Spore forming bacteria are special problems for the dairy industry. Heat treatments are insufficient to kill the spores. This is a continuously increasing problem for the industry, but we should be able to control it. In this context, we investigated the combined effect of nisin, monolaurin, and pH values on three heat resistant spores in UHT milk and distilled water and to select an optimal combination for the maximum spore inactivation. The inhibitory effect of nisin (between 50 and 200 IU/ml), monolaurin (ranging from 150 to 300 µg/ml), and pH (between 5 and 8) was investigated using a central composite plan. Results were analyzed using the response surface methodology (RSM). The obtained data showed that the inactivation of Bacillus spores by the combined effect of nisin-monolaurin varies with spore species, acidity, and nature of the medium in which the bacterial spores are suspended. In fact, Terribacillus aidingensis spores were more resistant, to this treatment, than Paenibacillus sp. and Bacillus sporothermodurans ones. The optimum process parameters for a maximum reduction of bacterial spores (∼3log) were obtained at a concentration of nisin >150 IU/ml and of monolaurin >200 µg/ml. The current study highlighted the presence of a synergistic effect between nisin and monolaurin against heat bacterial spores. So, such treatment could be applied by the dairy industry to decontaminate UHT milk and other dairy products from bacterial spores.

1. Introduction

The presence of highly heat-resistant spores of Bacillus in UHT milk has emerged as a major problem in the dairy industry as it affects the commercial sterility required for these products [1]. In addition, spore-forming bacteria become the main obstacle limiting the further extension of milk’s shelf life [2]. To prevent the growth of Bacillus species, there are different kinds of heat treatments, which are used such as pasteurization and ultrahigh-temperature processing (UHT). Despite these thermal treatments ensuring the commercial sterility of the finished product, many problems regarding the sterility of UHT milk appeared [3]. The poor quality of the finished product is caused by the presence of highly heat resistant spores produced by Bacillus species. Among these, spores are those of Bacillus sporothermodurans which are described for the first time by Petterson et al. [4].

The use of antibacterial agents has gained great attention with interesting results in the destruction of vegetative cells and inactivation of bacterial spores in various food systems [5]. Nisin, monolaurin, and lactoperoxidase are examples of natural preservatives currently used in the food industry. Nisin is an antimicrobial peptide known to inhibit the growth of a number of Gram-positive bacteria including outgrowth of spores of Bacillus and Clostridium. Also, it has been shown to be effective in the microbial control of a number of pasteurized dairy products [6] and milk [5]. Monoglycerides are used in the food industry as favoring and emulsifying agents. Monolaurin is the most compound that has received attention because of its antimicrobial properties [7, 8]. There are several treatments that are based on the combination of antimicrobial agents, but the appearance of species that resist this type of treatment leads us to look for other types of combinations. For that, several combinations were studied. Nisin, for example, has been used in combination with lactoperoxidase system [9], monolaurin [10], high pressure, and heat treatment [11]. Many authors showed the synergistic inhibitory effect of nisin and monolaurin, used in association, against Escherichia coli, Bacillus subtilis, and Staphylococcus aureus [12]. In a previous study, Mansour et al. [13] investigated the inhibitory synergistic effects of nisin-monolaurin used in association with pH on Bacillus licheniformis spores in milk. In another work, Mansour and Millière [10] studied the combined effect of these antimicrobial agents on spore reduction of three Bacillus species (Bacillus cereus, Bacillus subtilis, and Bacillus coagulans) in milk. Although the mechanisms of action of nisin and monolaurin are different, the fact that the cytoplasmic membrane is their primary action site could explain their inhibitory synergistic effect [14]. The combined effect of nisin-potassium sorbate against Bacillus sporothermodurans spores has been studied [15], but the effect of antimicrobials agents on the inactivation of B. sporothermodurans, Terribacillus aidingensis, and Paenibacillus sp. spores has not previously been investigated. The aim of our study was to evaluate the inhibitory activity of nisin-monolaurin-pH on the inactivation of Bacillus sporothermodurans, Paenibacillus sp., and Terribacillus aidingensis spores in distilled water and milk. For this purpose, response surface methodology (RSM) was used to describe the spore inactivation by combining nisin-monolaurin and pH.

2. Material and Methods

2.1. Bacterial Strains

In this study, three Bacillus species were used for evaluating the effect of antimicrobials agents on bacterial spores. Bacillus sporothermodurans strain was isolated from raw milk, while Paenibacillus sp. and Terribacillus aidingensis strains were isolated from UHT milk produced in Tunisia for the first time as previously described by Kmiha et al. [16].

Paenibacillus sp., B. sporothermodurans, and T. aidingensis were grown on brain-heart-infusion agar supplemented with 1 mg/L vitamin B12 (Sigma Aldrich) (BHI-vitB12) at 37°C.

2.2. Spore Suspensions

The spores were prepared as described by Aouadhi et al. [11]. The strains are cultured first in BHI agar-vitB12 broth at 37°C for 24 hours, from a single colony obtained on a solid medium. This preculture is diluted 1/10 in the sporulation broth described by Herman and others [17] (25 g/L nutrient broth, 1 mg/L vitamin B12 (Sigma), 8 mg/L of MnSO4 H2O, and 1 g/L of CaCl2 H2O) and incubated at 37°C for 7 days. After that, cultures were centrifuged at 8000 rpm for 10 min. The obtained pellet is suspended and washed vigorously three times with sterile distilled water. After the last washing, the bacterial pellet is suspended in 5 mL of sterile distilled water and heat-treated by incubation at 100°C for 10 minutes, in order to destroy the residual vegetative cells. The obtained sporulation suspensions are washed with distilled water and centrifuged three times. After final washing, the spores are suspended in sterile distilled water and stored at a concentration of 107 to 108 sp/mL at 4°C.

2.3. Antimicrobial Agents

Nisin and monolaurin were purchase from Sigma Aldrich. Standard stock solution of nisin containing 1 × 105 IU/ml was prepared by dissolving 1.25 g of nisin (2.5%) in 10 mL sterile 0.02 mol/L HCl. Monolaurin, with a purity of 99%, was dissolved in 95% ethanol to a final concentration of 50 mg/mL.

2.4. Experimental Design

The effect of these two antimicrobial agents on the spores has been tested. The spores, after preparation, are suspended in sterile distilled water containing different concentrations of nisin (10-50-100-500-1000–2000 IU/ml) or in monolaurin (50-100-250–500 µg/ml). Next, to assess the influence of pH on improving the efficacy of these antimicrobial agents on the spores of the three species studied, the spores are resuspended in sterile distilled water in the presence of nisin (100 IU/ml) and/or monolaurin (250 µg/ml) and at different pH values ​​(5, 6, 7 and 8). Based on the results of preliminary tests, it can be suggested that the inactivation of Bacillus spores by the nisin-monolaurin combination varies depending on the species studied and the pH value tested. For this, we are interested in optimizing this process of spore inactivation using a three-factor design of experiments (nisin-monolaurin-pH).

A rotatable central composite design with three independent factors was performed in order to study the inactivation of three Bacillus spores. The factors investigated were nisin (50–150 IU/mL), monolaurin (100–300 µg/mL), and pH (5–8) (Table 1).

The experiments were carried out in distilled water and UHT milk. The spores are added, after preparation, to an initial concentration (N0) of 107 spores/mL. After each treatment, the count of the spores was determined on the BHI agar-vitB12 medium. The experimental response is expressed as the decimal log reduction number (log N0/N1). It is estimated taking into account the influence of the experimental factors. Three experiments are carried out at the center of the experimental field, in order to estimate residual variance value. An analysis of variance and estimation of response surface by multiple linear regressions were performed using the software STATGRAPHICS Centurion XV version 15.2.06.

2.5. Response Surface Methodology (RSM)

Response surface methodology (RSM) is an empirical modeling technique used to estimate the relationship between a set of controllable experimental factors and observed results [18]. The results of fractional factorial experiment showed that nisin, monolaurin, and pH were the significant factors to inactivate the Bacillus spores [13]. Based on the results, response surface methodology was employed in the present work and was used to determine three log-cycles reduction of Bacillus spores in distilled water and in UHT milk under the combined treatment by nisin-monolaurin-pH. The experimental design of the investigation of experiments with three independent variables to obtain the combination of values that optimizes the response within the region of the three-dimensional observation space to allow the design of a minimal number of experimental runs [18]. The different parameters, such as nisin, monolaurin, and pH were chosen as key variables and designated as X1, X2, and X3, respectively.

2.6. Enumeration of Inactivated Spores

After decimal dilution, spores suspensions that were subjected to treatment with nisin and/or monolaurin were plated on BHI agar and counted after incubation for 24 h at 37°C. Log reductions were calculated as the difference between the logarithmic counts of colonies in untreated (N0) and treated (N) samples (log N0-log N).

3. Results and Discussion

3.1. Predictive Response Model

The combined effect of nisin, monolaurin, and pH on the inactivation of three Bacillus spores, isolated from milk, was studied using RSM, in distilled water and skim milk. The response (Y) measured in terms of log cycle reduction (log N0/N) was represented by the following second-order polynomial equation, containing 10 estimated coefficients, where x1 is nisin concentration, x2 is monolaurin concentration, and x3 is pH (Table 2).

The analysis of variance (ANOVA), realized by statgraphics, showed that the R2 values ranged between 0.95 and 0.99 indicating a high degree of correlation between the observed and predicted values. The values are used as a tool to check the significance of each coefficient, which in turn may indicate the pattern of interactions between variables. Table 3 demonstrated that the linear coefficients (x1, x2, and x3), the quadratic term coefficients (x12, x22, and x32), and the cross coefficients (x1x2 and x1x3) were significant with small values (), while the cross coefficient (x2x3) was not significant ().

3.2. Localization of Optimum Conditions

The maximum inactivation of three Bacillus spores by combined effect of nisin, monolaurin, and pH was determined using the response surfaces and their corresponding contours plots obtained by solving the regression equations (Figures 13). These graphical representations permit the visualization of the relationship between the response and experimental levels of each variable. In each figure, one factor is maintained constant at the center of the experimental value determined by used software.

Figure 1 summarizes the effect of nisin and monlaurin concentrations by keeping pH at constant value (7). The reduction of Bacillus spores varied among tested species, nisin, and monolaurin concentrations and inoculation medium. In distilled water (Figure 1(a)), a reduction of 1.85 log, 2 log, and 3.2 log was observed for, respectively, B. sporothermodurans, T. aidingensis, and Paenibacillus spores at a concentration of nisin above 150 IU/ml and of monolaurin above 150 µg/ml, whereas in UHT milk (Figure 1(b)) and in almost the same conditions (Nisin >80 IU/ml and monolaurin >200 µg/ml), there is a decreasing in the level of spore reduction with 1 log for T. aidingensis (from 2 to 1 log), 1.2 log for Paenibacillus (from 3.2 to 2 log), while for B. sporothermodurans, the reduction rate is almost the same in distilled water (1.85 log) and in the milk (1.5 log).

The combined effect of nisin (16–184 UI/ml) and pH (5.32–8.68), in presence of monolaurin at 250 µg/ml, on the inactivation of three Bacillus species demonstrated that the reduction rate of B. sporothermodurans and Paenibacillus spores is the same in both distilled water and milk (2 log) (Figure 2), but the reduction of T. aidingensis spores corresponds to 2 log in distilled water and only 1 log in milk. These results highlighted the presence of an important difference between conditions of spore reduction of the evaluated species. For B. sporothermodurans spores, the maximum reduction was obtained at acid pH (5–6) in presence of 90 IU/ml of nisin in both milk and distilled water, while the range of pH for which a maximum reduction of Paenibacillus sp. spores was observed at 5–9 in both media, in presence of 190 IU/ml of nisin. For T. aidingensis spores, the inactivation conditions depend on inoculation medium. So, in distilled water, the optimum of spore reduction was achieved in basic pH at a concentration above 150 IU/ml of nisin, but in milk, this reduction was observed in acid pH at a range of nisin concentrations between 50 and 200 IU/ml.

The effect of monolaurin and pH, at a fixed concentration of nisin (100 IU/ml), on the inactivation of the evaluated Bacillus spores is given in Figure 3. From the analysis of obtained data, we can notice that the rate of spore reduction is the same, which is calculated previously. Indeed, for B. sporothermodurans spores, the optimum reduction was obtained at acid pH with a concentration of monolaurin between 250 and 300 µg/ml in both media. For Paenibacillus spores, in the range of pH of 5 to 9 and monolaurin concentrations of 150 to 300 µg/ml, the reduction rate corresponds to 2 log, while for T. aidingensis, the optimum of spore reduction (2 log) was observed at basic pH and a concentration of monolaurin between 150 and 180 µg/ml in distilled water, and 1 log spore reduction was reached in milk at acid pH with concentrations of monolaurin ranging from 190 to 290 µg/ml.

By analyzing the nisin-monolaurin-pH plots, it can be noticed that the effect of nisin and monolaurin against Bacillus spores depends on the concentrations of these two antimicrobials agents and pH values. In fact, the spore inactivation found in UHT milk was not significantly different from that obtained in distilled water in the case where nisin and monolaurin concentrations maintained at 100 IU/ml and 250 µg/ml, respectively. This remark is different from that reported in previous studies, which demonstrated that food components may act on treatment efficiency by either decreasing or increasing the inactivation bacteria. Our results are in agreement with the findings of Aouadhi et al. [11] who reported that the inactivation of B. sporothermodurans spores by nisin, moderate heating, and high pressure is the same in both milk and distilled water, whereas when the pH was maintained at its optimum value (7), the level of spore reduction was higher in distilled water than in milk. This result confirms that when spores were treated in food matrices, a slight protective effect was noted for spores treated in milk.

In addition, the obtained results demonstrated that the nisin-monolaurin combination was more effective in reduction of Bacillus spores than either agent used alone. Since the preliminary tests of the sensibility of Bacillus spores showed that the maximum inhibition was 1.6 log for B. sporothermodurans spores by nisin (100 IU/ml) and 1.7 log with 2000 IU/ml of nisin, while in the presence of monolaurin, the reduction rate was 1.4 log for B. sporothermodurans and 1.7 log for Paenibacillus spores obtained in the presence of 250 µg/ml and 500 µg/ml, respectively (data not shown), whereas the combining effect of nisin-monolaurin increased the level of spore reduction for B. sporothermodurans (3 log), Paenibacillus, and T. aidingensis (2 log). So, this result showed that there is an inhibitory synergistic effect of nisin-monolaurin combination against Bacillus species. Also, several researchers reported that most of antimicrobial compounds studied showed inhibitory effects (prevention of microbial outgrowth and recovery during storage), some including monolaurin and nisin which have been capable of enhancing process lethality against bacterial spores [19].

The different effect of the tested inhibitors on Bacillus spore was due to their different mechanisms of action, though the cytoplasmic membrane is their primary site of action, which can explain their inhibitory synergistic effect. Nisin binds into the membrane, is inserted into it, and forms pores leading to proton motive force dissipation and to efflux of many vital intracellular compounds [20]. The effect of nisin against bacterial spores is likely to be sporostatic rather than sporicidal [6], and it has been suggested to be the result of its binding to sulfhydryl groups on protein residues [21]. Nisin prevents outgrowth of spores of C. botulinum and B. cereus [22], as spore outgrowth rather than spore germination is inhibited by the presence of nisin [23], whereas Delves-Broughton [24] suggested that the nisin action against spores is far less understood than for vegetative cells. It is predominantly sporostatic rather than sporicidal.

While according to Ababouch et al. [25, 26], monolaurin was able to inhibit the process of spore germination. Bacterial endospore germination has been defined as the degradation process by which the dormant state is irreversibly terminated. Germination is followed by an outgrowth, which is the process of synthesis of new bacterial macromolecules and conversion of germinated spores into a newly emerged vegetative cell. The mechanism of outgrowth inhibition by monoacylglycerols MAG was suggested as an inhibition of oxygen consumption, which indicates that the inner membrane and its enzymes responsible for oxygen transport are the possible sites of action. The usefulness of monolaurin in the heat inactivation of spores was substantiated by Kimsey et al. [27], who showed enhanced thermal inactivation of Geobacillus stearothermophilus in the presence of monolaurin. It was included that monolaurin could be used as food additive to reduce the heat treatment required to achieve commercial sterility of foods. The lipophilic nature of monolaurin, its emulsification properties, evidence of fatty acid inhibition of cells through interaction with the cell membranes, and the reports of spore membranes as a site of thermal damage in C. perfringens, C. botulinum, and B. stearothermophilus spores [28] suggested that monolaurin could be interacting with spore membranes and sensitizing them to heat. The antimicrobial activity of monolaurin is produced through its ability to destabilize the functions of the membrane and is enhanced with lactate, sorbate, ascorbate, and nisin. Monolaurin was partially sporicidal [29].

Many researchers suggested that simultaneous addition of nisin and monolaurin was reported to inhibit the growth of B. licheniformis [13] and in another study showed bactericidal activity against several Bacillus species, preventing both sporulation and regrowth in skimmed milk [10].

Moreover, pH influences the activity of these two antimicrobial agents. So, when combining nisin and monolaurin, their antimicrobial activity increased with increasing pH values (7.0) [13]. This same study showed that monolaurin and nisin acted synergistically on vegetative cells and outgrown spores, showing total inhibition at pH 6 of B. licheniformis spores in milk, although monolaurin enhanced thermal inactivation of B. cereus spores in pH7.2 phosphate buffer, inhibited outgrowth of Bacillus spp., Geobacillus stearothermophilus, Alicyclobacillus spp., and C. sporogenes spores in a model agar system, enhanced high pressure inactivation of B.subtilis spores in milk [7, 19, 30], and inhibited spore growth in milk [31], while other researchers demonstrated that the influence of monolaurin on B. stearothermophilus spores did not appear to be pH dependant over the range of pH 6 to 8 [27].

So, inactivation of Bacillus spores by combined treatment nisin-monolaurin depends on a range of factors, related to the species, the physical parameters of treatment (such as pH), and the medium in which bacterial spores are suspended. Furthermore, while combining nisin and monolaurin produces additive effects, more studies are necessary to determine if these agents might also work well in the presence of other antimicrobial agents or physical parameters to make them more effective to inhibit, completely, bacterial spores.

4. Conclusion

The obtained results illustrate the presence of a synergistic effect between nisin and monolaurin against three Bacillus spores. The synergistic effect related to this study could offer an original alternative for controlling these bacterial spores and could increase the scope for nisin and monolaurin usage within the food industry. The combination of these two antimicrobials would improve the shelf life of many food products. This result appears to be promising for the dairy industry and offer an alternative to high heat processing is desired. Nevertheless, further investigations and more refining of parameters of the combined treatment by nisin-monolaurin are necessary to attempt complete spore inhibition in dairy products.


Data Availability

No data were used to support this study.

Conflicts of Interest

The authors declare no conflicts of interest.


The authors acknowledge the financial support provided by the Tunisian Ministry of Higher Education and Scientific Research.