In-vitro Assessment of Antimicrobial, Antibiofilm and Antioxidant Potential of Essential Oil from Rosemary (Rosmarinus officinalis L.)

The present study was conducted to investigate in-vitro antimicrobial, antibiofilm and antioxidant efficacy of Rosemary essential oil (REO) for its potential application in meat products. The oil was tested against four Gram positive (Bacillus cereus, Listeria monocytogenes, Staphylococcus aureus and Enterococcus faecalis) and six Gram negative (Salmonella enterica serovar Typhi, Escherichia coli, Shigella flexneri, Pseudomonas aeruginosa, Proteus mirabilis and Klebsiella pneumoniae) organisms using zone of inhibition and Minimum Inhibitory Concentration (MIC) estimation. Widest inhibition zone was exhibited by Staphylococcus aureus whereas, Enterococcus faecalis and Salmonella enterica showed the largest MIC values. Antibiofilm activity (%) was determined by using pure culture of L. monocytogenes and S. aureus as positive control, whereas 1, 1 diphenyl-2picrylhydrazyl (DPPH) and 2-2-azinobis-3ethylbenthiazoline-6-sulphonic acid (ABTS) radical scavenging activity was taken as parameter for antioxidant activity. The results showed that with application of REO, biofilm formation of both L. monocytogenes and S. aureus was inhibited by 73.0 and 77.65 %, respectively in comparison to their respective controls. Six different concentrations of oil was used for determination of ABTS and DPPH radical scavenging activity and it was ranging from 8.1651.80% for DPPH whereas ABTS values ranged from 6.81-44.16% for rosemary oil under investigation. It can be concluded that rosemary essential oil possesses potent antimicrobial, antibiofilm and antioxidant activity, and may be used as a natural preservative to extend the storage stability of meat products.

A key source for high quality nutrients, meat and meat products occupy a prominent position in human food basket (Mehta et al., 2015). One of the prominent limitations is perishability and susceptibility of meat to bacterial spoilage and oxidative degradation (Fung and Toldra, 2010). To counter that, application of synthetic antimicrobials and preservatives in meat products is usually done. However, with increase in consumer awareness about their health and well-being, negative perception towards synthetic antimicrobials and preservatives has led to an extensive search for natural sources. Along with containment of growth of spoilage microbes, the prime consideration is to cease oxidation reactions that often occur during meat processing and storage which further leads to deterioration of quality as detected by off-flavours, decreased nutrient value and health concerns due to accumulation of toxic compounds (Lorenzo et al., 2014). Further, some food related pathogens are known to form stable biofilms in meat processing plants which may be a potential source for contamination that leads to meat spoilage and also allows pathogens to tolerate and withstand hostile and adverse environmental conditions. It is reported to render protection to bacteria against various antimicrobials and sanitizers, thereby increasing resistance in them by at least 1000 times than normal (Miladi et al., 2016).
Utilization of natural antimicrobials and antioxidants can be foreseen as one of the most important strategies for development of functional and healthier meat products. In this context, essential oils and extracts of medicinal herbs and spices have attained value due to the inherent antimicrobial as well as antioxidant properties (Fernandes et al., 2018;Poojary et al., 2017;Putnik et al., 2017). Many studies have explored the application of aromatic phytochemical preparations with two fold functionality against microbial degradation as well as lipid oxidation in meat and meat products, particularly essential oils (Govaris et al., 2005). Essential oils (EOs) are naturally-derived aromatic compounds, with broad range of biological activities (El-Asbahani et al., 2015). Up to now, EOs have been used as flavouring additives in food products but recently they are proving to be potent source for natural antimicrobials in food and beverage products (Burt, 2004).

Rosemary (Rosmarinus officinalis L.) belongs to
Lamiaceae family and chemically it can be classified as one of three chemotypes i.e. cineoliferum (high 1, 8-cineol content), camphoriferum (camphor >20%) and verbenoniferum (verbenone >15%) (Napoli et al., 2010). It has been reported to have many beneficial effects such as antiviral, anti-inflammatory and anti-carcinogenic. The aim of this study was to investigate and evaluate invitro antimicrobial, antibiofilm and antioxidant potential of rosemary essential oil for its possible application in meat products replacing synthetic antimicrobials and antioxidants.

Source of rosemary essential oil
Rosemary essential oil was procured from Kanta Enterprises Pvt. Ltd, Noida, UP, India. As per the certificate of analysis provided with the product, it has specific gravity in range of 0.894 to 0.912 and refractive index in range from 1.46 to 1.48. All the reagents and chemicals used in the study were of analytical grade and procured from reputed firms.  (Presti et al., 2015;Aneja et al., 2011). For anti-bacterial assay, streaking of fresh overnight culture of each pathogen over BHI agar plate was done and the plates were incubated at 37 o C for 16h. Under aseptic conditions, pure isolated colonies was transferred to sterile normal saline (0.85%) solution and the density of each microbial suspension was adjusted equal to 0.5 McFarland standard (Andrews, 2001). Thereafter, 100µl of the inoculum of each test pathogen was spread over pre-solidified MHA (Muller Hinton Agar) plates and 8mm wells were made using a sterile borer after drying. 100 µl of Rosemary essential oil (REO) was poured into each of the wells and in order to accelerate its diffusion in agar, the plates were pre-incubated at 4 o C for 1hour, followed by overnight incubation at 37 o C. The antibacterial activity, indicated by the zone of inhibition (ZOI) surrounding the well containing REO was recorded using zone scale (Hi-Media). All the tests were performed in triplicate, and the mean values of the diameter of inhibition zones were recorded.

Bacterial strains and growth conditions
The Minimum Inhibitory Concentration (MIC) values were determined by method followed by Jiang et al. (2011) with slight modifications. Final concentration of REO (0.5% v/v) was made by dissolving it in sterilized physiological saline solution (0.9% w/v) added with Tween 80. Serial double dilutions of the REO were prepared in a 96-well microtiter plate in the range 0.156% to 4.0% (v/v). The final concentration of each bacterial strain in nutrient broth was adjusted to 10 5 -10 6 CFU/ mL. Each REO dilution (100 μL) was dispensed into the wells of a microtiter plate and each well was then inoculated with 200 μL of the suspension which contained equal amounts of nutrient broth and bacterial culture. The resulting suspensions were mixed with a micro-pipette and microtiter plates were incubated at 37 °C for 24 h. After incubation, the wells were examined for growth of microorganisms and the MICs were determined. The MIC was computed as the lowest concentration of the essential oil at which the microorganism did not demonstrate visible growth.

Antibiofilm activity of Rosemary essential oil: Inhibition of biofilm growth and development and its visualization
Biofilm inhibition was achieved by following method of Galvao et al. (2012). 10 µL of a standardized (~10 6 CFU/ ml) L. monocytogenes and S. aureus culture was aliquoted into each 96-well microtitre plate followed by the addition of 30 µL of REO (1250 ppm), respectively. Following incubation at 37 o C for 24 h, the plates were rinsed three times with phosphate-buffered saline (PBS, pH 7.2) to remove loosely attached cells. The plates were air-dried and then the wells were stained with 250 µL of 0.1% crystal violet and incubated at room temperature for 30 min. After incubation, the plates were washed and then left to dry. Finally, 250 µL of 33% glacial acetic acid was added to solubilize the dye and the OD 570 was recorded using microplate reader. The per cent biofilm inhibition was estimated following the formula: anti-biofilm activity (%) = (Control OD570 nm − Test OD570 nm / Control OD570 nm ) × 100. The positive control was the amount of biofilm formed with a pure culture of L. monocytogenes and S. aureus.

Visualization of biofilms
L. monocytogenes and S. aureus biofilms, with or without treatment with REO, were grown on pre-sterilized glass cover slips (Musthafa et al., 2010). An aliquot of 80 μL/ well (~10 6 CFU/mL) of overnight grown test pathogens were added to 6 well tissue culture plate containing sterile glass cover slips, BHI medium (1680 μL/well) and REO (240 μL/well). The bacterial suspensions were used as controls. Biofilms were stained with crystal violet and the stained biofilms were visualized under light microscope at 40X magnification (Olympus Microscope, USA).

Antioxidant activity of Rosemary essential oil (REO)
Antioxidant activity of REO was evaluated as free radical scavenging activity (RSA). Ability of essential oil to donate hydrogen atoms or electrons was measured from bleaching of coloured methanoloic solution of 1, 1 diphenyl-2picrylhydrazyl (DPPH). More the number of hydroxyl groups, the higher is the possibility of free radical scavenging ability.

1, 1 diphenyl-2picrylhydrazyl radical scavenging activity (DPPH)
Radical scavenging potential of Rosemary essential oil (REO) was assessed using a methanolic solution of the stable free radical i.e. DPPH. The method of Blois (1958) was used in studying the effect of various oil concentrations on DPPH radicals with some modifications. A solution of DPPH (0.15 mmol/L) in methanol was prepared. Oil concentrations (0.125%, 0.25%, 0.5%, 1.0%, 1.25%, 1.5%, 2.0%, and 2.5%) were prepared in methanol and 200 µl of each dilution was mixed with 50 µl of DPPH solution in a 96-well microtitre plate. The mixture was allowed to stand at room temperature in dark for 30 min and the decrease in absorbance at 517 nm was measured. Butylated hydroxyanisole (BHA) was used as positive control. Inhibition of DPPH radical was calculated by the following formula: Where, A 0 and A 1 are the absorbance of control and the sample, respectively. The IC 50 value, which represents the concentrations of the sample required to cause 50% inhibition of DPPH radical, was estimated by linear regression analysis from the obtained RSA values and was expressed in μl of essential oil per ml.

2-2-azinobis-3ethylbenthiazoline-6-sulphonic acid radical scavenging activity (ABTS)
It was determined by method followed by Yang et al., (2010) and Kumar et al., (2017) with slight modifications. ABTS cation decolorization assay was conducted on various concentrations of Rosemary essential oil (REO) viz. 0.125%, 0.25%, 0.5%, 1.0%, 1.25%, 1.5%, 2.0% and 2.5%, made in methanol. 50 µl of various concentrations of REO and 150 µl of ABTS solution were mixed and after 1 min incubation at room temperature, absorbance was measured spectrophotometrically at 732 nm. Methanol was used as blank solution, and the ABTS solution without essential oil served as the control. The cation scavenging activity was measured same as with DPPH and A 0 and A 1 are the absorbance of control and the sample, respectively.

Statistical analysis
Data was analyzed statistically on 'SPSS-16.0' (SPSS Inc., Chicago, II USA) software package as per standard methods (Snedecor and Cochran, 1989). Whole set of experiment was repeated three times and the mean values were reported along with standard error. The statistical significance was estimated at 5% level (p<0.05) and evaluated with Duncan's Multiple Range Test (DMRT).

Antimicrobial activity and MIC of Rosemary essential oil
The results obtained from the well-diffusion assay indicated that REO exerted antibacterial activity against all the tested strains (Fig. 1). The size of the inhibition zone of REO varied from 12.5 to 36.0 mm and maximum inhibition diameter was observed for Staphylococcus aureus (36±0.68) followed by Listeria monocytogenes (26.5±0.50), whereas minimum zone diameters were observed in Salmonella enterica (12.50±0.26) followed by Enterococcus faecalis (12.75±0.41) (Fig. 2). The results of zone inhibition assays corresponded to the MIC values determined by microdilution broth assay. The MIC (ppm) values of rosemary essential oil against all the targeted organisms were found in range of 1250 to 25000 ppm (Fig. 3). A similar trend between MIC values and zone inhibition reveals that Staphylococcus aureus responded maximum to REO. Staphylococcus aureus is a common gram positive organism responsible for food borne illness around the globe. Further, it is a highly changeable pathogen in response to antibiotics and can put human health in jeopardy (Alfatemi et al., 2014). Santoyo et al. (2005) investigated antimicrobial activity of supercritical CO 2 extracted essential oil-rich fractions obtained from Rosmarinus officinalis L. They conducted their studies through disc diffusion and broth dilution methods against six microbial species viz. Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Candida albicans and Aspergillus niger. All components of the essential oil presented antimicrobial activity against tested microorganisms, with inhibition zones and minimal bactericidal concentration values varied from 17 to 33 mm and 2.25 to 0.25 mg/ml, respectively. Similar to our findings, they reported that S. aureus was the most sensitive microorganism, with maximal inhibition zones (27 to 33 mm) and the lowest MBCs (0.75 to 0.25 mg/ml). Fu et al. (2007) studied the antimicrobial activity of the clove (Syzygium aromaticum L.) and rosemary (Rosmarinus officinalis L.) essential oils alone and in combination and found that the MICs of clove oil and rosemary oil ranged from 0.062% to 0.500% (v/v) and 0.125% to 1.000% (v/v), respectively. Further, a synergism in antimicrobial action between these two oils was observed against individual tested microorganisms.
The results obtained in this study for MICs of rosemary essential oils are in accordance with findings of Fu et al. (2007) and Ojeda-Sana et al. (2013)   In the present study, a strong positive correlation between MIC values and zone inhibition diameters was observed. However, little discrepancy of obtained values as compared with previously conducted studies might be due to variation in origin and type of rosemary plant, method of extraction employed, final composition of rosemary essential oil, methodology adopted for evaluation of zone of inhibition and MIC etc. Zaouali et al. (2010) also demonstrated a significant variation in the composition of oils and their biological activity potentials according to varieties and populations of plants. Perusal to results presented in Fig. 2 and 3, tested REO was found to be more effective against gram-positive bacteria than gramnegative. These differences could be attributed due to fact that the cell membrane of hydrophobic compounds. It would have made them inherently resistant to these hydrophobic and lipophilic compounds (Rodriguez-Garcia et al., 2016;Tongnuanchan and Benjakul, 2014;Burt, 2004).

Antibiofilm activity of Rosemary essential oil
Some of the microorganisms like Listeria have a tendency to exist as communities enclosed in matrix, predominantly polysaccharides in nature, known as biofilms. It enables them to exhibit resistance against sanitizers, disinfectants and antimicrobial agents. They can be formed on wide range of surfaces in food processing industry and are of a great concern from safety point of view as bacteria from biofilms can be transferred to food products (Gandhi and Chikindas, 2007). In present study, antibiofilm potential of REO was tested against two organisms viz. L. monocytogenes and S. aureus. On exposure to essential oil, a significant reduction in biofilm formation by bacteria was observed. The results showed that in the presence of REO, biofilm formation of both L. monocytogenes and S. aureus was inhibited by 73.0% and 77.65%, respectively in comparison to their respective controls (Fig. 4). The correlation between anti-microbial efficacy and anti-biofilm activity was observed. Furthermore, with the addition of the REO, L. monocytogenes and S. aureus exhibited a lower degree of cluster cells with poorly developed biofilm architecture than control group. The control slides demonstrated a dense layer of biofilm formation and were easily stained with CV stain. The images clearly differentiated the biofilm architecture between treated and the untreated biofilms in both the tested pathogens. Results of microscopic analysis revealed the maximum degree of reduction in the number of micro colonies at the concentration of 1250 ppm against both the tested pathogens (Fig. 4).

Antioxidant efficacy of Rosemary essential oil
The results for antioxidant activity (DPPH and ABTS) of Rosemary essential oil are presented in Table 1.  (2013) reported a significant positive correlation between chemical composition of rosemary essential oil and its free radical scavenging capacity. Myrcene rich chemotype of rosemary essential oil exhibited better free radical scavenging capacity and IC 50 values ranging from 11 µL/mL and 25 µL/mL. Yang et al. (2010) evaluated the antioxidant activities of major components of six different herb essential oils and found that ABTS radical scavenging activity was 69.5± 2.82% with RC 50 value at 3% concentration of rosemary essential oil. The above evidence from previous studies suggest that some essential oils have potential antioxidant and free radical scavenging activity. Along with proven health benefits, these attributes can be helpful in preventing oxidative rancidity in high fat food products such as meat, thereby extending its storage stability.

CONCLUSION
In vitro antimicrobial, antibiofilm and antioxidant study of Rosemary essential oil reveals that it has shown strong antimicrobial action against common food spoilage microorganism with concurrent broad spectrum of activity against Gram positive organisms, capability to inhibit formation of biofilm and it also possess potential ability to scavenge free radicals. Owing to this action, it can be used as an alternative to the synthetic antimicrobials and antioxidants for control of foodborne pathogens, which can minimize the health risk and side effects. Further, it can be tried as a potential natural preservative in foodstuffs, particularly meat and meat products without affecting the organoleptic quality and can prevent the oxidative rancidity during storage at refrigeration temperature, thereby enhancing the storage stability and shelf life. Even so, there is a need to utilize essential oils for incorporation in food matrices for multifunctional benefits, its in-vivo toxicological studies in animal model systems can be carried out in future.