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. 2020 Sep 22;58(8):3010–3018. doi: 10.1007/s13197-020-04804-9

Antimicrobial activity of Baccharis dracunculifolia DC and its synergistic interaction with nisin against food-related bacteria

Palmira Penina Raúl Timbe 1, Amanda de Souza da Motta 2, Paolo Stincone 1, Cristian Mauricio Barreto Pinilla 1, Adriano Brandelli 1,✉
PMCID: PMC8249481  PMID: 34294963

Abstract

The antimicrobial activities of Baccharis dracunculifolia DC essential oil (EO) and hydroalcoholic extract (HE) were evaluated. The EO showed broad antimicrobial activity and its synergistic combination with nisin was tested. Major components of EO were nerolidol, beta-pinene and D-limonene, while artepillin C, rutin and cafeic acid were major phenolics of HE. EO and HE were tested by agar diffusion assay against several strains of bacteria and yeasts, and mixed cultures of bacterial strains. The EO presented the largest spectrum of antimicrobial activity inhibiting all Gram-positive bacteria tested. Yeasts were not inhibited. The effect of EO against mixtures of sensitive and non-sensitive bacteria was tested on milk agar, being the inhibitory effect only observed on mixtures containing susceptible strains. The combination of EO and nisin at ½ MIC was evaluated on the growth curve of Staphylococcus aureus, Bacillus cereus, Listeria monocytogenes and Salmonella Enteritidis during 24 h at 37 °C. The combination EO-nisin was effective and no viable counts of B. cereus, L. monocytogenes and S. Enteritidis was observed, while the individual antimicrobials caused no inhibition. The counts of S. aureus were about 4 log CFU/mL lower in comparison with EO or nisin alone. B. dracunculifolia DC may be a potential source of natural antimicrobials, and its synergistic effect with nisin would reduce the working concentration, minimizing the organoleptic effects associated with this plant antimicrobial.

Keywords: Baccharis dracunculifolia, Essential oil, Foodborne bacteria, Natural antimicrobial, Nisin

Introduction

The presence of pathogenic and spoilage microorganisms in foods of both plant and animal origin, including water, is a point of utmost importance. It has been estimated that about 25% of food products are degraded by microorganisms after harvest or slaughter (Petruzzi et al. 2017). The ingestion of food containing pathogenic microorganisms, or their toxins is considered the main cause of foodborne diseases, and therefore a serious public health problem. The control of undesirable microorganisms in food is necessary and can be accomplished by applying synthetic antimicrobials, used individually or in combination with other conservation methods (Pisoschi et al. 2018). However, in recent decades there has been an increase in the number of consumers seeking for more natural foods, mainly driven by health concerns. This growing demand encourages research on natural antimicrobials, including those isolated from plants.

Baccharis dracunculifolia DC, popularly known as alecrim-do-campo, is a 2–3 m high shrub, native to some countries of America, including Brazil (Gomes and Fernandes 2002), where it is used empirically in the treatment of some diseases (Oliveira et al. 2007). Nevertheless, it has been proven that its secondary metabolites have biological activities such as antimicrobial (Salazar et al. 2018; Cazella et al. 2019), antiparasitic (Assis Lage et al. 2015), anti-inflammatory (Figueiredo-Rinhel et al. 2017) and antiviral (Búfalo et al. 2009). Furthermore, it has been reported that bees use their resin to produce green propolis, a compound with therapeutic effect (Lemos et al. 2007). The antimicrobial properties of this plant have been associated with terpenes (Salazar et al. 2018; Cazella et al. 2019) and phenolic compounds (Veiga et al. 2017).

Despite the recognized biological activities of essential oils and plant extracts, one of their major limitations for food applications is related with very pronounced odors and tastes, which in some cases are undesirable. In addition, their antimicrobial action may be reduced due to interactions with food matrix components, requiring higher concentrations to obtain similar effect to that observed in vitro (Calo et al. 2015). The use of B. dracunculifolia DC essential oil associated with antibiotics results in synergism (Salazar et al. 2018), and this combination may be an effective strategy to reduce antimicrobial concentrations.

Nisin is a bacteriocin produced by Lactococcus lactis subsp. lactis that has activity against Gram-positive bacteria (Khan and Oh 2016). Nisin is considered Generally Recognized as Safe (GRAS), and is used as a natural food preservative in more than 50 countries (Pisoschi et al. 2018). Some studies demonstrate that nisin combination with plant-isolated antimicrobials results in a synergistic effect (Pinilla and Brandelli 2016; Shi et al. 2017). However, there are no reports on the effect of combining nisin with B. dracunculifolia DC bioactive compounds against foodborne pathogens.

In this context, this study evaluated the antimicrobial activity of B. dracunculifolia DC essential oil and hydroalcoholic extract against food-related microorganisms, and the antimicrobial effect of the combination of the essential oil with nisin.

Materials and methods

Essential oil and hydroalcoholic extract of B. dracunculifolia DC

The essential oil of B. dracunculifolia DC (EO) was obtained from Harmonia Natural (Canelinha, SC, Brazil). The composition of the EO was provided by the distributor, being the three main components nerolidol (21%), β-pinene (16%), and D-limonene (14%). Caryophyllene, elixene, α-pinene, germacrene D and spatulenol were present in minor amounts (Table 1). For broth tests, the essential oil solution was prepared with ultrapure water (obtained from Milli-Q purifier) and Tween 80 (5 mg/mL).

Table 1.

Composition of Baccharis dracunculifolia DC essential oil (EO) and hydroalcoholic extract (HE)

Compound Concentration (%)
EO
Nerediol 21.0
β-pinene 16.1
D-limonene 14.3
Caryophyllene 10.8
(-) Spatulenol 7.6
Germacrene D 7.3
α-pinene 7.2
Elixene 3.5
HE
Artepillin-C 27.7
Rutin 17.9
Cafeic acid 7.2
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The hydroalcoholic extract of B. dracunculifolia DC (HE) was obtained from Ciclo Farma Indústria Química Ltda. (Serrana, SP, Brazil). All solvent was evaporated under vacuum at 30 °C and the material was reconstituted in 10% (v/v) DMSO as described previously (Gouvinhas et al. 2018). The composition of HE was determined by HPLC as described elsewhere (Cassol et al. 2019), after extraction with 80:20 (v/v) methanol–water.

Nisin

Nisin (Nisaplin®) was supplied by Danisco Brasil Ltda. (Pirapozinho, SP, Brazil). According to the manufacturer, the formulation contains 2.5% pure nisin. The stock solution was prepared by dissolving Nisaplin® in 0.01 M HCl to obtain 2 mg/mL nisin, which was further diluted in 10 mM phosphate buffer (pH 7.0) to reach working concentrations.

All antimicrobials were stored at 4 °C, and prior to testing they were sterilized by filtration, using 0.22 μm filter membranes (Millipore, Burlington, MA, USA).

Microorganisms

Different strains of Gram-positive and Gram-negative bacteria and yeasts were evaluated (Table 2). The cultures were obtained from the American Type Culture Collection (ATCC), commercial sources, food samples, food processing environment and clinical sources.

Table 2.

Antimicrobial activity of B. dracunculifolia DC essential oil (EO) and hydroalcoholic extract (HE)

Microorganisms Origin Inhibition diameter (mm)
EO HE
Gram-positive bacteria
Staphylococcus aureus ATCC 1901 Culture collection 20.8 ± 1.6baA 12.7 ± 0.4bcB
Bacillus cereus ATCC 9634 Culture collection 17.0 ± 2.2fecdA 20.5 ± 2.1aA
Bacillus subtilis FTC01 Commercial probiotic 23.6 ± 0.5aA 15.7 ± 3.0baB
Bacillus amyloliquefaciens I3 Soil 17.8 ± 1.3ecdA 17.8 ± 1.3baA
Listeria monocytogenes ATCC 7644 Culture collection 16.5 ± 0.4fecdA 7.7 ± 5cB
Listeria monocytogenes 17D78/03 Food 15.7 ± 0.9 fedA 13.0 ± 0.7bcA
Listeria monocytogenes 4C Bovine carcasse 15.8 ± 0.6 fecdA 15.6 ± 0.9baA
Listeria monocytogenes 4B Bovine carcasse 13.8 ± 1.0 fghA 0 dB
Listeria monocytogenes QF Oxford-6 Sliced cheese 13.7 ± 0.5fghA 0 dB
Listeria innocua 6B Food 10.3 ± 0.9iA 0 dB
Listeria innocua L07 Buffalo milk 10.0 ± 0.8iA 0 dB
Listeria innocua L10 Buffalo milk 11.0 ± 1.6ihA 12.6 ± 1.0bcA
Listeria innocua L11 Buffalo milk 12.3 ± 0.5ighA 0 dB
Listeria innocua L13 Buffalo milk 10.3 ± 1.2iA 0 dB
Listeria seeligeri BP Palcam-2 Ham countertop 15.3 ± 1.2fegA 0 dB
Listeria sp. BQ Oxford-1 Ham countertop 14.7 ± 0.5fegA 0 dB
Listeria seeligeri MP Oxford-4 Food handler hands 14.3 ± 0.5fgA 0 dB
Listeria sp. BP Oxford-5 Cheese countertop 15.0 ± 0.8fegA 0 dB
Listeria seeligeri PF Oxford-3 Sliced ham 14.8 ± 0.2fegA 0 dB
Gram-negative bacteria
Aeromonas hydrophila ATCC 7966 Culture collection 18.6 ± 0.94bcdA 17.0 ± 2.0bcA
Salmonella enterica serovar Enteritidis ATCC 13076 Culture collection 19.0 ± 0.82bcA 8.7 ± 2.4cB
Pseudomonas aeruginosa ATCC 27853 Culture collection 0jA 0dA
Erwinia psidii Phytopathogen 0jA 0dA
Escherichia coli ATCC O157:H7 Culture collection 0jA 0dA
Escherichia coli ATCC 35,218 Culture collection 0jA 0dA
Escherichia coli ATCC 8739 Culture collection 0jA 0dA
Escherichia coli ESBL Clinical isolate 0jA 0dA
Escherichia coli S1 Clinical isolate 0jA 0dA
Escherichia coli S2 Clinical isolate 0jA 0dA
Escherichia coli S12 Clinical isolate 0jA 0dA
Escherichia coli NDM Clinical isolate 0jA 0dA
Escherichia coli KPC Clinical isolate 0jA 0dA
Yeast
Candida krusei ATCC 6258 Culture collection 0jA 0dA
Candida parapsilosis ATCC 22019 Culture collection 0jA 0dA
Saccharomyces cerevisiae Commercial strain 0jA 0dA
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Values are means ± standard deviations of three independent experiments (n = 3). a−j Different letters represent significant differences within the same column; A−B different letters represent significant differences within the same row (p < 0.05)

Antimicrobial susceptibility testing

The sensitivity of all microorganisms to EO and HE was evaluated in order to verify the spectrum of action and select the most active antimicrobial for subsequent testing. The initial screening was performed by the disk-diffusion method (CLSI 2012), where the microbial suspension was adjusted in saline solution (0.85 g/L NaCl) to obtain a turbidity equivalent to 0.5 McFarland, which corresponds to approximately 1.5 × 108 CFU/mL for bacteria and 106 cells/mL for yeasts. The assay consisted of placing 6 mm diameter cellulose discs on the surface of plates containing Mueller–Hinton or Sabouraud agar, previously seeded with swab of the bacterial or yeast suspensions. An aliquot of 5 μL EO, or 10 μL HE at a concentration of 25 mg/mL, was applied onto the discs. The plates were incubated at 30 °C or 37 °C for 24 h for yeast or bacterial growth, respectively. After incubation, the inhibition diameters were measured with a caliper rule, and the results were expressed in mm. The assay was performed in triplicate and 10% DMSO, Tween 80 (5 mg/mL), and 0.01 M HCl were used as controls.

Susceptibility of mixed bacterial cultures to the EO

Milk agar was used as a growth medium to simulate a food matrix (Lopes et al. 2017), and the activity of EO (antimicrobial with greater spectrum of action) was tested in triplicate against three bacterial mixtures by the disk-diffusion method described above. The mixed cultures were obtained by preparing individual suspensions of approximately 1.5 × 108 CFU/mL, followed by mixing equal volume of each bacterial suspension. The first mixed culture consisted of three bacteria of the same genus sensitive to EO (Listeria monocytogenes ATCC 7644, Listeria seeligeri MP and Listeria innocua 6B); the second by bacteria of different genus and sensitive to EO (L. monocytogenes ATCC 7644 and S. aureus ATCC 1901); and the third by two bacteria of different types, being L. monocytogenes ATCC 7644 sensitive and E. coli ATCC 8739 not sensitive to EO.

Evaluation of synergistic potential between EO and nisin

The possible synergistic interactions between the antimicrobial activity of EO and nisin was evaluated. For this purpose, some microorganisms sensitive to EO were selected: S. aureus ATCC 1901, B. cereus ATCC 9634, L. monocytogenes ATCC 7644, and S. enterica serovar Enteritidis ATCC 13076. The verification of synergism initially consisted of determining the Minimum Inhibitory Concentration (MIC), and lastly it was evaluated the synergistic interactions through the microbial growth curve (time-kill curve).

MIC of EO and nisin was determined in triplicate by the broth macrodilution method (CLSI 2012). MIC values were obtained by testing 12 serial dilutions of each antimicrobial, where the EO was evaluated at concentrations between 4.4 and 9000 μg/mL, and nisin at concentrations between 0.007 and 15 μg/mL. This assay consisted of preparing culture tubes containing the antimicrobial at the desired concentration in 9 mL of BHI broth, to which 1 mL of the bacterial inoculum at approximately 105 CFU/mL was added. The tubes were then incubated in a rotary shaker (150 rpm) at 37 °C for 24 h. After incubation, the visual observation of turbidity of the culture medium was used as an indicative of bacterial growth. The MIC value was defined as the lowest antimicrobial concentration in which no medium turbidity was observed and was expressed in μg/mL. Controls of culture medium with bacterial inoculum without antimicrobial, and medium without inoculum were performed.

The synergistic effect between EO and nisin was evaluated through the microbial growth curve as described elsewhere (Pinilla and Brandelli 2016), with minor changes. The antimicrobial concentrations tested were ½ MIC of the EO and ½ MIC of nisin, either individually or in combination against each bacterium. Cultures were incubated in a rotary shaker (150 rpm) at 37 °C for 24 h and aliquots were taken at time 0, and after 2, 4, 6, 8, 10, and 24 h. They were then serially diluted in saline, inoculated onto BHI agar plates by the plate spread method and incubated at 37 °C for 24 h. After incubation, the CFU/mL values were determined and expressed on the logarithmic scale. A synergistic effect was considered when microbial counts decreased by ≥ 2 log after 24 h in combined antimicrobial treatments compared to individual antimicrobial treatments (Shi et al. 2017). The detection limit of this method is 2 log CFU/mL (Mpofu et al. 2016). The test was performed in duplicate, and the control consisted of culture medium with bacterial inoculum without antimicrobials.

Statistical analysis

The statistical analyzes were performed using SAS software (Version 9.0, SAS Institute Inc., Cary, NC), and data were presented as mean ± standard deviation (SD). The differences between averages were tested by the Analysis of Variance (ANOVA) test, applying the Tukey test, and were considered significant when p < 0.05. MIC values were presented as the mode, the value that appears most often in the set of data values.

Results and discussion

Antimicrobial susceptibility testing

The results of the disk-diffusion test to evaluate the antimicrobial spectrum of EO and HE against different microorganisms are shown in Table 2. The inhibitory spectrum of EO and HE revealed antibacterial but not antifungal activity. EO inhibited all Gram-positive bacteria tested while HE did not inhibit 11 strains of Listeria (Table 2). In this test, the Gram-negative bacteria were found to be less sensitive, and both EO and HE inhibited only two strains, S. enterica serovar Enteritidis ATCC 13076 and A. hydrophila ATCC 7966, from the thirteen Gram-negative strains tested. Controls performed with 10% DMSO, HCl (0.01 M) and Tween 80 (5 mg/mL), did not inhibit microbial growth.

It has been reported that Gram-negative bacteria are less susceptible to essential oils or plant extracts because their cell wall structure is more complex than Gram-positive bacteria, presenting an outer membrane, that serves as a barrier for active substances to reach the cytoplasmic membrane (Nazzaro et al. 2013). In this analysis, EO presented a broader spectrum of action compared to HE, and this finding could be associated with the difference in the chemical composition of these antimicrobials. Furthermore, EO was tested as received, while HE was tested after reconstitution at a concentration of 25 mg/mL.

The composition analysis showed that artepillin C (27.7%), rutin (17.9%) and cafeic acid (7.2%) were the major hydroxycinnamic acids in the HE (Table 1). These results are in agreement with previous reports of phenolic profile of B. dracunculifolia DC and B. trimera species (Guimarães et al. 2012; Sabir et al. 2017). Particularly, artepillin C has been already reported as the main bioactive phenolic compound responsible for the antibacterial effect of B. dracunculifolia DC extracts (Veiga et al. 2017), and the flavonoid rutin has shown a wide range of pharmacological activities including antimicrobial activity (Gullón et al. 2017). The essential oils of B. dracunculifolia DC are basically composed of terpenes (Assis Lage et al. 2015; Salazar et al. 2018; Cazella et al. 2019). As mentioned earlier, the three major EO terpenes of this study were nerolidol (21%), beta-pinene (16%), and D-limonene (14%), with other terpenoid compounds being at lower concentrations (Table 1). Nerolidol is a sesquiterpene present in essential oils of numerous plants and a strong antimicrobial activity was documented for this compound, including the activity against the important food spoilage Lactobacillus fermentum (Ephrem et al. 2019). The monoterpene β-pinene was also considered for its broad antimicrobial activity, inhibiting S. aureus, Staphylococcus epidermidis, Klebsiella pneumoniae and Enterobacter aerogenes (Salehi et al. 2019). In general, the antimicrobial action of the EO probably results from synergistic interactions of the different constituents, as both major and minority components exhibit this property, and some of the components were associated with antimicrobial effect, such as nerolidol, α-pinene, spatulenol, germacrene D and artepillin C (Veiga et al. 2017; Cazella et al. 2019).

The inhibitory activity of B. dracunculifolia DC EO against S. aureus, B. cereus and L. monocytogenes agrees with the results obtained in other studies regarding Gram-positive bacteria (Salazar et al. 2018; Cazella et al. 2019). However, inhibition of A. hydrophila and S. enterica serovar Enteritidis was observed in this work, while P. aeruginosa ATCC 27853 and E. coli ATCC 35218 (Cazella et al. 2019) and Candida spp. of clinical origin, including C. parapsilosis (Pereira et al. 2011), were inhibited in other studies. The discrepancy in antimicrobial spectrum has been associated with differences in the microbial strains tested, and also the variation in the chemical composition of this essential oil. Although B. dracunculifolia DC EO is essentially composed by terpenes, different lots may have qualitative and quantitative variations (Cazella et al. 2019). Extracts or essential oils of any plant are recognized to present a natural variation in their composition according to plant age, plant part, geographical location, chemical soil composition, water availability, temperature variations, exposure to sunlight, bacteria, viruses, fungi or parasites, extraction method, and storage conditions (Verma and Shukla 2015; Khayyat and Roselin 2018); such variation in the chemical composition of the essential oils could influence antimicrobial activity.

Some studies indicate that the hydroalcoholic extracts of B. dracunculifolia DC harvested in the Brazilian territory consist essentially of phenolic compounds, mentioning artepillin C, caffeic acid, ferulic acid, p-coumaric acid, aromadendrin-4-methyl ether, baccharine, drupanine, isosakuranetin, 2,2-dimethyl-6-carboxyethenyl-2H-1-benzopyran (Lemos et al. 2007; Búfalo et al. 2009). The antimicrobial action of HE is probably associated with these phenolic compounds. Other studies investigating the antimicrobial activity of B. dracunculifolia DC HE also reported that strains of E. coli, P. aeruginosa and Candida spp. (Oliveira et al. 2007) are not sensitive, whereas S. aureus (Oliveira et al. 2007; Veiga et al. 2017) and B. cereus (Fabri et al. 2011) are often inhibited. Furthermore, the present study showed the activity against B. subtilis FTC01 and A. hydrophila ATCC 7966, which was not previously described. The antimicrobial activity of both B. dracunculifolia DC HE and EO was first reported against B. amyloliquefaciens, S. enterica serovar Enterididis, and several Listeria strains, broadening the knowledge on the action spectrum of these antimicrobials. The EO was the antimicrobial that inhibited the largest number of microorganisms, being selected for the subsequent tests.

Susceptibility testing of mixed bacterial cultures

In foods, the occurrence of microorganisms is often not isolated, and several genera can coexist in a food matrix. In any ecosystem that microorganisms inhabit, they create survival mechanisms through intra and interspecific interactions that can induce resistance to antimicrobials, including resistance gene transfer and biofilm formation (Verraes et al. 2013). In this context, a qualitative test was used to verify whether the presence of various microorganisms would affect antimicrobial activity of EO, using milk agar as food simulating growth medium. With this test, it was observed that the presence of more than one microorganism did not interfere with antimicrobial susceptibility, as EO inhibited mixed cultures formed by sensitive bacteria, but had no effect on that containing a non-susceptible strain (Table 3). The absence of inhibition could be associated to the presence of a non-susceptible strain, because around the disc it was possible to observe isolated colonies, suggesting that the sensitive strain was inhibited, suggesting that there was no transfer of resistance genes.

Table 3.

Antimicrobial activity of B. dracunculifolia DC essential oil (EO) in milk agar against mixed bacterial cultures

Mixed culture Microorganisms Inhibition diameter (mm)
I L. monocytogenes ATCC 7644, L. innocua L11, Listeria seeligeri MP 13.3 ± 1.7a
II L. monocytogenes ATCC 7644, S. aureus ATCC 1901 12.1 ± 1.0a
III L. monocytogenes ATCC 7644, E. coli ATCC 8739 0 ± 0b
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Values are means ± standard deviation of three independent experiments (n = 3). a,b Different letters represent significant differences (p < 0.05)

Evaluation of synergistic potential between EO and nisin

For the study of synergism, the MIC values of EO and nisin were initially determined. The results are presented in Table 4. Regarding susceptibility to EO, S. Enteritidis ATCC 13076 was found to be the least sensitive bacterium with a MIC value of 563 μg/mL, whereas B. cereus ATCC 9634 was more sensitive with a MIC value of 70 μg/mL. Regarding susceptibility to nisin, S. Enteritidis ATCC 13076 was also the least sensitive and was not inhibited at all. On the other hand, nisin had a strong antimicrobial effect against L. monocytogenes ATCC 7644 and inhibited its growth at a concentration of 0.015 μg/mL. These findings corroborate with those available in the literature, which state that Gram-negative bacteria are generally not inhibited by nisin (Chen and Hoover 2003) or less sensitive to some essential oils (Nazzaro et al. 2013). However, the antimicrobial activity is related with multiple variables including the composition of the essential oils that influences the mode of action on different target microorganism (Chouhan et al. 2017).

Table 4.

Minimum inhibitory concentration (MIC) of B. dracunculifolia DC essential oil (EO) and nisin.a

Microorganism EO (μg/mL) Nisin (μg/mL)
Listeria monocytogenes ATCC 7644 141 0.015
Staphylococcus aureus ATCC 1901 141 0.93
Bacillus cereus ATCC 9634 70 3.75
Salmonella Enteritidis ATCC 13076 563  > 15b
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a Values are presented as the mode (the value at which the probability mass function takes its maximum value) of at least six independent determinations

b Not inhibited by the highest concentration tested (15 g/mL)

The synergistic effect between the EO and nisin was investigated by testing the antimicrobials at ½ MIC values individually and in combination against B. cereus, L. monocytogenes, S. aureus and S. Enteritidis by monitoring the microbial growth curves. In the combined use of natural antimicrobials, the compounds can act independently or interact with each other resulting in antagonistic or synergistic effects (Semeniuc et al. 2017). The microbial growth curve is considered one of the most appropriate method to verify the synergistic effect, as it allows to visualize the effect over time (Albano et al. 2016).

The results of the microbial growth curves are shown in Fig. 1. After 24 h incubation, the combination of EO with nisin was effective and no viable counts were detected for B. cereus, L. monocytogenes, and S. Enteritidis, while these bacteria reached more than 8 log CFU/mL in the presence of individual antimicrobials. The counts for S. aureus were 2.81 log CFU/mL for the combined treatment, showing about 4 log CFU/mL decrease in comparison with EO or nisin alone (Fig. 1). Thus, the combination of EO with nisin had a synergistic effect on all bacteria tested. This result fits the criterion that considers the synergistic effect occurs when bacterial counts are reduced by ≥ 2 log CFU/mL after 24 h in treatments with combined antimicrobials relative to the individual compounds (Shi et al. 2017). On the other hand, the EO and nisin alone had no significant effect, being the number of bacteria present in these treatments close to the control (medium without antimicrobial) after 24 h, excepting for S. aureus ATCC 1901 where individual antimicrobials showed a difference of 2.1 log CFU/mL compared to the control (Fig. 1).

Fig. 1.

Fig. 1

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Effect of nisin and B. dracunculifolia DC (EO) essential oil used individually or in combination on growth of L. monocytogenes ATCC 7644 (a), B. cereus ATCC 9634 (b), S. aureus ATCC 1901 (c) S. Enteritidis ATCC 13076 (d). Viable counts were monitored in the presence of ½ MIC EO + ½ MIC nisin (—○—), ½ MIC EO (•••▲•••), ½ MIC nisin (–-□–-), control (—●—). Detection limit (—). Values are means ± standard deviation of three independent experiments (n = 3)

In general, the antimicrobial action of plant essential oils is not associated with a specific mechanism of action due to the large number of compounds present. However, the essential oil of B. dracunculifolia DC is composed mainly by terpenes (Salazar et al. 2018). Due to the high lipophilicity, terpenes react easily with the cell membrane, causing structural and functional damage resulting in its expansion, and increased permeability allowing the release of intracellular constituents (Guimarães et al. 2019). In the other hand, the effect of nisin on bacterial membranes is well recognized. Nisin is a cationic molecule, which facilitates its interaction with the negatively charged bacterial membrane. On the cell membrane, nisin specifically binds to the peptidoglycan precursor, lipid II, resulting in inhibition of cell wall synthesis and formation of pores that cause cell constituents to overflow (Khan and Oh 2016). Some studies report the synergistic effect between essential oils and nisin, but the mechanism of action involved is not established (Shi et al. 2017). However, terpenes as well as nisin target the cell membrane, suggesting that terpenes may increase the size and number of pores formed by nisin, resulting in greater membrane damage. Moreover, the association with nisin has the advantage that it has no effect on food properties because it is an odorless, colorless, and tasteless substance (Lopes et al. 2017). The synergistic effect observed in the present study is important because it reduces the amount of EO required to produce the antimicrobial effect, therefore lower concentrations may be employed in food applications, reducing the intense aroma of the EO.

Conclusion

The antimicrobial spectra of B. dracunculifolia DC EO and HE was evaluated in this study and both antimicrobials were found to have high antibacterial but not antifungal activity. Gram-positive bacteria were more sensitive to both EO and HE, and the EO was the antimicrobial with broader spectrum of action because it inhibited all 19 strains of Gram-positive bacteria tested. In addition, the sensitivity of mixed bacterial cultures to EO in milk agar revealed the presence of multiple sensitive bacterium and milk constituents did not interfere with antimicrobial activity. The combined use of EO and nisin showed a synergistic activity against S. aureus ATCC 1901, B. cereus ATCC 9634, L. monocytogenes ATCC 7644, and S. enterica serovar Enteritidis ATCC 13076, being more effective than EO alone. Based on these results, EO and nisin could be used simultaneously in food as a strategy to reduce organoleptic effects of B. dracunculifolia DC EO.

Acknowledgements

Authors thank Dr. P.M. Reque for technical support on HPLC analysis of the extracts. This work received financial support from Ministério da Ciência e Tecnologia, Ensino Superior e Técnico Profissional (MCTESTP, Mozambique) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil) [Grant No 306936/2017-8].

Compliance with ethical standards

Conflicts of interest

Authors declare no conflicts of interest.

Footnotes

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