error bars presented indicate the standard deviation of the mean (shown only in the positive direction).was confirme 6538 (Figure 3 while nisin ffect (Figure 3 reus ATCC 65 pathogens.Ho s selection of r od preservation 320 AU ml Another issue to consider is the variation of resistance to bacteriocins found among strains of the same species.For example, we noted that bovicin HC5 and nisin have exerted an inhibitory effect on L. monocytogenes but strain ATCC 7644 was more sensitive to nisin while L. monocytogenes Scott A was more sensitive to bovicin HC5.The sensitivity variation to nisin among different strains of S. aureus isolated from dairy products was also observed (Sudagidan & Yemenicioglu, 2012).Mantovani and Russell (2003) found that some bacteria resistant to nisin showed no significant resistance to bovicin HC5, even after repeated treatments with sublethal doses of that bacteriocin.Higher concentrations of bacteriocins could increase the bactericidal effect and reduce the chances of selecting resistant bacteria and to have an inhibitory effect on several strains that could contaminate food.

Effect of Bovicin HC5 and/or Nisin on
Another alternative to reduce the selection of resistant cells would be the combined use of bacteriocins.In this study we showed that the impact of bovicin HC5 and nisin can be greatly enhanced if both bacteriocins are applied in combination.In fact, concentrations as low as 10 AU ml -1 of each bacteriocin in TSB resulted in reduction of the absorbance of cultures of L. monocytogenes Scott A and L. innocua LMA83 indicating cell lysis.The results showed that the combined effect of bovicin HC5 and nisin allow the use of lower dosages compared to the individual application.
According to Gálvez et al. (2007) when cells are exposed to a combination of antimicrobial factors, the intensity of the damage can be increased since these factors may act on different sites of the same target.The repair of multiple damages may require a high energy expenditure, resulting in energy depletion and cell death.The addition of nisin (0.5% w/v) in ready to eat salad did not achieved the complete inactivation of L. monocytogenes but the combination of this bacteriocin with enterocin AS-48 reduced the concentration of viable cells below the detection limit of the technique after 24 h of incubation (Molinos et al., 2009).
Although the addition of bacteriocins in milk used for consumption is not permitted, this product serves as an important system to evaluate the influence of chemical composition of milk on the activity of bacteriocins.Milk is a complex mixture of substances such as water, protein, lactose and fat, which can affect the effectiveness of bacteriocins.The addition of nisin at concentrations of 62.5, 125, 250 and 500 IU ml -1 in skim milk had a significant effect on L. monocytogenes, while in raw milk, the inhibitory activity was only moderate, with a rapid reduction of the L. monocytogenes population during two days of incubation before growth was resumed (Kim, Choi, Bajpai, & Kang, 2008).These results demonstrate that the activity of bacteriocin is dependent on the fat content in milk, and the interaction between the lipids of milk and nisin may limit the application of this bacteriocin in dairy fat products (Sobrino-Lopez & Martin-Belloso, 2008).
No research to assess the synergistic effect of bovicin HC5 with another antimicrobial agent in milk was carried out so far.However, results from the synergistic effect of nisin and other antimicrobial substances can be exemplified.Nisin combined with garlic extract showed potential antilisterial activity with a synergistic effect in reducing the viable cell number of L. monocytogenes in milk and skim milk in 14 days (Kim et al., 2008).Synergistic effect was also found by Arqués et al. (2008) evaluating the combination of nisin and reuterin, antimicrobial produced by Lactobacillus reuteri, in milk.These authors reported a reduction of approximately two log cycles of S. aureus treated with these antimicrobials in combination after 24 h of incubation.

Conclusions
The present study demonstrates that the combination of bovicin HC5 and nisin against Listeria and S. aureus in TSB and milk was more effective than the addition of these bacteriocins individually.These results indicate their potential use as biopreservatives in food.However, the molecular basis of synergistic effect between bovicin HC5 and nisin are now under investigation.Furthermore, the combination of bovicin HC5 with others antimicrobial agents against foodborne pathogens is focus of further research.Aknowledgments N. J. Pimentel-Filho was supported by a fellowship from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and this research has been supported by Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), Brazil.

Table 1 .
Listeria and S. aureus in TSB Effect of bovicin HC5 or nisin on the growth of Listeria and S. aureus -, no growth; AU, activity units.