CONTRIBUTION TO THE KNOWLEDGE ABOUT THE PRESENCE AND ROLE OF ENTEROBACTER GERGOVIAE IN SENSORY CHARACTERISTICS OF DAIRY PRODUCTS

Enterobacter gergoviae KGPMF 20 was found in traditionally made cheese from Sokobanja (South-eastern Serbia). In this paper, the characteristics of the species were evaluated by investigation of adhesion to diff erent solvents and co-aggregation ability with other species. Moreover, its enzymatic activity was evaluated by using spectrophotometric method, with the intention to detect the role of the isolate in the sensory characteristic of cheese. Th e results of enzymatic activity indicated that E. gergoviae KGPMF 20 has low, almost no enzymatic activity. It could be concluded that this isolate did not aff ect the sensory characteristic of cheese.


INTRODUCTION
Cheese from Sokobanja (Southeastern Serbia) is made from unpasteurized cow milk. Since potentially pathogenic bacteria are not eliminated by high temperatures during the pasteurization process, they may be found in cheese. Enterobacter gergoviae is a member of the Enterobacteriaceae family and it was isolated from the cheese originating from Sokobanja (Mladenovic et al., 2018). Enterobacter gergoviae can be isolated from fresh cow's milk (Wahyuni and Budiarso, 2009), and from traditional cheese as well (Ethiopian cottage cheese) (Melkamsew et al., 2012).
Based on its occurrence in dairy products, the objective of this study was to investigate E. gergoviae KGPMG 20 isolated from traditionally produced cheese from Sokobanja (Serbia). Th e ability of adhesion in the presence of solvents, co-aggregation ability with Enterococcus faecalis KGPMF 49, and the potential to produce extracellular enzymes were examined.

MATERIALS AND METHODS
Th e tested cheese was produced in a traditional way, in countryside households around Sokobanja (Southeastern Serbia). Th e methods of production, sampling, and analyses of chemical characteristics of cheese were described in Mladenović et al. (2018). E. gergoviae species KGPMG 20 was found in cheese. Biochemical characteristics and identifi cation of the species were described in Mladenović et al. (2018).

Th e adhesion to solvents
Th e adhesion to solvents of E. gergoviae KGPMG 20 was measured in accordance with the method described in Rosenberg (1980), with some modifi cations (Bellon-Fontaine et al., 1996; Kos et al., 2003). Aft er incubating the bacteria in TSB for 24 h, the bacteria were centrifuged at 5000 rpm for 15 minutes, and then washed twice and suspended in 0.1 M KNO 3 (pH 6.2) to approximately 108 CFU mL -1 . Th e absorbance of the bacterial suspension was measured at 600 nm (A 0 ). 1 mL of solvent was added to 3 mL of bacterial suspension. Aft er 10 minutes of incubation at room temperature, the two-phase system was mixed using a vortex for 2 minutes. Th e aqueous phase was removed aft er 20 minutes of incubation at room temperature and its absorbance was measured at 600 nm (A 1 ). Th e percentage of bacterial adhesion in the presence of solvent was calculated as follows: Th ree diff erent solvents were tested in this study: xylene (Sineks, Belgrade, Serbia), which is a polar solvent; chloroform (Alkaloid, Skoplje, Macedonia), a monopolar and acidic solvent, and ethyl acetate (Zorka, Sabac, Serbia), a monopolar and basic solvent. Only bacterial adhesion to xylene demonstrated cell surface hydrophobicity or hydrophilicity (Ocaña and Nader-Macías, 2002). According to Ocaña and Nader-Macías (2002), the percentage of hydrophobicity is expressed as 0 -35% -low hydrophobicity; 36 -70% -medium hydrophobicity; 71 -100% -high hydrophobicity.

Th e co-aggregation ability
Th e co-aggregation of E. gergoviae KGPMF 20 with Enterococcus faecalis KGPMF 49 isolated from the same Sokobanja cheese was examined. E. faecalis KGPMF 49 was isolated from the same cheese (Muruzović et al., 2018). Th e coaggregation was monitored by using a modifi ed procedure described in Ocaña and Nader-Macías (2002). Overnight bacterial cultures were centrifuged at 5000 rpm for 15 minutes, and then washed twice in PBS buff er (Alfa Aesar GmbH & Co, Karlsruhe, Germany) followed by resuspension in 4 mL of the same buff er and the cell number was approximately 10 8 CFU mL -1 . 2 mL of each suspension of both bacteria whose coaggregation was monitored was mixed well on a vortex. Aft er mixing, 200 μL from the surface of the suspension was transferred to a microtube containing 1800 μL of PBS, and the absorbance values were read at 600 nm (A 0 ). Th e same procedure was repeated aft er 2 h (At). Th e percentage of coaggregation of the species was calculated in the following way:

Th e preparation of fermentation liquid and detection of enzymatic activity
In order to investigate the enzymatic activity, it is necessary to obtain fermentation liquids of the isolate. 100 μL of overnight bacterial culture was separately inoculated in 10 mL of TSB (Torlak, Belgrade, Serbia) and MH (Torlak, Belgrade, Serbia). Inoculated broths were incubated for 24 h at 37°C. Aft er the incubation, the samples were centrifuged at 10.000 rpm/30 min/ 4°C. Th en, the supernatant, which represented the fermentation liquid, was separated. Fermentation liquid was kept in the refrigerator at 4°C until the experiment was performed. Two broths were used as they had a diff erent composition and there was a possibility that they could aff ect the enzymatic activity of bacteria.
Aft er the preparation of fermentation liquid, the enzymatic activity was evaluated. Th e acid and alkaline invertase activity, the activity of alkaline phosphatase, α-amylase activity, proteolytic activity and the total concentration of protein were all determined by the methods described in Jakovljević (2014).

RESULTS AND DISCUSSION
In this paper, the adhesion and co-aggregation ability, as well as the enzymatic activity of E. gergoviae KGPMF 20 were demonstrated for the fi rst time. Th e isolate originated from Sokobanja cheese which was produced in the households around Sokobanja (southeastern Serbia), from fresh and unpasteurized cow's milk. According to the results from Mladenović et al. (2018), E. gergoviae KGPMF 20 is a gram-negative, oxidase negative and catalase-positive bacteria. It showed the fermentation ability of glucose and lactose with production of acid and gas, while the ability of H 2 S production (hydrogen sulphide) form triple sugar was not detected. It formed a light pink colony on HiChrome coliform agar. Th e investigated isolate was sensitive to antibiotics (streptomycin, chloramphenicol and tetracycline).
Th e adhesion ability of E. gergoviae KGPMF 20 was detected in the presence of chloroform (13.85%), and ethyl acetate (13.89%). Th e species demonstrated no ability of adhesion in the presence of xylene.
Adequate hydrophobic/hydrophilic properties of microorganisms can contribute to benefi cial processes such as degradation of hydrocarbons or biodegradable polyesters during milk fermentation (Obuekwe et al., 2009). Hydrophobic microorganisms may have the ability to form a biofi lm to various abiotic and biotic surfaces (Krasowska and Karel, 2014). According to Tresse et al. (2006), cellular hydrophobicity is crucial for biofi lm formation. Del Re et al. (2000) and Giaouris et al. (2009) indicated that bacteria with a hydrophobic surface have a higher binding affi nity for epithelial cells and solid surfaces. Bacterial biofi lm can lead to bacterial resistance to antibiotics and thus contributes to the pathogenicity of one species. Bacterial adhesion in the presence of xylene is an indicator of hydrophobicity or hydrophilicity of cell surface. Th e ability to adhere in the presence of two other solvents, chloroform and ethyl acetate is an indicator of the ability of bacterial cell as a donor of base or acid electron acceptors (Bellon-Fontaine et al., 1996). Based on the results, it can be concluded that E. gergoviae KGPMF 20 had low hydrophobicity. It showed no ability of adhesion to xylene, meaning that it had a low potential to attach and form biofi lm in abiotic surfaces.
Th e co-aggregation of E. gergoviae KGPMF 20 and E. faecalis KGPMF 49 was investigated for the fi rst time in this study. Th e percentage of co-aggregation between these bacteria was 14.2%. Enterobacteria and enterococci are a member of the normal fl ora of the human gastrointestinal tract (Silva et al., 2012;Pugin et al., 2017) and of the dairy products made from unpasteurized cow's milk (Muruzović et al., 2018;Mladenović et al., 2018). Th erefore, their interaction needs to be investigated and evaluated.
Th e enzymatic activity of E. gergoviae KGPMF 20 was investigated in two diff erent broths and the results are shown in Table 1. Th e results indicated that small activity of amylase and alkaline phosphatase was observed, while the activity of acid and alkaline invertase was not detected (except for the very small activity of acid invertase in MH). Th e protease activity was observed only in TSB. According to the results of the screening method, E. gergoviae KGPMF 20 does not have the proteolytic or lipolytic activity . According to a study by Kamaladevi et al. (2014), E. gergoviae isolated from the areas of Vaippar, Th oothukudi District and Tamil Nadu in India demonstrated the ability to produce lipase. In this paper, it was shown that E. gergoviae KGP-MF 20 had a very low or no enzymatic activity, so it had no role in sensory properties of cheese.
In the Sokobanja cheese, the presence of the Enterobacter genus with only E. gergoviae KGPMF 20, was determined . Based on the available literature, it was found that the appearance and description of

CONCLUSION
Based on the results from this study, E. gergoviae KGPMF 20 can interact with other species isolated from cheese. Th e species had a low hydrophobicity and showed no adhesion ability in the presence of xylene. It produces a very low concentration of extracellular enzymes, so it had no eff ect on the sensory