Abbreviations
EOs: Essential oils
MIC: minimum inhibitory concentration
MBC: minimum bactericidal concentration
E. coli: Escherichia coli
S. aureus: Staphylococcus aureus
S. agalactiae: Streptococcus agalactiae
Introduction
Bovine mastitis is a universal disease caused by many agents and affects the quality and quantity of milk. Mastitis is the cause of most economic losses in dairy cattle in many countries [ 1 ]. For a long time, antibiotics have been the most important cure and control program for mastitis. The appearance of resistant strains of common bacteria in lactating animals has been due to the usage of antibiotics [ 2 ]. EOs are secondary metabolites of plants and possess antimicrobial activity. After long-term use, no side effects in humans and resistance in bacteria have been observed, making them a potential weapon in the fight against bacterial diseases [ 3 ]. Antimicrobial effect of EOs is not due to one specific mechanism because there are several different chemical groups in the structure of EO. Hydrophobicity of essential oils or their components helps them to target the cell membranes of bacteria that contain lipid. This property increases the permeability of membranes, thus contents of cell leak [ 4 ].
The Mentha species is the most common plant for medicinal and health purposes [ 5 ]. Most studies have shown the antibacterial ability of Mentha pulegium (pennyroyal) and Mentha piperita (peppermint), two of the Mentha species, against different bacteria [ 6 , 7 ].
Escherichia coli, Staphylococcus aureus, and Streptococcus agalactiae cause various diseases. Moreover, these bacteria are the most important causes of bovine mastitis [ 8 ]. The most common treatment for mastitis is intramammary antibiotics. Nutrients of milk, such as fat and protein, may interfere with the antibacterial agents and decrease the bioavailability of EOs [ 9 ]. It is essential to examine the antibacterial activity of essential oils in milk before using them as an intramammary infusion. In previous studies, we tested the antimicrobial effects of essential oils from four other plants on these bacteria in milk [ 10 , 11 ]. Although many studies have been conducted on the antibacterial effect of various EOs in laboratory media, few studies exist investigating this antimicrobial effect in milk. Therefore, in this study, we decided to study the impact of peppermint and pennyroyal essential oils in milk.
Result
GC/MS analysis
The chemical constituents of peppermint and pennyroyal EO are given in Tables 1 and 2. Eight constituents were determined in peppermint, which represented 98.57% of the total oils. Carvone (63.02%) and limonene (24.48%) were found as the major components. GC/MS analysis of the pennyroyal EO showed 21 various constituents (99.51% of its chemical composition). Three major compounds were: pulegone (48.16%), eucalyptol (14.57%), and piperitenone (10.09%).
| No. | Components | Retention time (min) | Area sum% |
|---|---|---|---|
| 1 | (-)-β-Pinene | 4.931 | 0.91 |
| 2 | D-Limonene | 6.007 | 24.48 |
| 3 | p-Menth-8-en-2-one, trans- | 10.177 | 2.88 |
| 4 | (±)-Pulegone | 11.23 | 2.13 |
| 5 | (-)-Carvone | 11.438 | 63.02 |
| 6 | Carvyl acetate E | 14.357 | 1.08 |
| 7 | (-)-β-Bourbonene | 15.039 | 0.88 |
| 8 | Caryophyllene | 15.939 | 3.19 |
| No. | Components | Retention time (min) | Area sum% |
|---|---|---|---|
| 1 | 3-Carene | 4.105 | 1.58 |
| 2 | Sabinen | 4.817 | 1.43 |
| 3 | β-Pinene | 4.936 | 2.12 |
| 4 | β-Myrcene | 5.103 | 0.76 |
| 5 | Cymene | 5.892 | 0.8 |
| 6 | D-Limonene | 6.003 | 2.37 |
| 7 | Eucalyptol | 6.08 | 14.57 |
| 8 | 1-Cyclohexene-1-methanol, α,α,4-trimethyl- | 8.939 | 2.61 |
| 9 | Menthone | 9.092 | 0.79 |
| 10 | .p-Menthan-3-one | 9.323 | 0.78 |
| 11 | endo-Borneol | 9.516 | 0.96 |
| 12 | Isopulegon | 9.592 | 0.92 |
| 13 | L-.alpha.-Terpineol | 10.112 | 0.95 |
| 14 | Pulegone | 11.285 | 48.16 |
| 15 | Carvone | 11.381 | 1.55 |
| 16 | Piperitone | 11.64 | 1.45 |
| 17 | Thymol | 12.601 | 1.02 |
| 18 | Piperitenone | 13.837 | 10.09 |
| 19 | Terpinyl acetate | 14.058 | 1.01 |
| 20 | 3-Methoxy-5-propylphenol | 14.395 | 4.17 |
| 21 | Caryophyllene | 15.942 | 1.42 |
MIC and MBC
The effects of peppermint, pennyroyal EOs, and positive control (lincospectinomycin) on bacteria are declared in Table 3. The antibacterial effect was shown on the microorganisms. The MIC and MBC were 0.62% and 1.25% for pennyroyal, 0.31-1.25% and 0.62-2.5% for peppermint, 0.31-0.62% and 0.62-2.5% for peppermint and pennyroyal, respectively.
| Bacterium | MIC (%V/V) | MBC (%V/V) | |
|---|---|---|---|
| Peppermint | Escherichia coli | 1.25 | 2.5 |
| Staphylococcus aureus | 0.62 | 1.25 | |
| Streptococcus agalactiae | 0.31 | 0.62 | |
| Pennyroyal | Escherichia coli | 0.62 | 1.25 |
| Staphylococcus aureus | 0.62 | 1.25 | |
| Streptococcus agalactiae | 0.62 | 1.25 | |
| Peppermint+pennyroyal | Escherichia coli | 0.62 | 1.25 |
| Staphylococcus aureus | 1.25 | 2.5 | |
| Streptococcus agalactiae | 0.31 | 0.62 | |
| Lincospectinomycin | Escherichia coli | 0.078 | 0.156 |
| Staphylococcus aureus | 0.039 | 0.078 | |
| Streptococcus agalactiae | 0.039 | 0.078 | |
| MIC: minimum inhibitory concentration; MBC: minimum bactericidal concentration. | |||
Time kill assay
The influence of peppermint and pennyroyal on the bacteria is depicted in Figures 1, 2, and 3. The bacterial counts of the treatment and control groups were initially about 6.0 log10cfu/ml for all three bacteria. At 6-h, the bacterial reduction of treatments compared to the control group was significant for E. coli (p = 0.00) and S. agalactiae (p = 0.01) bacteria. The S. agalactiae (p = 0.00) and S. aureus (p = 0.01) counts significantly decreased in peppermint and pennyroyal group at 24-h.
Figure 1. Time-kill curve for E. coli exposed to 0% (◆red, control) and sub-MIC of peppermint (•green) and pennyroyal (■blue) EOs in milk. a-c Values with different letters are significantly (p < 0.05) different within the same time.
Figure 2. Time-kill curve for S. aureus exposed to 0% (◆red, control) and sub- MIC of peppermint (•green) and pennyroyal (■blue) EOs in milk. a-c Values with different letters are significantly (p < 0.05) different within the same time.
Figure 3. Time-kill curve for S. agalactiae exposed to 0% (◆red, control) and sub-MIC of peppermint (•green) and pennyroyal (■blue) EOs in milk. a-c Values with different letters are significantly (p < 0.05) different within the same time.
Discussion
In our study, the main components of peppermint were carvone (63.02%) and limonene (24.48%). The Mentha piperita EO extracted from Poland included menthyl acetate (19.2%), menthone (22.7%), and menthol (29.0%) [ 12 ]. In another study from Saudi Arabia, the main constituents of peppermint were menthofuran (6.88%), menthyl acetate (8.95%), menthone (24.56%), and menthol (36.02%) [ 5 ]. Major chemical compositions of peppermint EO from Brazil were terpin-4-ol (8.00%), 3-octanol (10.1%), carvone (23.42%), and linalool (51.0%) [ 13 ]. In several studies from Iran, major components of peppermint were reported as 1,8-cineole (2.15%), menthyl acetate (4.61%), menthofuran (6.49%), and menthol (25.16%) [ 14 ]; menthofuran (11.18%), menthyl acetate (15.1%) and menthol (53.28%) [ 6 ]; isomenthone (10.3%), trans-carveol (14.5%), pipertitinone oxide (19.3%), and α-terpene (19.7%) [ 15 ]. The major components of peppermint in our study were similar to a previous study from Iran [ 15 ] but different from other Iranian studies [ 6 , 14 ].
Carvone is an oxygenated monoterpene and limonene is a monoterpene hydrocarbon. Most of the antimicrobial activity in the oils has been attributed to the oxygenated monoterpenes [ 16 ]. The mechanism of monoterpene’s antimicrobial action is mainly associated with the lipophilic nature of their aglycones, which allows them to rapidly cross biological membranes and then interact with a plethora of biomolecules [ 17 ].
Major constituents of pennyroyal in the present study were pulegone (48.16%), eucalyptol (14.57%), and piperitenone (10.09%). These results are similar to EO from Iran [ 18 ] that report (E)-p-mentha-2-en- 1-ol (12.157%), 3,3'-dimenthol (18.859%), and cis-pulegone oxide (45.676%) as major components and essential oil from Portugal [ 19 ] represents pulegone (86.64%), isomenthone (4.60%), and piperitenone (2.58%) as main components. Also, another study from Portugal reported the main components of pennyroyal as neo-menthol (9.2%), pulegone (23.2%), and menthone (35.9%) [ 20 ]. The difference in principle components may be the result of climate, the effect of sunlight, and geographical conditions.
The strong antibacterial activity of pulegone have been demonstrated against a set of bacteria, including S. typhimurim and E. coli. Cytotoxicity of this essential oil appears to include a bacterial membrane damage that occurs when the essential oil passes through the cell wall and cytoplasmic membrane, and disrupts the structure of their different layers of polysaccharides, fatty acids and phospholipids [ 20 ].
To the best of our knowledge, there is no study about the antibacterial effects of peppermint and pennyroyal oil against these three bacteria in milk. Nevertheless, various MIC and MBC values have been declared against S. aureus and E. coli in laboratory mediums. In one study, the MIC value ranged from 0.39 to 3.12 mg/ml, and the MBC value ranged from 0.78 to 12.48 mg/ml for peppermint against multidrug-resistant strains of S. aureus [ 12 ]. In other studies, MIC values of peppermint were reported against reference strains of S. aureus 0.5 mg/ml [ 21 ], 0.75 ± 0.03 μg/ml [ 5 ], 0.10% [ 22 ] and against E. coli 0.20 ± 0.09 μg/ml [ 5 ] and 0.25% [ 22 ]. The MIC and MBC of pennyroyal oil reported by Luis and Domingues (2021) against E. coli and S. aureus were %8. In another study, MIC and MBC of pennyroyal against S. aureus and E. coli were reported 0.004% and 0.0005%, respectively [ 23 ]. In our study, the MIC of peppermint against E. coli and S. aureus was 1.25% and 0.62%, respectively and the MIC of pennyroyal on S. aureus and E. coli was 0.62% which was higher than most of other studies in the laboratory mediums.
Based on findings of the present study, the antibacterial activity of peppermint and pennyroyal was lower than lincospectin in milk, but in a previous study, the antibacterial effect of peppermint was the same (Streptococcus agalactiae) or higher (S. aureus and E. coli) than lincospectin and pennyroyal effect on Streptococcus agalactiae was higher than lincospectin in laboratory synthetic media [ 24 ].
According to the MIC and MBC results of the present study, E. coli (Gram-negative bacteria) was more resistant than S. aureus and S. agalactiae (Gram-positive bacteria) to peppermint EO. The resistance of the three bacteria to pennyroyal EO was the same. The resistance of Gram-negative bacteria is due to the outer membrane surrounding the cell wall which restricts the diffusion of hydrophobic compounds through the lipopolysaccharide. In addition, the periplasmic space contains enzymes, which are able to break down foreign molecules introduced from outside [ 25 ].
Significant reductions in bacterial population were observed at 6 and 24-hour in the time-kill assay. The antibacterial activity of peppermint and pennyroyal oil appears promising based on their activity at sub-MIC concentrations. To the best of our knowledge, we did not find any other studies comparing the impact of these essential oils on the bacteria in milk.
Though much information exists about the antibacterial activity of EOs, many studies have been done in model broth systems. The essential oil concentrations for antibacterial effect are higher in complex foodstuffs such as vegetables, dairy products, fish, and meat than in laboratory media [ 26 ]. For the application of essential oils in the treatment of bovine mastitis, oils must have miscibility in milk and obtain in vivo antibacterial effect in milk. In this study, we investigated the antibacterial effect of peppermint and pennyroyal EOs using the MIC&MBC method and time-kill assay in milk. According to MIC and MBC results, the antibacterial effects of our EOs were lower in milk than those effects in laboratory mediums in other studies. In a few studies, the activity of essential oils in milk and laboratory medium has been compared. Zhue et al. (2016) studied the antibacterial effect of Cinnamon cassia on major bacterial bovine mastitis in culture media and its miscibility in milk [ 9 ]. They showed that 4 MBC of oil of C. cassia was eliminated and had similar activity against S. aureus and E. coli 29 in milk within 8 hours [ 9 ]. In another study, milk composition, especially fat content, decreased the antibacterial efficacy of eugenol in milk [ 27 ].
In conclusion, peppermint and pennyroyal essential oils exhibited antibacterial properties against the three bacteria tested, indicating potential as a supplement or alternative to antibiotics in treating bovine mastitis.
Materials and Methods
Essential oils Analysis
EOs of peppermint and pennyroyal were obtained from Dorrin Golab Company, Kashan, Iran. Chemical composition determination was conducted with gas chromatography coupled to GC/MS (Model 5977A, Agilent Technologies, USA) using an HP-5MS capillary column with an internal diameter of 0.25 mm and film thickness of 0.25 µm. The temperature of the injection port was set to 260 ºC, while the oven temperature was programmed to increase from 50°C to 250°C at a rate of 4°C per minute. Helium was used as the carrier gas, with an injection volume of 1μl and a flow rate of 1 mL/min.
Cultures and Medias
The antibacterial effects were assessed against three major mastitis reference bacteria purchased from Persian Type Culture Collection, Tehran, Iran (PTCC) as a lyophilized culture: one gram-negative bacteria (Escherichia coli ATCC 25922) and two gram-positive bacteria (Streptococcus agalactiae ATCC 13813 and Staphylococcus aureus ATCC 9144). Cultures were prepared with twice the growth of the lyophilized cultures in tryptic soy broth (TSB) (Biolife, Milano, Italy) for 18 - 20 h at 37 ºC. Sterile glycerin (1:5) was used for dilution of the cultures, and these cultures were kept at - 20 ºC. Stock cultures were obtained by twice culture in TSB and streaking on tryptic soy agar (TSA) (Biolife, Milano, Italy) slant. After incubation at similar conditions, these cultures were kept at 4 ºC and subcultured once a month [ 28 ].
Inoculum Preparation
Bacterial inoculum was obtained by transferring cells from working cultures to TSB tubes, and after incubation for 18 h at 35 ºC, the subcultures were performed. The optical density (OD) (absorbance) of the bacterial broth cultures was adjusted to an optical density at 600 nm of 0.1 with a spectrophotometer (Libra S12, Biochrom Ltd., Cambridge, London). The cell concentration of these cultures was 1.64 × 1011 cfu/ml of S. agalactiae, 3.4 × 1010 cfu/ml of S. aureus, and 2.4 × 1011 cfu/ml of E. coli [ 28 ].
Preparation of Milk
Free antibiotic residue milk was obtained and sterilized with autoclavation (121 ºC, 15 min) [ 4 ].
Determination of MIC and MBC
DMSO (Sigma, Germany) was used to increase the solubility of EOs in milk with the preparation of a 1:1 dilution of the oils. Antibacterial analysis was done using this dilution. Autoclaved milk was applied as the growth medium rather than the laboratory medium. MIC was determined using double serial dilutions (10, 5, 2.5, 1.25, 0.62, 0.31, 0.15, and 0.07%) of the oils in milk. These tubes containing 1 mL of liquid were vortexed for 90 s. 100 µl of 1:400 dilution of bacterial inoculums were inoculated in tubes, and after 15 s vortex, the cultures were incubated at 37 ºC for 24 h. After the vortex, eight dilutions (10-fold) were made with 0.1 mL of culture and normal saline. Eighths of a TSA plate were inoculated with dilutions and incubated at 37 ºC for 24 h. Then, bacterial counts were determined. The lowest concentration that did not result in the growth of bacteria after subculture on TSA was regarded as MBC. MIC was considered the Ante-MBC concentration [ 9 ].
For assessment of synergism/antagonism in antibacterial action between the EOs of peppermint and pennyroyal, the essential oils were mixed volume to volume (1:1). Some controls were employed in experiments. The negative control was milk to assess autoclavation. Positive control was milk containing the bacteria to evaluate the growth of bacteria in the milk. Also, bacterial culture and DMSO without the EOs were other positive controls. Another control was the antibiotic Linco-Spectin (Linco-Spectin 5% + spectinomycin 10%) (Lincoject, Rooyan Darou, Semnan, Iran) to compare the antibacterial activity of essential oils. Each treatment was done in duplicate. Each trial was repeated at two separate times.
Time-kill assay
Sub-MIC of EOs and each bacteria were inoculated to milk. Inoculated media lacking in EO were regarded as control. After 0, 6, 10, and 24 h of incubation at 37 ºC nine 10-fold dilutions using sterile normal saline were created. TSA plates were used to plate on dilutions, and after incubation for 24 h at 37 ºC, a count of bacteria in dilutions was carried out. Each trial was repeated at two separate times. Plotting log10 cfu/ml versus time (hour) was used to construct curves [ 9 , 28 ].
Statistical analysis
The data were analyzed utilizing one-way ANOVA along with Tukey’s test employing the SPSS (18) software (IBM Corp., Armonk, NY, USA) at p <0.05).
Authors' Contributions
R.R.: study design and preparation of manuscript. S.N.: conduct the study and data collection. J.B.K.: analysis and interpretation of the data.
Acknowledgements
The authors would like to thank Gonbad Kavous University for funding this project.
Competing Interests
The authors declare that there is no conflict of interest regarding to publication of this paper.
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