Antimicrobial activity of Satureja Khuzestanica Jamzad and Satureja bachtiarica Bunge essential oils against Shigella flexneri and Escherichia coli in table cream containing Lactobacillus plantarum LU5

Marzeh khuzestani and Marzeh bakhtiari EOs were added in probiotic table creams. Marzeh khuzestani EO exhibited the highest antibacterial/bactericidal activity. The combination of EOs had a synergism effect to inhibit pathogenic bacteria. Table creams containing different mixtures of EOs showed high total acceptability.

based and naturally occurring antimicrobials with a high potential biological activity. The antimicrobial effects of EOs in order to control the food spoilage have been reported in many researches (Hashemi, Niakousari, Saharkhiz, & Eskandari, 2011;Mohammadi & Aminifard, 2013;Olmedo, Nepote, & Grosso, 2013;Sumalan, Alexa, & Poiana, 2013;Tian et al., 2012). Recently, the demand for potential natural food preservatives with a broad spectrum of anti-oxidant and antimicrobial activities to enhance the shelf life of perishable foods is increasing (Fratianni et al., 2010). Marzeh Khuzestani (Satureja Khuzestanica Jamzad) is an aromatic endemic plant growing wildly in the southern-west side of Iran with a wide application as a traditional medicine due to its antiseptic and sedative properties (Hashemi et al., 2012;Siavash Saei-Dehkordi, Fallah, Heidari-Nasirabadi, & Moradi, 2012). Marzeh Khuzestani EO is rich in natural monoterpenoid and carvacrol with diverse biological activities such as antimicrobial, antitumor, analgesic, anti-inflammatory, anti-parasitic, anti-hepatotoxic, and hepatoprotective activities which makes it an excellent antimicrobial agent in food industry (Can Baser, 2008).
In the term of food preservation, natural preservatives such as EOs are more suitable than chemical preservatives but the dosage and the costs for natural preservatives limit their application in food industry (Ju et al., 2019). Hence, evaluation of synergistic effect of combined EOs may be an effective way to solve this issue (Ju et al., 2020). Kong et al. (2016) reported a synergistic effect against Fusarium solani from combination thymol and salicylic acid which it was more than two-fold higher than when it used alone.
The synergistic effect of thymol and carvacol against Pseudomonas aeruginosa and Staphylococcus aureus has been reported by Lambert, Skandamis, Coote, and Nychas (2001). Ju et al. (2020) observed that the combination of eugenol and citral significantly improved the antifungal effects (3.4-fold) against the main bread spoilage fungi (Penicillium roqueforti and Aspergillus niger) compared to each agent used separately.
Lactic acid bacterias (LABs) are one of the major groups of probiotics and they are widely used in the production of dairy products (Hashemi & Gholamhosseinpour, 2019). Some species of LABs, such as Lactobacillus plantarum, are commonly isolated from dairy products as well as fruits and vegetables and their role as probiotics in human health has been confirmed (Amin, Jorfi, Khosravi, Samarbafzadeh, & Farajzadeh Sheikh, 2009). In the case of the addition EOs into probiotic food, it is necessary that EOs exhibit a low inhibitory effect on the probiotic culture with an acceptable inhibitory activity on the pathogens. Table or coffee cream is an extremely viscous product contains 18% milk fat and can be fermented by the defined strains of LAB. This cream can be used as condiment along with the snacks or vegetables or also can be used as an ingredient in sauces or dressings (Tamime, 2009

| Plant materials and essential oil extraction
Marzeh khuzestani (Satureja khuzestanica) and Marzeh bakhtiari (Satureja bachtiarica Bunge) with the moisture content of 13% (dry basis) were purchased from a local market in Kohgiluyeh and Boyer-Ahmad province, Iran and kept at 4°C prior extraction. For essential oil extraction, hydrodistillation of 35 g of each dried plant in 500 ml distilled water was applied for about 3.5 hr by an all-glass Clevengertype apparatus (British Pharmacopoeia, 1980).

| Minimal inhibitory concentration (MIC) and Minimal bactericidal concentration (MBC)
Briefly, inoculums were prepared at 10 6 CFU/ml and concentration range of each essential oil was adjusted at 0.8-100 µg/ml (dimethyl sulfoxide as solvent). Consequently, 95 µl of nutrient broth or MRS broth, 5 µl of each microbial inoculum, and 100 µl of each essential oil concentration were added to each well of the 96-well plates. Afterward, incubation was performed at 37°C for 24 hr. For measurement of MBC, 100 μl of the microbial suspensions from MIC was cultured in nutrient agar and the lowest concentration with no growth was determined as MBC (Clemente, Aznar, & Nerín, 2019).

| Fractional inhibitory concentration (FIC) and fractional bactericidal concentration (FBC)
Briefly, the different microorganism's suspensions in PBS, 20 μl of each EO concentration based on the MIC values and 1,760 μl of yeast extract were prepared. Afterward, the incubation was done at 37°C for 24 hr. The combination effect of both essential oils based on FIC value was reported as: antagonism, indifferent, additive, and synergy according to the method of Hyldgaard, Mygind, and Meyer (2012). For determination of the minimal bactericidal combination of essential oils, a method suggested by Mosquera, Sharp, Moore, Warn, and Denning (2002) was carried out to measure the FBC indicator. The following formula used for calculation of FIC and FBC; The antimicrobial interaction was calculated as FIC or FBC ≤ 0.5: synergic effect (where the combined antibacterial activity is greater than the sum of activity of both EOs when used separately); 0.5 < FIC or FBC ≤ 1: additive effect (where the combined antimicrobial activity is equal to sum of the activity of EOs acting jointly); 1 < FIC or FBC ≤ 4: no interactive effect; FIC or FBC > 4: antagonistic effect (where the combined antibacterial activity is lower than the sum of activity of both EOs used separately).

| Combinatorial agar diffusion assays
Both essential oils were mixed at 25, 50, and 75% (v/v) concentration, and agar diffusion test was carried out for 100 μl of each bacterial suspension (10 6 CFU/ml). Subsequently, 10 μl of each essential oil mixture was added to sterile filter disk (10 mm diameter) and placed on the nutrient agar or MRS + nutrient agar medium. After that, plates were incubated at 37°C for 24 hr (Hashemi et al., 2018).

| Antibacterial activity of essential oils in table cream
The antibacterial activity of mixture of essential oils was measured on table cream. Table cream was purchased from Pegah Dairy Company and autoclaved for 15 min. After cooling, table cream was inoculated with 9.1-9.3 log CFU/g of each target strain separately and the mixture of essential oils (1 μl) was added to the samples.
Samples were placed in polystyrene plastic tray covered by a lid and sealed with parafilm. Then, samples were kept at 4°C for 10 days and the count of each microorganism was detected during storage.
For microbial determination, each sample was blended with peptone water in a stomacher (BagMixer 400 W, Interscience Co.) and following serial dilution; plating was carried out onto suitable medium.

| Sensory evaluation
Sensory assessment of table cream supplemented with essential oils was done by using a twelve-member (six male and six female) semitrained panel. The panelists scored the overall acceptability aspects by using a 9-point hedonic scale, where 1 = unacceptable and 9 = very acceptable, whereas the limit of acceptability was 6 (Hashemi, Amininezhad, Shirzadinezhad, Farahani, & Yousefabad, 2016).

| Statistical analysis
Statistical analyses were performed with ANOVA and significant differences at p < .05 were carried out by Duncan's multiple range tests using SPSS package program (Version 22, SPSS Inc.).

| Minimal inhibitory concentration (MIC) and Minimal bactericidal concentration (MBC) of EOs
The antimicrobial activity of Marzeh khuzestani and Marzeh bakhtiari EOs was determined against L. plantarum as a common food probiotic strain and against S. flexneri and E. coli as food-borne bacteria. The MIC and MBC obtained are presented in  Although a higher concentration of EOs required to exhibit bactericidal activity (MBC) than MIC, a similar pattern observed for the MBC of the EOs and it ranged from 6.25-25 and 12.5-100 for the Marzeh khuzestani and Marzeh bakhtiari, respectively. A higher bactericidal activity observed for the Marzeh khuzestani EO and S. flexneri was the most sensitive strain.

| Fractional inhibitory concentration (FIC) and fractional bactericidal concentration (FBC)
The combination effect of the Marzeh khuzestani and Marzeh bakhtiari EOs evaluated, and the fractional inhibitory effects (FIC) and fractional bactericidal concentration (FBC) against the selected bacteria are presented in Table 3.

| Combinatorial agar diffusion assays
Three different combinations of Marzeh khuzestani:Marzeh bakhtiari (1:1, 1:2, and 2:1) were prepared, and their antimicrobial inhibition zone against selected bacteria is presented in Table 4 coli cells up to 1.9 and 2.3 log CFU/g by the end of storage time.
Govaris, Botsoglou, Sergelidis, and Chatzopoulou (2011) reported that oregano and thymol EOs showed strong antimicrobial activity against the L. monocytogenes and E. coli in feta cheese.

| Sensory analysis
Sensory evaluation scores of the creams treated with different combination ratios of Marzeh khuzestani and Marzeh bakhtiari during refrigerated storage at 4°C are shown in Figure 4. Results showed that by increase in the EOs addition in the cream, a slight decrease in overall acceptability was observed. So that, the lowest acceptability

| CON CLUS ION
Marzeh khuzestani and Marzeh bakhtiari EOs were taken to evaluate their antibacterial efficiency alone or in combination against L.
plantarum, S. flexneri, and E. coli. GC-MS analysis showed that the major compounds in Marzeh khuzestani EO were carvacrol, while thymol, carvacrol, borneol, and linalool were the major compounds  properties of the product.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.

I N FO R M E D CO N S E NT
Written informed consent was obtained from all study participants.