Inhibitory Effects of Nisin Combined with Plant-derived Antimicrobials on Pathogenic Bacteria and the Interaction with Complex Food Systems

Abstract

This study aimed to evaluate the synergistic effects of nisin combined with cinnamaldehyde (Nis+Cin) and nisin combined with carvacrol (Nis+Car) on food-borne bacteria. The minimum inhibitory concentrations for Nis, Cin and Car ranged from 2,500 to 10,000 IU/mL, 0.78–6.25 mg/mL and 0.78–3.13 mg/mL, respectively. Nis+Cin displayed a total synergism against all test bacteria, showing a marked release of bacterial intracellular constituents. In a food model, high protein concentration, low starch and oil concentration as well as low pH, positively influenced the antimicrobial activity. Moreover, in sandwich spread, Nis+Cin enhanced the inhibition of S. typhimurium at the greatest level by reducing cell numbers by more than 4 log CFU/g from 6 days of storage. These results suggest that the dose used for each antimicrobial compound can be minimized by combination, thereby decreasing the possibility of antimicrobial resistance and also reducing food processing costs.

Introduction

Food safety with regard to microbiological contamination is a major topic worldwide. Food contamination by spoilage and/or pathogenic microorganisms can survive even severe levels of thermal processing in the food industry, and there are an increasing number of antibiotic-resistant gram-negative pathogenic bacteria. Despite the introduction of many novel advanced food processing technologies, antimicrobials must still be widely used during food processing. Black et al. (2008) suggested synthetic antimicrobial agents as an attractive approach against spoilage and pathogenic microorganisms in food. However, many of these chemical agents are thought to possess carcinogenic and teratogenic attributes. Furthermore, repeated use of synthetic chemicals or antibiotics can induce microbial resistance (Hou et al., 2007). Nonetheless, continued widespread use of these synthetic preservatives has increased public awareness of potential health concerns. As a result, alternative methods are required. At present, the world appears to be facing an environmentally friendly ‘green’ consumerism trend, and manufacturers and consumers aim to search for natural alternatives to render food safe. The use of natural antimicrobial preservatives has been introduced as an attractive method for the antimicrobial inactivation. Recently, there has been interest in the development of preservatives such as lacto-antimicrobials, ovo-antimicrobials, phyto-antimicrobials, bacto-antimicrobial and various combinations of these to improve the quality and safety of agro-industrial products (Klangpetch et al. 2013). Many of these compounds have been demonstrated to be safe for use in food. This study focused on the antimicrobial effects and application of several bacto- and phyto-antimicrobials regarded as GRAS (generally recognized as safe); nisin, cinnamaldehyde and carvacrol.

Nisin is an antibacterial peptide produced by the lactic acid bacteria Lactococcus lactis subsp. lactis. Nisin is the only bacteriocin that has been approved as a food additive, and has thus gained interest for use as a food preservative throughout the world (Food and Drug Administration, 1998). In addition, nisin is particularly effective against gram-positive microbials, including Staphylococcus, Clostridium, Listeria and Bacillus (Mozzi et al., 2010). As nisin is heat-stable and not harmful to humans, it is used to inhibit the outgrowth of Clostridium and Bacillus spores during the production of many food products, such as canned vegetables, dairy products including processed cheeses (Delves-Broughton et al., 1996), and its effects are primarily seen against gram-positive organisms. Deegan et al. (2006) proposed that nisin forms a pore in microbial lipid membranes and inhibits cell wall synthesis by binding and inducing the mislocalization of lipid II. In order to enhance food safety and prolong shelf-life, the use of nisin has been combined with other processes, such as thermal processing and other bacteriocins (Gao et al., 2011). As the effect on vegetative cells is limited to gram-positive bacteria, the combination of nisin with other natural antimicrobials to broaden its antimicrobial spectrum should be studied.

Plant extracts have been conventionally applied to preserve foods and improve food flavor. In the past decade, a number of scientific studies have identified several plant-essential oils with antimicrobial properties (Zeng et al., 2012). Some studies have investigated the benefits of plant essential oils having anti-inflammatory, antimicrobial and antioxidant activities (Alma et al., 2003). Various bioactive compounds present in these oils, including cinnamaldehyde and carvacrol, have been identified and some are reported to have notable antimicrobial properties. Both of these compounds inhibited both gram-positive and gram-negative bacteria (Mitsch et al., 2004). Cinnamaldehyde is an aromatic aldehyde present as a major component of bark extract of cinnamon, an important ingredient in Thai food, whereas carvacrol is a monoterpenoid phenolic compound obtained from oregano oil and basil leaf oil, popular herbs in Thai food. Both are classified as GRAS by the U.S. Food and Drug Administration. Cinnamaldehyde and carvacrol have been reported to be effective in Salmonella spp. inactivation on tomatoes (Mattson et al., 2011), and inactivation of the main pathogens in milk (Ananda et al., 2009). Ravishankar et al. (2009) revealed that cinnamaldehyde and carvacrol showed the potential antimicrobial effects against antibiotic resistant S. enterica in vitro and in foods. Carvacrol was found to be effective at concentrations of 1 to 7.5 mM against S. typhimurium and E. coli (Juneja and Davidson, 1993; Poi et al., 2000).

As mentioned above, plant-derived antimicrobials exhibited potent effects on inhibition of gram-negative bacteria, and in this study, the synergistic effects of these compounds with nisin were expected to lower the effective dose of each antimicrobial. Although there have been numerous reports on the antimicrobial activities of a variety of plant-derived antimicrobials against a board spectrum of microorganisms in culture media, their specific applications for enhancing food safety under different food processing systems needs to be explored.

Among the various ingredients in a complex food system, essential oil activity reportedly decreases considerably. Nutmeg and oregano were previously found to be effective against E. coli O157:H7 in culture medium while the effects were not noticeable in ready-to-cook chicken (Firouzi et al., 2007). It was also revealed that the inactivation potential of spices against S. typhimurium was reduced when applied to ground beef when compared to direct contact (Uhart et al., 2005). Therefore, to optimize the application of these natural antimicrobials, determination of their potential within model systems designed to simulate food complexes or in real food products is needed. In the present study, changes in the antimicrobial efficiency of nisin when combined with plant-derived antimicrobials in actual food components were assessed and quantified using four different model media. In addition, a sandwich spread consisting of animal and vegetable-origin materials was used as an actual complex food system.

This study was conducted to investigate the combined effects of nisin and the plant-derived antimicrobials cinnamaldehyde and carvacrol. The ultimate goal was to characterize the synergistic effects and their action mechanisms individually and in combination on food-borne bacteria, thereby minimizing the dose used for each compound, and reducing both the possibility of antimicrobial resistance and processing costs. In addition, information on appropriate food models for utilizing these antimicrobials was summarized and a real complex food system was examined.

Materials and Methods

Bacterial culture conditions    The following bacterial strains used in this study: Bacillus cereus NBRC 13494 and Staphylococcus aureus TISTR 118 for gram-positive bacteria; and Escherichia coli TISTR 117 and Salmonella Typhimurium NBRC 105726 for gram-negative bacteria. Strains with NBRC and TISTR numbers were obtained from the Biological Resource Center of National Institute of Technology and Evaluation, Japan, and the Thailand Institute of Scientific and Technological Research, respectively.

Stock cultures of bacteria were grown in tryptic soy broth (TSB, Difco Inc., Detroit, MI) media at 30 °C for 24 h. Cultivated cell suspensions were centrifuged, and then the harvested pellets were washed twice in NaCl solution (0.85 % w/v) to obtain working suspensions. The bacterial concentration of each cell suspension was then adjusted to 106 CFU/mL for experiments.

Preparation of nisin and plant-derived antimicrobial solutions    Nisin (Nis, Sigma Aldrich from Lactococcus lactis, potency ≥1,000,000 per IU/g, CAS Number 1414-45-5) standard stock solution containing 10⁴ IU/mL was prepared by dissolving 10 mg of nisin in 1 mL of sterile 0.02 N HCl. Cinnamaldehyde (Cin, Sigma Aldrich, from cassia oil, purity ≥95 %, natural and food grade, CAS Number 104-55-) and carvacrol (Car, Sigma Aldrich, purity 99 %, natural and food grade, CAS Number 499-75-2) stock solutions (50 mg/mL) were prepared using distilled water, and were filtered using a 0.20 µm membrane filter (Rajkovic et al., 2004; Ye et al., 2013).

Minimum inhibitory concentration (MIC) determination    The standard broth microdilution method was applied to determine the MIC of each antimicrobial (Nedorostova et al., 2009). Serial 2-fold dilutions of the Nis stock solution were performed to obtain working concentrations from 20,000 to 39 IU/mL in Mueller Hinton Broth (MHB, Difco Inc.). Plant-derived antimicrobials, Cin and Car, were also diluted in 2-fold serial dilutions from 5 mg/mL to 0.04 mg/mL. These antimicrobials were added to the prepared bacterial suspensions and incubated for 24 h before recording the MICs. Optical density was determined by absorbance at 630 nm using a microplate reader (SpectraMax M2 plate reader; Molecular Devices, CA, USA). Controls for this experiment were samples incubated in the absence of any antimicrobial.

Checkerboard assay    The synergistic interaction between Nis and Cin (Nis+Cin) and Nis and Car (Nis+Car) was evaluated using the checkerboard assay, as described by Mackay et al. (2000). Firstly, a 25-µL aliquot of 2-fold serial dilutions of each antimicrobial were mixed in a 96-well microplate to obtain a fixed concentration of the first anitimicrobial and increasing concentrations of the second antimicrobial in each row and column. Then, 50 µL of prepared bacterial suspension (10⁶ CFU/mL) was added to each well and cultured at 30 °C for 24 h. Optical density was determined as described above. The fraction inhibitory concentration index (FICi) was determined according to the following formula:   

where, MICA is the MIC of antimicrobial A, MICB is the MIC of antimicrobial B and MICA/B is the MIC of antimicrobial A in combination with antimicrobial B. The FIC_i results for each combination were determined as: total synergism; FIC_i ≤ 0.5, partial synergism; 0.5 < FIC_i ≤ 0.75), no effect; 0.75 < FIC_i ≤ 2 and antagonism; FIC_i > 2.

Antibacterial dynamics determination    The antibacterial dynamics of the antimicrobials were measured using a liquid culture inhibition assay. A total of 50 µL of the cell suspension and 50 µL of the antimicrobial agent were added to 96-well microplates. After incubation at 30 °C, absorbance was measured as described above every 2 h. The bacterial suspension without antimicrobials (added with 50 µL of TSB instead) was used as a control. The antibacterial dynamic was determined using the following formula (Hancock and Lehrer, 1998):   

where, A₀ and A are the absorbance of the control and tested samples, respectively.

Estimation of cell constituents' release from membrane damage    The antimicrobial combination providing the best inhibitory effect against the tested bacteria was used as a representative to evaluate the mechanisms of action. Typically, the primary toxicity target of plant-derived antimicrobials is the microbial cell membrane, leading to the leakage of crucial intracellular constituents (Juven et al., 1994; Ultee et al., 1999). Cell membrane damage should therefore be studied to describe the mechanism of action of the antimicrobials and their combination.

The release of treated-bacterial cell constituents into supernatant was measured according to the method described in author's previous study (Klangpetch et al., 2011). One hundred milliliters of each bacterial suspension was filtered with a 0.20 µm pore size syringe filter. The absorbance of the filtrate was measured at 260 and 280 nm using a spectrophotometer (Genesys, Thermo Scientific, USA). Untreated cells were also examined.

Interactive effects of food constituents and pH    The effects of food ingredients and pH value on the antimicrobial efficacy of the antimicrobials were assessed using a range of model media and the most susceptible bacteria as indicator strains according to Gutierrez's method (Gutierrez et al., 2008) with slight modification. Antimicrobials were fixed factors at the MIC being independently assessed. Model media in this study consisted of: (i) water soluble starch from potato (Sigma-Aldrich) prepared in TSB at the concentration of 0, 1, 5 and 10 %; (ii) beef extract (Difco Inc.) prepared in deionized water at the concentration of 1.5, 3, 6 and 12 %; and (iii) sunflower oil prepared in TSB (with 0.1 % Tween 80 (Merck)) at the concentration of 0, 1, 5 and 10 %. The pH of prepared media was adjusted to 7.2. TSB was adjusted to a pH range of 4 to 7 with 10 % citric acid solution to study the effects of pH on antimicrobial efficiency (Horsch et al., 2014). Growth of the model bacteria in each model medium with antimicrobials was observed in 96 well-microplate, as described above. Sterile media inoculated with microbial suspensions were used as positive controls, and sterile media with antimicrobials were used as negative controls. Survival curves of the bacteria were monitored and recorded at 630 nm during 24 h of incubation. Lag time (λ) refers to the duration from inoculation to the onset of the log-phase growth. Bacterial specific growth rates (µ_max) were computed with SoftMax Pro Software and were determined as the slope of the linear portion of the log-phase growth curve according to Kim et al. (2018).

Application of antimicrobials in real complex food system    Sandwich spread consisting of mayonnaise, tuna, chopped carrot, onion and cucumber with pH 4.5 was obtained from a local market. One hundred milliliters of cell suspension (S. typhimurium) and antimicrobials at a predetermined MIC (alone and combined) were added to 25 g of sandwich spread and mixed for 2 min by stirring. Samples were aseptically packed and stored at 4 °C. Twenty-five grams of each sample was transferred and homogenized in a sterile stomacher bag with 50 mL sterile peptone water using a stomacher for 2 min (Stomacher 400 Circulator; Seward Laboratory Systems Inc., USA). Enumeration of S. typhimurium was performed at 2, 4, 6, 8, 10, 12 and 14 days of storage by spread-plating onto Xylose lysine desoxycholate agar (Difco). Plates were incubated at 30 °C for 24 h prior to enumeration.

Statistical analysis    Three independent trials were conducted for each treatment. Mean values and standard deviations of the data were calculated. Statistical analyses were carried out using SPSS 11 software. Duncan's one-way multiple comparisons were performed to determine significant differences (p < 0.05)

Results and Discussion

Individual effects of Nis, Cin and Car against tested bacteria    Nis, Cin and Car exhibited different antimicrobial potentials against the examined 2 Gram-negative bacteria (E. coli and S. typhimurium) and 2 Gram-positive bacteria (S. aureus and B. cereus) based on the MIC shown in Table 1. The MICs of Nis, Cin and Car ranged from 2,500 to 10,000 IU/mL, 0.78–6.25 mg/mL and 0.78–3.13 mg/mL, respectively. As expected, the maximum MIC values of Nis as well as Cin were found against both gram-negatives. Gram-negative bacteria can cause many types of infection and are spread to humans in a variety of ways. Several species are common causes of food-borne diseases. They exhibited resistance against antibacterial because of their outer membrane existing. Certain types possess efflux pumps that export antibacterials to prevent them from reaching the intracellular space. Therefore, lowering the dose of antibacterials used to inactivate gram-negative bacteria is required. Recently, combining antimicrobials with other processes or other antimicrobials has been studied. Synergistic effects are a positive interaction occurring when two antimicrobials are combined to exert an inhibiting effect, resulting in a higher activity than the sum of the individual effects (Kumar et al., 2014). Nis is known to act synergistically with several antimicrobials, for instance, chelators, small substances from plants, reuterin, proteins such as lactoferrin, milk-derived peptides, lysozyme (Shi et al., 2017) and lipids with anionic characteristics, such as rhamnolipids (Magalhaes and Nitschke, 2013). However, little information is available with respect to the antibacterial activity and mechanisms of Nis in combination with Cin or Car against gram-negative or even gram-positive bacteria.

Table 1. MIC values of Nis, Cin and Car against target food-borne bacteria

Bacteria	MIC
	Nis (IU/ml)	Cin (mg/ml)	Car (mg/ml)
E. coli	10000	1.56	0.78
S. Typhimurium	10000	6.25	1.56
S. aureus	2500	0.78	3.13
B. cereus	2500	0.78	3.13

Combined effects of Nis+Cin and Nis+Car    The quantitative effects of Nis in combination with Cin and Car are described with FIC_i in Table 2. The activity of Nis+Cin displayed a synergism on all tested bacteria, particularly S. Typhimurium and B. cereus, evaluated as Total Synergism. Najjar et al. (2007) showed that the FIC_i obtained for Nis combined with ε-Polylysine against B. cereus vegetative cells was 0.56, indicating partially synergistic activity. The Total Synergism was also evaluated when combined Nis with Car, but only against gram-positive bacteria. Moreover, there were no synergistic effects found against E. coli with this combination. From these results, it may be noted that to inhibit the growth of B. cereus, combining Nis with Cin could minimize the concentrations by 64 and 2 times, respectively, and by 8 and 4 times both for inhibition of S. typhimurium. Therefore, S. typhimurium and B. cereus were used as the representative bacteria to determine the synergistic antimicrobial dynamics and mechanism of action.

Table 2. Combination effects of Nis+Cin and Nis+Car

	Bacteria	MICc (IU/ml) + (mg/ml)	FICi	Conclusion
Nis+Cin	E. coli	5000+0.39	0.75	Partial synergism
	S. Typhimurium	1250+1.56	0.50	Total synergism
	S. aureus	39+1.56	0.52	Partial synergism
	B. cereus	39+0.39	0.25	Total synergism
Nis+Car	E. coli	10000+0.78	2.00	No effect
	S. Typhimurium	2500+0.78	0.75	Partial synergism
	S.aureus	156+0.78	0.31	Total synergism
	B. cereus	156+0.78	0.31	Total synergism

MICc: MIC in combination

When compared to the combination of the main active components of essential oils in this study, the combination of essential oils themselves showed weaker effects. For instance, oregano combined with other essential oils had only partially synergistic effects against B. cereus, and had no synergistic effects against L. monocytogenes (Rajkovic et al., 2005). Furthermore, in contrast to this study, combining Nis with essential oils, for instance, cinnamon essential oil, possessing Cin as a main component caused no interactive effects against E. coli and S. typhimurium, and adversely performed antagonistic effects against B. cereus. Oregano essential oils possessing Car as a main component also showed no interactive effects against E. coli, S. typhimurium and S. aureus when combined with Nis (Turgis et al., 2012).

Antimicrobial dynamic    The correlation between antibacterial ability and incubation time for Nis and Cin when applied individually at each MIC when combined (MICc) and when used in combination against S. typhimurium and B. cereus is presented in Fig. 1. For S. typhimurium, the result showed that the ability of Cin increased gradually, reaching a peak at 8 h, showing almost 2 times greater activity when compared with Nis (Fig. 1a). The antibacterial ability of Nis reached a peak at 6 h and decreased after 8 h. Nis+Cin reached a peak at 8 h and was gradually enhanced after 10 h, tending to reflect the function of Cin. These results suggested that the activity peak of Nis+Cin is closely associated with Cin, synergistically achieving a low concentration. On the other hand, for B. cereus, Nis showed more distinct activity, reaching a peak earlier at 4 h, giving more than 2 times greater activity when compared to Cin (Fig. 1b). However, the activity sharply decreased from 4 to 8 h, and then continued steadily until the end of the test period. The antibacterial activity of Cin gradually increased to reach a peak at 18 h, showing greater activity than Nis. The activity of Nis+Cin reached a peak early at 6 h, relating to Nis activity at the beginning, and unlike Nis, the activity was stable along the incubation period. Therefore, the results suggested that the combination activity dynamic against B. cereus was related to Nis followed by Cin. The results on B. cereus are related to a previous study that investigated Nis combined with ε-poly-L-lysine (ε-PL), and found that the first activity peak against B. subtilis is closely related to Nis, while the second peak is related to ε-PL (Liu et al., 2015).

Fig. 1.

Antimicrobial dynamic of Cin (1.56 mg/mL), Nis (1,250 IU/mL) and Nis+Cin (1,250 IU/mL+1.56 mg/mL) against S. Typhimurium (a) and Cin (0.39 mg/mL), Nis (39 IU/mL) and Nis+Cin (39 IU/mL+0.39 mg/mL) against B. cereus (b) during incubation at 30 °C for 0–24 h

Nazer et al. (2005) found that thymol existing with other aromatic compounds improved inhibition, but demonstrated no real synergistic effects against Salmonella. With similar composition, the combination of essential oils may exhibit additive rather than synergistic effects. Consequently, combining with other antimicrobials containing different active compounds may enhance the potential of essential oils. To control pathogenic and spoilage microorganisms in food, essential oils or their active compounds may be combined at low doses with other food preservation techniques. The results from present study are similar to previous studies proposing that essential oil constituents could be improved for their antimicrobial properties in combination with other natural preservatives, such as bacteriocins or fatty acids (Grande et al., 2007; Yamazaki et al., 2004).

Cell constituents' release from membrane damage    The primary target for the toxicity of plant-derived antimicrobials is the microbial cell membrane, leading to leakage of important intracellular constituents (Ultee et al., 1999). Cell membrane damage was studied in order to assess the mechanism of action of the antimicrobials and their combination. To study the mechanisms of Cin and Nis, S. typhimurium and B. cereus were used as representative gram-negative and gram-positive bacteria. Firstly, bacterial cell membrane damage was estimated by evaluating the release of cell constituents. The release was determined by measuring the absorbance of the resulting filtrates at 260 and 280 nm. In the case of S. typhimurium, measuring absorbance at 260 and 280 nm confirmed that individually using Cin at the MIC (3.12 mg/mL) caused the greatest release of nucleic acid (260 nm) and protein (280 nm) for 30 and 33 times, respectively, followed by Cin at the MICc (1.56 mg/mL), when compared to the No addition sample (Fig. 2a). Although individual use of Nis neither at MIC (10,000 IU/mL) nor MICc (2,500 IU/mL) showed any release, combining Nis with Cin induced a great release of intracellular constituents. For B. cereus, Nis at the MIC (2,500 IU/mL) as well as MICc (39 IU/mL) caused a release similar to the control (physical disruption by vortexing the cells with glass beads) and combination showed a similar result as for S. typhimurium (Fig. 2b).

Fig. 2.

Absorbance at 260 and 280 nm of bacterial supernatant of S. Typhimurium (a) and B. cereus (b) after treated with Cin and Nis individually at MIC and MICc (MIC when combined) and in combined. (+) Control indicated the physically disrupted sample.

The use of essential oils combined with bacteriocins was reported to play an important role in forming membrane pores. Changing cytoplasmic permeability and irritating the proton motive force and the pH gradient of bacterial cell (Liu et al., 2015). Nis has been revealed for its synergistic effect in the presence of thyme essential oil against S. typhimurium and in the presence of oregano essential oil against Listeria. Apart from Nis, in the presence of satureja essential oil, the pediocin also exhibited a synergistic effect on E. coli O157:H7 inactivation (Turgis et al., 2012).

The release of cell constituents confirmed the antibacterial activity of Nis and plant-derived antimicrobials could be attributed to their interaction with anionic lipids on the cytoplasmic membrane of bacterial cells. The interaction may lead to perturbation of the plasma membrane. The pore formed by interactions between Nis-anionic lipids causes an efflux of adenosine triphosphate, amino acids, preaccumulated rubidium, or the collapse of vital ion gradients, leading to cell death (Tong et al., 2014).

Interaction with food constituents and pH    In order to study the effects of food constituents and pH on the activity of the antimicrobials, S. typhimurium was used as a representative in the model food systems consisting of protein, carbohydrate and fat at different concentrations. The lag time (λ) and maximum specific growth rate (µ_max) are indicated in Table 3.

Table 3. Lag time (λ) and maximum specific growth rate (µ_max) of S. Typhimurium grown in model media containing Cin (1.56 mg/mL) or Nis (1,250 IU/mL)

Model media	λ (h)			µmax (h−1)
	Cin	Nis	Control	Cin	Nis	Control
Beef extract (%)
0	4.1	4.9	4.12	0.19	0.28	0.33
1	3.33	3.48	3.11	0.21	0.29	0.31
5	6.13	4.11	2.91	0.16	0.39	0.42
10	6.89	4.09	2.88	0.08	0.33	0.48
Starch media (%)
0	13.01	5.34	3.32	0.23	0.28	0.32
1	11.21	5.11	3.51	0.28	0.28	0.29
5	9.13	4.42	3.46	0.29	0.31	0.31
10	4.11	4.01	3.67	0.28	0.33	0.26
Sunflower oil media (%)
0	12.9	5.43	3.41	0.21	0.26	0.31
1	12.1	5.67	3.89	0.22	0.22	0.29
5	8.71	5.53	3.98	0.2	0.21	0.27
10	5.12	5.11	3.88	0.21	0.21	0.22
pH
4	-	-	10.91	-	-	0.07
5	-	-	8.75	-	-	0.18
6	12.09	5.52	2.85	0.18	0.27	0.32
7	11.12	5.11	3.51	0.22	0.28	0.34

(−) : not detected

Effects of protein    In order to assess the interaction of proteins with Nis and Cin on antimicrobial efficiency, S. typhimurium was cultured in beef extract at concentrations of 0, 1, 5 and 10 %. It was apparent that increasing beef extract (protein) content shortened the lag time (λ) of S. typhimurium. However, when Cin was present in the media, the lag time of S. typhimurium grown in the media at all concentrations was longer when compared to controls. The degree of increase by addition of Cin was enhanced with increases in beef extract content. This suggests that the antimicrobial efficiency of Cin was greater in higher protein concentrations, resulting in a longer lag time, particularly at 5 % and 10 % protein, when compared to controls. This was confirmed by the growth rate, which was lower at higher protein concentrations. Peptones, the main constituent in beef extract medium displaying hydrophobic properties, may consequently interact with these hydrophobic plant-derived antimicrobials; thus, the antimicrobials readily dissolved in this medium. Baranauskien et al. (2006) revealed that proteins have a high binding capacity, particularly with volatile compounds. However, Nis appeared to lack activity against S. typhimurium showing shorter lag time and higher specific growth rate when compared to Cin.

Effects of starch    The interaction of carbohydrates with the activity of Nis and Cin against S. typhimurium was evaluated in a potato starch media at 0, 1, 5 and 10 %. The lag time and specific growth rate of S. typhimurium grown in starch model media containing Cin decreased with increasing concentration. Low concentrations positively influenced the antimicrobial potential, with longer lag times by comparison with the control. However, no differences were observed in the growth rate of S. typhimurium at any concentration of starch model media, regardless of the presence of antimicrobials (p < 0.05). The results were similar to a previous report studying the influence of carbohydrates on the efficacy of oregano and thyme essential oils on L. monocytogenase (Gutierrez et al., 2008).

Fig. 3.

Effect of Cin (1.56 mg/mL) or Nis (1,250 IU/mL), and combination of Cin and Nis on S. Typhimurium viable count during storage (4 °C) of sandwich spread.

Effects of sunflower oil    In order to evaluate the effects of oil on antimicrobial potential, S. typhimurium was cultured and monitored in sunflower oil model media with concentrations of 0, 1, 5 and 10 %. When Cin existed in the oil media, with increasing oil concentration, S. typhimurium had a shorter lag time. The addition of antimicrobials did not affect the growth rate with respect to controls at any oil concentration. A previous study also reported that the antibacterial effect against C. botulinum of Nis combined with fatty acids was reduced in the presence of soybean oil or 20 % milk fat (Glass and Johnson, 2004). It was suggested that the lipophilic properties of the essential oils led to less availability of the antibacterial effects in the aqueous phase (Mejlholm and Dalgaard, 2002).

Effects of pH    The effects of pH on the antimicrobial activity were determined using TSB adjusted to a pH of 4 to 7. With antimicrobials, S. typhimurium showed no growth at pH 4 and 5. The longer lag time of S. typhimurium grown in the pH model was demonstrated when compared to the controls, particularly with the presence of Cin. The growth rate increased at higher pH values, irrespective of the presence of antimicrobials. It was also revealed previously that under acidic conditions, the antimicrobial effects of plant extracts were increased (Del Campo et al., 2000) because the hydrophobicity of the extracts increase under low pH, enabling dissolution in the bacterial phospholipid membrane (Juven et al., 1994).

Application of antimicrobials in real complex food systems    The sandwich spread consisting of mayonnaise, tuna, chopped carrot, onion, cucumber with pH 4.5 obtained from local market (prepared with no preservatives) was used as a model real complex food system to determine the effects of antimicrobials individually and in-combination against S. typhimurium. Although existing in an acidic environment, S. typhimurium was not inactivated during storage, maintaining the number a value of 7 log CFU/g without antimicrobials. Nis had a weaker effect on inactivation of S. typhimurium, showing inactivation of 1.5 log cfu/g from 4 days until the end of the storage period. Cin notably inactivated cells for 4 log CFU/g at 6 days of storage, but the cells recovered and increased for 1 log CFU/g at 8 days, and then the number remained steady throughout the storage. Combination uses of Nis with Cin enhanced inactivation at the greatest level by reducing cells for more than 4 log CFU/g from 6 days of storage and the number did not increase throughout the storage period. Pajohi et al. (2011) previously reported the efficacy of a combination of essential oil from Cuminum cyminum L. seeds and Nis on two strains of bacillus in barley soup, which significantly decreased cell number for more than 4 log reductions after 5 days of storage. However, this combination did not stop the growth of the vegetative form of B. cereus.

Conclusions

Combinations of Nis with plant-derived antimicrobials were assessed for synergistic effects, as this would lower the effective concentrations necessary for these antimicrobials, thereby successfully achieving the ultimate goals of controlling food-borne pathogens and spoilage bacteria in food, while reducing the undesirable organoleptic impact of each compound and cost of production. Total synergistic effects were found with a combination of Nis with Cin and Nis with Car. The combination of Nis and Cin was most effective against a broad spectrum of both gram-negatives and gram-positives. As the mechanism of action was thought to be the damage and collapse of bacterial cell membrane integrity, leakage of vital intracellular constituents by this combination was investigated. Retention of the antimicrobial efficacy of Nis+Cin within real food systems was observed to take the hurdle effects of other preservation techniques into account.

Acknowledgements    The authors gratefully acknowledge financial support from the Thailand Research Fund for the project No. TRG5880052.

References

•  Alma,  M.H.,  Mavi,  A.,  Yildirim,  A.,  Digrak,  M., and  Hirata,  T. (2003). Screening of chemical composition and in vitro antioxidant and antimicrobial activities of the essential oil from Origanum syriacum L. growing in Turkey. Biol. Pharm. Bull., 26, 1725-1729.
•  Ananda,  B.S.,  Kazmer,  G.W.,  Hinckley,  L.S.,  Andrew,  M., and  Venkitanarayanan,  K.S. (2009). Antibacterial effect of plant-derived antimicrobials on major bacterial mastitis pathogens in vitro. J. Dairy Sci., 92, 1423-1429.
•  Baranauskien,  R.,  Venskutonis,  P.R.,  Dewettinck,  K., and  Verhe,  R. (2006). Properties of oregano (Origanum vulgare L.), citronella (Cymbopogon nardus G.) and marjoram (Majorana hortensis L.) flavors encapsulated into milk protein-based matrices. Food Res. Int., 39, 413-425.
•  Black,  E.P.,  Linton,  M.,  McCall,  R.D.,  Curran,  W.,  Fitzgerald,  G.F.,  Kelly,  A.L., and  Patterson,  M.F. (2008). The combined effects of high pressure and nisin on germination and inactivation of Bacillus spores in milk. J. Appl. Bacteriol., 105, 78-87.
•  Burnett,  S.R. and  Beuchat,  L.R. (2001). Human pathogens associated with raw produce and unpasteurized juices, and difficulties in decontamination. J. Ind. Microbiol. Biotechnol., 27, 104-110.
•  Deegan,  L.H.,  Cotter,  P.D.,  Hill,  C., and  Ross,  P. (2006). Bacteriocins: biological tools for biopreservation and shelf-life extension. Int. Dairy J., 16, 1058-1071.
•  Del Campo,  J.,  Amito,  M.J., and  Nguyen-The,  C. (2000). Antimicrobial effect of rosemary extracts. J. Food Prot., 63, 1359-1368.
•  Firouzi,  R.,  Shekarforoush,  S.S,  Nazer,  A.H.K.,  Broumand,  Z., and  Jooyandeh,  A.R. (2007). Effects of essential oils of oregano and nutmeg on growth and survival of Yersinia enterocolitica and Listeria monocytogenes in barbecued chicken. J. Food Prot., 70, 2626-2630.
• Food and Drug Administration, 1998. Nisin preparation: affirmation of GRAS status as a direct human food ingredient. Fed. Reg., 53, 11247-11251.
•  Gao,  Y.,  Qiu,  W.,  Wu,  D., and  Fu,  Q. (2011). Assessment of Clostridium perfringens spore response to high hydrostatic pressure and heat with nisin. Appl. Biochem. Biotechnol., 164, 1083-1095.
•  Glass,  K.A. and  Johnson,  E.A. (2004). Antagonistic effect of fat on the antibotulinal activity of food preservatives and fatty acids. Food Microbiol., 21, 675-682.
•  Grande,  M.J.,  Lopez,  R.L.,  Abriouel,  H.,  Valdivia,  E.,  Ben Omar,  N.,  Maqueda,  M.,  Martinez-Canamero,  M., and  Galvez,  A. (2007). Treatment of vegetable sauces with enterocin AS-48 alone or in combination with phenolic compounds to inhibit proliferation of Staphylococcus aureus. J. Food Prot., 70, 405-411.
•  Gutierrez,  J.,  Barry-Ryan,  C., and  Bourke,  P. (2008). The antimicrobial efficacy of plantessential oil combinations and interactions with food ingredients. Int. J. Food Microbiol., 124, 91-97.
•  Hancock,  R.E. and  Lehrer,  R. (1998). Cationic peptides: a new source of antibiotics. Trends Biotechnol., 16, 82-88.
•  Horsch,  A.M.,  Sebranek,  J.G.,  Dickson,  J.S.,  Niebuhr,  S.E.,  Larson,  E.M.,  Lavieri,  N.A.,  Ruther,  B.L., and  Wilson,  L.A. (2014). The effect of pH and nitrite concentration on the antimicrobial impact of celery juice concentrate compared with conventional sodium nitrite on Listeria monocytogenes. Meat Sci., 96, 400-407.
•  Hou,  L.X.,  Shi,  Y.H.,  Zhai,  P., and  Le,  G.W. (2007). Inhibition of foodborne pathogens by Hf-1, a novel antibacterial peptide from the larvae of the housefly (Musca domestica) in medium and orange juice. Food Control., 18, 1350-1357.
•  Hsieh,  P.C.,  Mau,  J.L., and  Huang,  S.H. (2001). Antimicrobial effect of various combinations of plant extracts. Food Microbiol., 18, 35-43.
•  Juneja,  V.K. and  Davidson,  P.M. (1993). Influence of altered fatty acid composition on resistance of Listeria monocytogenes to antimicrobials. J. Food Prot., 56, 302-305.
•  Juven,  B.J.,  Kanner,  J.,  Schved,  F., and  Weisslowicz,  H.. (1994). Factors that interact with the antibacterial action of thyme essential oil and its active constituents. J. Appl. Bacteriol., 76, 626-631.
•  Kim,  C.,  Wilkins,  K.,  Bowers,  M.,  Wynn,  C., and  Ndegwa,  E. (2018). Influence of pH and temperature on growth characteristics of leading foodborne pathogens in a laboratory medium and select food beverages. Austin Food Sci., 3, 1031.
•  Klangpetch,  W.,  Nakai  T.,  Noma  S.,  Igura,  N., and  Shimoda,  M. (2013). Combined effects of carbonation with heating and fatty acid esters on inactivation and growth inhibition of various Bacillus spores, J. Food Prot., 76, 1568-1574.
•  Klangpetch,  W,  Noma  S.,  Igura,  N., and  Shimoda,  M. (2011). The effect of low pressure carbonation on the heat inactivation of Escherichia coli. Biosci. Biotech. Biochem., 75, 1945-1950.
•  Kumar,  S.N.,  Lankalapalli,  R.S., and  Kumar,  B.S. (2014). In vitro antibacterial screening of six proline-based cyclic dipeptides in combination with b-lactam antibiotics against medically important bacteria. Appl. Biochem. Biotechnol., 173, 116-128.
•  Liu,  H.X.,  Pei,  H.B.,  Han,  Z.N.,  Feng,  G.L., and  Li,  D.P. (2015). The antimicrobial effects and synergistic antibacterial mechanism of the combination of ε-polylysine and nisin against Bacillus subtilis. Food Control, 47, 444-450
•  Mackay,  M.,  Milne,  K., and  Gould,  I. (2000). Comparison of methods for assessing synergic antibiotic interactions. Int. J. Antimicrob. Agents, 15, 125-129.
•  Magalhaes,  L. and  Nitschke,  M. (2013). Antimicrobial activity of rhamnolipids against Listeria monocytogenes and their synergistic interaction with nisin. Food Control, 29, 138-142.
•  Mattson,  T.E.,  Kollanoor,  J.K.,  Amalaradjou,  M.A.,  More,  K.,  Schreiber,  D.T., and  Patel,  J,  Venkitanarayanan  K. (2011). Inactivation of Salmonella spp. on tomatoes by plant molecules. Int. J. Food Microbiol., 144, 464-468.
•  Mejlholm,  O. and  Dalgaard,  P. (2002). Antimicrobial effect of essential oils on the seafood spoilage micro-organism photobacterium phosphoreum in liquid media and fish products. Lett. Appl. Microbiol., 34, 27-31.
•  Mitsch,  P.,  Zitterl-Eglseer,  K.,  Kohler,  B.,  Gabler,  C.,  Losa,  R., and  Zimpernik,  I. (2004). The effect of two different blends of essential oil components on the proliferation of Clostridium perfringens in the intestines of broiler chickens. Poult. Sci., 83, 669-75.
•  Mozzi,  F.,  Raya,  R.R., and  Vignolo,  G.M., (Eds.), (2010). Biotechnology of Lactic Acid Bacteria: Novel Applications. John Wiley & Sons, Oxford, pp. 233-249.
•  Najjar,  M.B.,  Kashtanov,  D., and  Chikindas,  M.L. (2007). Epsilon-poly-L-lysine and Nis A act synergistically against Gram-positive food-borne pathogens Bacillus cereus and Listeria monocytogenes. Lett. Appl. Microbiol., 45, 13-18.
•  Nazer,  A.I.,  Kobilinsky,  A.,  Tholozana,  J.L., and  Dubois-Brissonneta,  F.. (2005). Combinations of food antimicrobials at low levels to inhibit the growth of Salmonella sv. Typhimurium: a synergistic effect? Food Microbiol., 22, 391-398.
•  Nedorostova,  L.,  Kloucek,  P.,  Kokoska,  L.,  Stolcova,  M., and  Pulkrabek,  J. (2009). Antimicrobial properties of selected essential oils in vapour phase against foodborne bacteria. Food Control, 20, 157-160.
•  Pajohi,  M.R.,  Tajik,  H.,  Farshid,  A.A., and  Hadian,  M. (2011). Synergistic antibacterial activity of the essential oil of Cuminum cyminum L. seed and nisin in a food model. J. Appl. Microbiol., 110, 943-951.
•  Poi,  I.E.,  Mastwijk,  H.C.,  Bartels,  P.V., and  Smid,  E.F. (2000). Pulsed-electric field treatment enhances the bactericidal action of nisin against Bacillus cereus. Appl. Environ. Microbiol., 66, 428-430.
•  Ravishankar,  S.,  Zhu,  L.,  Olsen,  C.W.,  McHugh,  T.H., and  Friedman,  M. (2009). Edible apple film wraps containing plant antimicrobials inactivate foodborne pathogens on meat and poultry products. J. Food Sci., 74, 440-445.
•  Rajkovic,  A.,  Uyttendaele,  M.,  Courtens,  T., and  Debevere,  J. (2005). Antimicrobial effect of nisin and carvacrol and competition between Bacillus cereus and Bacillus circulans in vacuum packed potato puree. Food Microbiol., 22, 189-197.
•  Shi,  C.,  Zhang,  X.,  Zhao,  X.,  Meng,  R.,  Liu,  Z.,  Chen,  X., and  Guo,  N. (2017). Synergistic interactions of nisin in combination with Cin against Staphylococcus aureus in pasteurized milk. Food Control, 71, 10-16.
•  Tong,  Z.,  Ling,  J.,  Lin,  Z.,  Li,  X., and  Mu,  Y. (2014). Antibacterial peptide nisin: a potential role in the inhibition of oral pathogenic bacteria. Peptides, 60, 32-40.
•  Turgis,  M.,  Vu,  K.D.,  Dupont,  C., and  Lacroix,  M. (2012). Combined antimicrobial effect of essential oils and bacteriocins against foodborne pathogens and food spoilage bacteria. Food Res. Int., 48, 696-702.
•  Uhart,  M.,  Maks,  N., and  Ravishankar,  S. (2006). Effect of spices on growth and survival of Salmonella Typhimurium DT 104 in ground beef stored at 4 and 8 °C. J. Food Safety, 26, 115-125.
•  Ultee,  A.,  Kets,  E.P.W., and  Smid,  E.J. (1999). Mechanisms of action of carvacrol on the food-borne pathogen Bacillus cereus. Appl. Environ. Microbiol., 65, 4606-4610.
•  Yamazaki,  K.,  Yamamoto,  T.,  Kawai,  Y., and  Inoue,  N.. (2004). Enhancement of antilisterial activity of essential oils constituents by nisin and diglycerol fatty acid ester. Food Microbiol., 21, 283-289.
•  Ye,  H.,  Shen,  S.,  Xu,  J.,  Lin,  S.  Yuan,  Y., and  Jones,  G.S. (2013). Synergistic interactions of cinnamaldehyde in combination with carvacrol against food-borne bacteria. Food Control. 34, 619-623.
•  Zeng,  W.C.,  He,  Q.,  Sun,  Q.,  Zhong,  K., and  Gao,  H. (2012). Antibacterial activity of water-soluble extract from pine needles of Cedrus deodara. Int. J. Food Microbiol., 153, 78-84.