In vitro synergistic antibacterial activity of Melissa officinalis L . and some preservatives

The aim of this study was to investigate the antibacterial activity of aqueous, ethanol and ethyl acetate extracts of the species Melissa officinalis L. and their in vitro synergistic action with preservatives, namely: sodium nitrite, sodium benzoate and potassium sorbate against selected food spoiling bacteria, for a potential use in food industry. Synergistic action was noticed in almost every combination between plant extracts and preservatives. This work showed that the active compounds from ethanol, ethyl acetate and aqueous extracts of Melissa officinalis L. significantly enhanced the effectiveness of tested preservatives. Synergism was established at plant extract and preservative concentrations corresponding to 1/4 and 1/8 minimal inhibitory concentration values, which indicated the possibility of avoiding the use of higher concentrations of tested preservatives. Additional key words: minimal inhibitory concentration, plant extracts, synergism.


Introduction
Melissa officinalis L. (fam.Lamiaceae) is a perennial, aromatic herb native to southern Europe.Due to intense lemon aroma and flavor of leaves, M. officinalis is used widely in food and cosmetics (Enjalbert et al., 1983).It can be used as a herbal tea for its aromatic, digestive and antispasmodic properties in gastrointestinal disorders (Auf 'mkolk et al., 1985).Beneficial effects of ethanol, ethyl acetate and aqueous extracts of M. officinalis are attributed mainly to the presence of the phenol compounds (rosemary, protocatecuic, coffee acid and their methyl esters) and are related to their antioxidant activity (Hener et al., 1995;Tagashira et al., 1995;Canadanovic ´ Brunet et al., 2008).
Various medicinal properties have been attributed to principal constituents.Rosemary acid, a derivative of coffee acid, is the most abundant component of the El objetivo de este estudio fue investigar, para un uso potencial en la industria alimentaria, la actividad antibacteriana de extractos en agua, en etanol y en acetato de etilo de Melissa officinalis L. y su acción sinergística in vitro con los conservantes nitrito sódico, benzoato sódico y sorbato potásico sobre una selección de bacterias que dañan los alimentos.Se detectó una acción sinergística en casi todas las combinaciones entre los extractos de las plantas y los conservantes.Este trabajo muestra que los componentes activos de los extractos en etanol, acetato de etilo y agua de Melissa officinalis L. elevaron significativamente la eficacia de los conservantes analizados.El sinergismo fue establecido a concentraciones de los extractos de plantas y de los conservantes correspondientes a 1/4 y 1/8 de los valores de concentración inhibitoria mínima, lo que indica que se puede evitar utilizar concentraciones más elevadas de estos conservantes.
There is a major drive towards natural rather than chemical or synthetic additives in food, as they are perceived by consumers to be safe and better (Bagamboula et al., 2003).The traditional herbal medicines offer a novel source of antioxidants for the food industry, for boosting both the shelf-life and nutritional content of food (Hinneburg et al., 2006).Use of plant extracts as natural preservatives have been especially highlighted since The European Food Safety Authority (EFSA, 2008) said the rosemary extract is safe for use as an antioxidant in food.Essential oils are becoming increasingly popular as natural antimicrobial agents to be used for food preservation (Tzortzakis and Economakis, 2007;Pazos et al., 2008).Potential use of M. officinalis as natural antimicrobial agents have been less explored (Cosentino et al., 2003).
Different kinds of preservatives are used to prevent bio-deterioration of food products.In our test we choose three, commonly used in food industry: sodium benzoate, sodium nitrite, potassium sorbate.Sodium benzoate has proved a controversial additive, as recent studies have highlighted health concerns from its use (Haws et al., 2007;McCann et al., 2007).Also the commonly used preservative sodium nitrite has been under the spotlight since 2007 (Jiang et al., 2007).
Therefore, the aim of this work was to establish antibacterial activity of M. officinalis grown up in Serbia and to estimate the eff iciency of combined action of the plant extracts and preservatives (sodium benzoate, sodium nitrite and potassium sorbate) against selected food spoilers and thereby expand the possibilities for more effective and safe preservation of food.

Plant material
Melissa officinalis L. was collected during the summer of 2006 on Mt.Suvobor (Serbia).Identification and classification of the plant material was performed at the Faculty of Science of the University of Kragujevac.The voucher specimen of the plant is deposited in the herbarium of the Faculty of Science.

Preparation of the plant extracts
The aqueous extracts were obtained by cooking dry ground plant material (leaves) (50 g for each solvent) in a water bath at 80°C.The ethanol and ethyl acetate extracts were obtained in a Soxhlet apparatus.Following f iltration, the aqueous extract was evaporated in a water bath, while the ethanol and ethyl acetate extracts were evaporated under a vacuum at 40°C.Solutions of different concentrations of all dried extracts were resuspended in 5% DMSO.

Preparation of preservatives
The preservatives used in the experiment were as follows: sodium benzoate (C Product, Belgrade, 2007); sodium nitrite (Biochemistry Laboratory, Faculty of Science, University of Kragujevac); and potassium sorbate (C Product, Belgrade, 2007).Different concentrations of preservatives were created by dissolving them in liquid Mueller-Hinton broth (Torlak, Belgrade).Before testing, preservatives were heat-treated at 80°C for 15 min.

The minimal inhibitory concentrations
The MIC was determined by the tube macro-dilution method (NCCLS, 1997).Solution of each extract was serially diluted twofold in Mueller-Hinton broth so the final concentrations of the extracts in the medium were ranged from 40 to 0.15 mg mL -1 .Initial inoculants were prepared by suspending growth in a sterile saline and turbidity was adjusted to yield 0.5 McFarland standard and then diluted to 1:10 ratio.Prepared inoculum (0.1 mL) was added into each tube to obtain the final turbidity (approximately 10 4 colony-forming units (CFU) mL -1 ).
The MIC was defined as the lowest concentration of the plant extract at which visible growth is inhibited.The test tubes were incubated at 24°C/ 24 h.Each test included control, consisting of the substrate with the solvent.MIC of preservatives was determined the same way, final concentrations of the preservatives ranged from 10 to 0.03 mg mL -1 .

Synergism
The synergism between water, ethanol and ethyl acetate extracts with chosen preservatives was assessed by the checkerboard assay method (Satish et al., 2005).The following combinations were tested: ethanol extract with sodium benzoate, sodium nitrite, potassium sorbate, aqueous extract with sodium benzoate, sodium nitrite, potassium sorbate and ethyl acetate extract with sodium benzoate, sodium nitrite, and potassium sorbate.From the first to the sixth horizontal column, each plant extract of the combination was doubly diluted in Mueller-Hinton broth (MIC value of 32), while each of the tested preservatives of the combination was double diluted (MIC values of up to 32) and added in a quantity of 0.1 mL from the first to sixth vertical row.The MIC was defined as the lowest concentration of the plant extract at which visible growth is inhibited.The synergism between plant extracts and preservatives was determined by calculating the fractional inhibitory index (FIC) according to the formula: ΣFIC = FIC A + FIC B = [A]/MIC A + [B]/MIC B , where FIC A is the MIC of drug A in the combination/ MIC of drug A alone, and FIC B is the MIC of drug B in the combination/ MIC of drug B alone.Types of effects were classified as follows: FIC ≤ 0.5, synergism; FIC 0.5-1, additive effect; FIC 1-4, indifferent effect; and FIC > 4, antagonism (Berenbaum, 1981).

Results
Antibacterial activity of aqueous, ethanol and ethyl acetate extract of M. officinalis was quantitatively estimated by MIC values and results are represented in Table 1.The MIC varied, depending on the type and concentration of plant extracts and taxonomic characteristics of the species of microorganism tested.All tested extracts exerted antibacterial effects.
The strongest antibacterial activity was exhibited by ethanol extract.MIC was 5 mg mL -1 on all tested bacterial species except S. aureus (MIC = 10 mg mL -1 ), Proteus sp. and E. coli (MIC = 40 mg mL -1 ).The MIC of ethyl acetate extract on all tested bacterial species was 10 mg mL -1 , while MIC of aqueous extract fluctuated, depending on tested bacterial species from 5 to 20 mg mL -1 (Table 1).The most resistant bacterial species to all tested plant extracts and preservatives were E. coli and Proteus sp.
The MIC values of tested preservatives ranged from 5 to 10 mg mL -1 for sodium benzoate, from 0.5 to 2 mg mL -1 for sodium nitrite and from 5 to 10 mg mL -1 for potassium sorbate (Table 1).
Synergism was established in almost every combination of plant extract with preservatives.By the checkerboard method combinations (ethanol extract/ sodium benzoate, ethanol extract/sodium nitrite and  3).
Aqueous extract manifested synergism with sodium nitrite and potassium sorbate.Synergism was established in relation to B. mycoides, B. subtilis and P. fluorescens.FIC range was 0.375 to 0.50.MIC of aqueous extract, reduced to 1/4, while MICs of sodium nitrite and potassium sorbate were respectively reduced to 1/4 and 1/8 (Table 4).
Apart from synergism, other types of interaction between plant extracts and preservatives noticed in this study as deduced from calculation of the FIC index were additive and indifferent effects, as represented in Table 5.

Discussion
The antibacterial activity of M. officinalis has been reported in previous investigations (Friedman Henika et al., 2004;Mimica Djukic et al., 2004).In this study, the strongest antibacterial activity was manifested by the ethanol extract.Earlier works showed that the ethanol extract of M. officinalis can possess an antioxidant (Yanishlieva and Marinova, 2006) and antinociceptive effect (Guginski et al., 2009).
The strongest antibacterial activity among preservatives was exhibited by sodium nitrite.
Canadanovic ´ Brunet et al. (2008) tested different kinds of M. officinalis extracts.They showed that M. officinalis can be used as a source of natural antioxidants and as a possible food supplement.The preservative effect of M. officinalis was also showed in the work by Akarpat et al. (2008).Due to the results of this test, for the significant antibacterial activity of  M. officinalis, along with recent studies which have highlighted health concerns about the use of artificial preservatives, we tested the potential synergism between M. officinalis and the preservatives.The synergistic effect, by checkerboard method of M. officinalis, was tested in an earlier study (Gutiérrez et al., 2008) but with other plant extracts.To our knowledge, there are no studies that tested synergism M. officinalis with preservatives.
We established a synergism for the combination of all three extracts of M. officinalis with all the tested preservatives.When tested alone or in combination with preservatives, the ethanol extract was most effective.It showed synergism in relation to seven bacterial species and with all the tested preservatives.The MIC values of the three preservatives in combination with ethanol extract were reduced up to four times.Comparing to ethanol, ethyl acetate and aqueous extracts were less effective.They exerted synergy in relation to less bacterial species then the ethanol extract and the MIC value of one preservative in combination was reduced four times (sodium benzoate in case of ethyl acetate and potassium sorbate in case of aqueous extract).
The most sensitive on combinations of tested agents were B. subtilis and B. mycoides, common food spoilage bacterial species.In relation to these species the synergism was established in the combination of all the three plants extract with almost every tested preservative.
Escherichia coli showed some resistance to plant extracts when they were tested separately, but in combination: ethanol extract/sodium benzoate, ethanol extract/sodium nitrite and ethyl acetate/sodium benzoate manifested sensitivity.For these combinations synergism was detected.
According to the FIC index Proteus sp. and S. aureus exerted the greatest resistance to all combinations plant extract/preservative.No synergism was detected.
The combination of ethanol, ethyl acetate and aqueous extracts respectively with sodium nitrite, sodium benzoate, potassium sorbate exhibited synergism by inhibiting the growth of significant number of bacterial species at a lower concentration than when each agent was assayed separately.
The combination of ethanol, ethyl acetate and aqueous extracts with sodium nitrite, sodium benzoate and potassium sorbate inhibited the growth of significant number of bacteria species at a lower concentration  than when the single agents were assayed separately.
The synergism was recorded at 1/4 and 1/8 MIC values of preservatives which indicated the possibility of avoiding the use of higher concentrations of tested preservatives that could lead to accumulation of toxic products in conserved food.The compounds of ethanol, ethyl acetate and aqueous extract did not decrease the activity of preservatives because antagonism was not indicated.
The results obtained herein, confirmed that Melissa officinalis L. possess antimicrobial activity and according to exhibited synergism with sodium benzoate, potassium sorbate and sodium nitrite, suggest that it may be used in biotechnological f ields as natural preservative ingredients in food.

Table 1 .
Minimal inhibitory concentration (mg mL -1 ) values of plant extracts and preservatives

Table 4 .
Fractional inhibitory index (FIC) showing synergism for combination aqueous extract/preservatives