ANTIMICROBIAL ACTIVITY OF COMPOSITE CHITOSAN BIOFILMS WITH BEESWAX AND CARAWAY ESSENTIAL OIL

In this paper, influence of increasing concentrations of beeswax on the antimicrobial activity of chitosan-caraway essential oil biofilm was investigated. Selected microorganisms for the test were Salmonella Typhimurium and Listeria monocytogenes. Reduction of S. Typhimurium and L. monocythogenes viable counts in bacterial suspension in the presence of chitosan films with or without added beeswax amounted over 80 % after 24 h contact time, and over 95 % after 3 h of contact time, respectively and influence of added beeswax on growth reduction was not detected. However, when number of S.Typhimurium viable cells was analyzed (log CFU/ml), it was shown that addition of beeswax led to increased activity of films, especially for the films with 36 kg/m and 54 kg/mof beeswax. When films were transferred, after 24 h in bacterial suspension, onto Petri dishes with nutrient agar and incubated for 24 h on 37 °C, results also showed beeswax contribution to growth inhibition of S.Typhimurium.


INTRODUCTION
Growing consumer demand for food without chemical preservatives, directed great amount of research towards finding natural alternative substances that could be used for food preservation (Popović et al., 2015).Natural biopolymer chitin and its derivatives showed pronounced antimicrobial activity followed with other positive properties, which all led to extensive research in widening these biopolimers field of application (Chi, 2004;Hromiš et al., 2014).
Chitosan in solid form, in the form of fibers, films, membranes, hydrogels, microspheres and nanoparticles comes only in the surface contact with substrate and its antimicrobial activity depends on surface properties like hydrophilicity, zeta potential and morphology: particle size and shape, membrane thickness (Kong et al., 2010;Kong et al., 2008;Sadeghi et al., 2008;Takahashia et al., 2008).In order to intensify antimicrobial activity of chitosan films, different antimicrobial compounds are being added to chitosan film.This addition is important because film can manifest its antimicrobial activity only in direct contact with a substrate surface.In order to achieve antimicrobial activity that protects mass of the substrate it is necessary to have antimicrobial compound able to migrate into the substrate.Accordingly, low molecular weight active compounds are added into the chitosan matrix, being subsequently released to migrate into the substrate (Altiok et al., 2010;Coma et al., 2002;Pranoto et al., 2005).Plant essential oils and extracts can be considered as natural alternative in food protection and their use is in accordance with consumer tendency towards natural, minimally chemically treated food (Burt, 2004;Kocić-Tanackov et al., 2013).Two basic limitations in essential oil usage in food industry are pronounced organoleptic properties and loss of activity due to interactions with food components (Gutierrez et al., 2008;Rojas-Graü et al., 2009).
Caraway essential oil and its basic compounds, carvone and limonene showed antimicrobial activity towards different moulds and bacteria (Aggarwal et al., 2002;Dimić et al., 2009;Simić et al., 2008).Literature data about caraway essential oil antimicrobial activity vary, from pronounced activity over moderate to literature data of lack of activity towards most Gram positive and Gram negative bacteria (Alboofetileh et al., 2014;Cvijović et al., 2010;Mohamed et al., 2013).
In paper which analyzed methanol and ethanol extract of beeswax influence on the growth of five pathogenic bacterial species, three species of microscopic fungi and seven species of yeasts, activity of medium intensity was observed (Kacániová et al., 2012).In paper where effect of coating based on chitosan, beeswax and lime essential oil on E. coli DH5a and R. stolonifer "in vitro" and "in vivo" was analysed, it was shown that in both experiments, "in vitro" and "in vivo", coating based on chitosan and beeswax totaly inhibited bacterial growth (Ramos-García et al., 2012).Also, Al-Waili (2005) showed moderate growth of S. aureus on the nutrient agar with certain content of beeswax.
In this paper, influence of increasing concentrations of beeswax on the antimicrobial activity of composite biofilm based on chitosan with added caraway essential oil was investigated.Selected microorganisms for the test were two pathogenic bacteria, Salmonella Typhimurium as Gram-negative and Listeria monocytogenes as Gram-positive bacteria.

Material
Commercial chitosan powder from crab shells, highly viscous (deacetylation degree approximately 80 %) was purchased from Sigma-Aldrich Chemical Co.(St.Louis, Missouri, USA).Caraway essential oil was purchased from the manufacturer: Herba doo (Belgrade, Serbia) and beeswax was obtained from a local beekeeper.Other used chemicals were of analytical grade.

Film preparation
Chitosan film forming solution was prepared by dissolving chitosan powder in acetic acid (1 % volume concentration) to reach chitosan mass per volume ratio of 10 kg/m 3 .Solution was left stirring over night on a magnetic stirrer to dissolve chitosan.Caraway essential oil in volume concentration of 1 % and wetting agent Tween 20 (50 % of essential oil volume) were then added, solution was heated in a water bath to 60 °C and stirred with a laboratory stirrer to obtain white homogenous emulsion.In the same water bath, beeswax was melted in 30 mL of deionized water heated to 60 °C.Hot film forming emulsion was added to molten wax and six new different film forming emulsions with beeswax mass per volume ratio of: 0, 18, 36, 54, 72 and 90 kg/m 3 were obtained.Emulsions were casted on Petri dishes covered with teflon coating and left to air dry (23±2 °C, 50 ±10 % RH) on a horizontal table surface.Films were labeled according to beeswax mass per volume ratio, as 0, 18, 36, 54, 72 and 90, while 0 (C) was label used for the film without beeswax or caraway essential oil (Table 1).

Antimicrobial activity
Activity was tested according to the ASTM E2149 (2001) method, which is a quantitative antimicrobial test method, performed under dynamic contact conditions.Gram-positive (Listeria monocytogenes) and Gram-negative bacteria (Salmonella Typhimurium) were used as test organisms.Fresh inoculants for antibacterial assessment were prepared on nutrient agar (Merck, Germany) at 37 ˚C for 24 h.The incubated test culture was diluted using a sterilized 3 mM phosphate buffer (KH 2 PO4; pH 6.8) to give a final concentration of 1.5-3×10 5 colony forming units (CFU)/mL.This solution was used as a working bacterial dilution.
Before analyzing antimicrobial activity of films, cell viability of test organisms was determined in set experimental conditions.Cell viability determination in phosphate buffer showed no significant effect on S. Typhimurium cells viability, because there was no decrease or increase in viable cell number during 24 h.However, considerable decrease in L.monocytogenes cell number was recorded after 24 h.This is why film antibacterial effect against L.monocytogenes was tested only after 3 h (Fig. 1).

Fig. 1. Cell viability of test organisms in phosphate buffer after 3 h and 24 h
Film samples were treated with UV lamp (Kruss, Germany): 254 nm, ½ h for one side of film, cut into test pieces, which had contact surface area of 58 cm 2 and transferred to a 250 mL Erlenmeyer flask containing 50 mL of the working bacterial dilution.All flasks were capped loosely and shaken for 24 h at room temperature and 120 rpm using a Wrist Action incubator shaker.At different contact periods: 3 h and 24 h, 1 mL of the bacterial dilution was withdrawn, serial diluted and plated in Trypton soya agar (Merck, Germany).The inoculated plates were incubated at 37 °C for 24 h, and colonies were counted.The average values of the duplicates were converted to CFU/mL in the flasks by multiplying with the dilution factor.The antimicrobial activity was expressed as R % reduction of the organism after contact with the test specimen compared to the number of microorganisms cells surviving after contact with the control (Eq.1): Reduction, % (CFU/mL) = (B−A)

B
× 100 where A represents CFU per mL for the flask containing the treated substrate after the specified contact time and B represents "0" contact time CFU per mL for the flask used to determine "A" before the addition of the treated substrate.Reduction (%) was calculated using average of 4 replicates for A and B.
After 24 h in bacterial solution, in additional test, films were transferred from bacterial suspension onto Petri dishes with nutrient agar and incubated for 24 h on 37 °C to obtain further insight in antimicrobial activity of the films.Statistical analysis Statistical analysis was carried out using OriginPro 8 (OriginLab Corporation, Northampton, MA, USA).All data were presented as mean value with their standard deviation indicated (mean ± SD).Variance analysis (ANOVA) was performed, with a confidence interval of 95 % (p < 0.05).Means were compared by the Tukey test.

RESULTS AND DISCUSSION
Reduction of S. Typhimurium viable counts in bacterial suspension in the presence of chitosan film, chitosan film with caraway essential oil, as well as in the presence of films with added beeswax is shown in Fig. 2. For chitosan film without added caraway essential oil and beeswax, 0(C), bacterial growth reduction of 26.96 % (after 3 h contact) and 63.97 % (after 24 h contact) was observed.Considering that in this experiment we tested chitosan in solid form of the film, only surface of the film could come into contact with bacterial suspension (Kong et al., 2010;Kong et al., 2008;Lagaron et al., 2007;Sadeghi et al., 2008;Takahashia et al., 2008).In addition, test was performed in neutral pH 6.8, where ratio of charged amino groups on C-2 chitosan atom is low.This could be one of the reasons for lower antimicrobial activity (Kong et al., 2010).When activity of chitosan film with caraway essential oil against S. Typhimurium growth is analyzed, it can be seen that activity increases as the contact time increases, reaching about 80 % after 24 h.When beeswax was added to the film, no significant change could be observed in the film activity (P>0.05),although for films with beeswax concentrations 36, 54 and 72 kg/m 3 tested antibacterial activity reached and exceeded 90 %.For the film with highest amount of beeswax low activity was detected.It is possible that high amount of beeswax interacted with other lipophilic compound, caraway oil, and as a result of interaction oil release from the film was slowed or blocked.

Fig. 2 . Reduction (%) of viable cell number after 3 h and 24 h of film contact time in S. Typhimurium bacterial suspension
If inhibition of S.Typhimurium growth in the presence of tested composite films is presented as number of viable cells (log CFU/ml) after selected contact periods of 3 h and 24 h (Fig 3), instead of reduction (%) (Fig. 2), some differences between films with and without beeswax can be observed, especially after longer contact period.Similar to the results for reduction, chitosan film without added beeswax or caraway essential oil, 0 (C) did not show decrease in viable cell number.After 24 h contact with chitosan film with caraway essential oil, but without beeswax, CFU/ml decreased for about 0.7 log units.Addition of beeswax led to increased activity of films, especially for the films with 36, 54 and 72 kg/m 3 of beeswax, where inhibition was 1.9 log cycle, 1.3 log cycle and 1.6 log cycle.

Fig. 3. Number of viable cells, log CFU/ml after after 3 h and 24 h of film contact time in S. Typhimurium bacterial suspension
After 24 h in bacterial suspension, films were transferred onto Petri dishes with nutrient agar and incubated for 24 h on 37 °C.Results are presented in Fig. 4. For the film 0 (C), bacterial growth was observed both in contact with the film and in film surrounding.For all the films with added beeswax, bacterial growth was observed in surrounding of films, but there was no growth observed in direct contact with films.Effect of increasing concentration of beeswax on the growth inhibition of S.Typhimurium was not clearly noticeable.Lack of bacterial growth in direct contact with films with added beeswax may be due to bacteriostatic effect of films.On the other hand, mechanism of activity of film without beeswax is not clearly bactericidal or bacteriostatic, due to observed growth of bacteria in contact with the film, as well as in the film surrounding.In contact with chitosan film with added caraway essential oil, reduction was above 90 % and similar reduction (P>0.05) was observed in all films with added beeswax, except for the film with highest concentration of beeswax, 90 kg/m 3 , similar to the results obtained for S.Typhimurium.

Fig. 5. Reduction (%) of viable cell number after 3 h of film contact time in L.monocytogenes bacterial suspension
Inhibition of L.monocytogenes in the presence of tested composite films is presented in Fig. 6, as number of viable cells (log CFU/ml) after contact period of 3 h.No contribution of added beeswax to film activity against L.monocytogenes was observed, but test could not be performed after 24 h of contact time, where possibly different results might be obtained, as in the case of S.Typhimurium.

CONCLUSION
Chitosan-caraway essential oil film resulted in over 80 % inhibition of S.Typhimurium after 24 h contact period and over 90 % of inhibition of L. monocythogenes after 3 h of contact period, compared to no or 20-30 % reduction of bacterial growth by the chitosan film.However, contribution of beeswax to antimicrobial activity of composite film was also detected after longer contact period in S.Typhimurium suspension (1 log cycle lower CFU/ml after 24 h of contact period).Moreover, for the film without beeswax, bacterial growth was observed in contact with the film, as well as in the film surounding after transfering film from bacterial suspension into nutrient agar and incubation, while for films with added beeswax, absence of bacterial growth in direct contact with films was detected.Due to reduction in cell viability of L. monocytogenes in experimental conditions after 24 h, only results after 3 h of contact period could be tested and in these conditions no contribution of beeswax to antilisterial activity of films could be detected.

Fig. 4 .
Fig. 4. Images of S.Typhimurium bacterial growth on nutrient agar after film transfer from bacterial suspensionDue to reduction in cell viability of L. monocytogenes in experimental conditions after 24 h, only results after 3 h of contact period with films are presented in Fig.6.It can be

Fig. 6 .
Fig. 6.Number of viable cells, log CFU/ml after 3 of film contact time in L.monocytogenes bacterial suspension

Table 1 .
Composition of films