BROTH DEPENDING PRODUCTION OF EXTRACELLULAR ENZYMES BY ENTEROBACTERIA ISOLATED FROM DAIRY FOOD

In this paper, we investigated, for the first time, the enzyme activity of enterobacteria from cheese from Southeastern Serbia (Sokobanja), which was produced in a traditional way. The tested bacteria were selected from the spring, summer, and autumn collections and grown in two different broths. The enzyme activity was investigated in the two broths and measured using spectrophotometry method. It was concluded that there were differences in the amount of extracellular enzymes depending on the broths. A statistically significant rise in the amount of enzymes was demonstrated in Tripton soy broth. Bacteria in cheese do not possess the ability to produce extracellular enzymes in large amounts. Therefore, they do not change the organoleptic characteristics of the cheese. There is a possibility that if they are found outside their natural environment (cheese), without interactions with antagonists, they may exhibit stronger enzyme activity.


INTRODUCTION
Gram-negative bacteria release proteins (extracellular enzymes) into the growth medium (PUGSLEY et al., 1986). One of the most interesting aspects of secretion in gram-negative bacteria is how the protein crosses the membrane (EISELE et al., 1972). The characterization of extracellular bacterial enzymes is very important since they have a role in causing infections, cell cytotoxicity, and their possible applications in biotechnology (MARTY et al., 2002). Extracellular enzymes of the pathogen increase its virulence (SHARMA and TIWARI, 2005). inoculated (10 8 -10 9 CFU/ml). 10 ml of MH was inoculated in the same way. Inoculated broths were incubated 24 h at 37°C. After the incubation, samples were centrifuged at 10.000 rpm for 30 min at 4°C. Then, the supernatant, which represented the fermentation liquid, was separated. Fermentation liquid was kept in the refrigerator at 4°C until the experiment was performed.

Determination of acid and alkaline invertase activity
Invertase enzyme activity was determined by SUMNER and HOWELL (1935). To determine the activity of alkaline invertase in the fermentation liquid of the bacteria, it is necessary to prepare a reaction mixture containing 0.5 ml of crude enzyme extract, 0.5 ml of 0.02 mol/l phosphate buffer (pH 8.0) and 1 ml of 1% (w/v) ) sucrose. The contents of the tube (sample) were mixed and incubated in a water bath at 37°C for 15 min. In the second control tube, all the ingredients were added as in the test tube, and that tube was placed in the ice until the incubation of the test was completed. 1 ml of the contents of both tubes were taken and 2 ml of the dinitrosalicyl reagent were added. The test tubes were incubated at 100°C for 5 min. The tubes were cooled at room temperature and then the amount of liberated reducing sugars was spectrophotometrically determined at 540 nm (MILLER, 1959). The enzymatic activity of acid invertase was determined in the same way in a reaction mixture containing 0.5 ml of crude enzyme extract, 0.5 ml 0.01 mol/l sodium acetate buffer (pH 4.5), and 1 ml of 1% (w/v) sucrose.
To construct a standard curve, it was necessary to make a 1 mg/ml solution of 5.56 x 10 -3 mol/l glucose standard (Sigma Aldrich). 0.05, 0.10, 0.20, 0.30, 0.40, and 0.50 ml of standard glucose solution were measured into six tubes and the tubes were filled up with distilled water to 1 ml. As a blank standard, 1 ml of water was taken. The contents of the tubes were mixed and the tubes were incubated at 55°C for 20 min. The further procedure was the same as the one for determining invertase activity.
The invertase activity unit (IU) is defined as the amount of enzyme which catalyzes the production of 1 μmol of glucose per minute at 37°C.

Determination of alkaline phosphatase activity
The enzymatic activity of alkaline phosphatase was determined based on the amount of inorganic phosphorus using Allen's method (ALLEN, 1940). Enzymatic activity was measured in a reaction mixture containing 1 ml of glycol buffer (pH 9.0) with Mg 2 + , 1 ml of enzyme extract, and 1 ml of β-glycerophosphate (substrate). The tubes containing the reaction mixture were incubated at 37°C for 30 min. After the incubation, the enzyme reaction was interrupted with the addition of 3 ml of 10% TCA (trichloroacetic acid). The tubes were placed in ice for 15 minutes after which the contents of the tubes were filtered and the filtrate was collected. Control tubes were prepared in the same way, but without incubation, TCA was immediately added, and the tubes were held in ice for 15 min and filtered. In order to determine the concentration of phosphorus in the sample, Allen's reaction was performed. 1 ml of the filtrate of the test and control tube were poured into two tubes, as well as 0.4 ml of amide (amidol), 0.4 ml of 60% PCA (perchloric acid), 0.2 ml of NH4-molybdate and 3 ml of distilled water. The tubes were incubated at room temperature for 11 min. At the same time, a series of dilutions of the standard phosphorus solution were prepared and the above procedure 126 was repeated, which served to construct a standard phosphorus curve. The absorbance solution was measured at 720 nm.

Determination of alkaline protease activity
The proteolytic activity of the fermentation liquid was determined based on the presence of enzymes in 1 ml of the liquid medium by the Anson method (Anson, 1938), based on the amount of tyrosine liberated by the hydrolysis of casein under the action of proteolytic enzymes. The proteolytic activity of the fermentation liquid was determined as follows: in one tube, 1 ml of fermentation fluid was added, while in another 5 ml of 2% (w/v) solution of casein. Both tubes were heated in a water bath at 37°C for 15 min. After that, 2 ml of heated casein in a test tube with a fermentation liquid (test), were mixed and returned to incubate for another 10 min. The enzyme reaction was stopped by the addition of 5 ml of cold 5% (w/v) trichloroacetic acid (TCA). The mixture was blended and then proceeded through the filter paper. In another control tube, 1 ml of the fermentation fluid was pipetted and 2 ml of cold casein and 5 ml 5% (w/v) TCA were immediately added. The mixture was blended and then proceeded through the filter paper. 2 ml of the filtrate were taken from the test and control tubes and 5 ml of 6% (w/v) Na2CO3 and 1 ml of Folin-Ciocalteu reagent were added. The mixture from the tubes was blended and left at room temperature for 30 min until the blue color was developed. In a separate tube (blank), 1 ml of distilled water was measured instead of tyrosine and all other ingredients were added. The absorbance value was read on the spectrophotometer at 660 nm.
To determine the amount of tyrosine, constructing a standard curve was required. It was necessary to make a 1.1 x 10 -3 mol/l standard tyrosine solution (prepare it in 100 ml of deionized water and heat carefully until it is dissolved and cooled at room temperature). 0.05, 0.10, 0.20, 0.30, 0.40, 0.60, and 0.60 ml of a standard solution of tyrosine were measured into six tubes and the tubes were supplemented with distilled water to the volume of 2 ml. As a blank standard, 2 ml of water were taken. All tubes were incubated at 37°C for 10 minutes, and a further procedure was the same as the one for the determination of proteolytic enzyme activity. After constructing a standard tyrosine curve, the tyrosine absorber values contained in the sample filtrate (a) and the control filtrate (b) should be inserted.

Determination of α-amylase activity
Invertase enzyme activity was determined using the Bernfeld method (BERNFELD, 1955) with modifications. The activity of α-amylase in the fermentation liquid was determined by the preparation of a reaction mixture containing 0.5 ml of crude enzyme extract, 1.5 ml of buffer (pH 6.9) (20 mmol sodium phosphate and 6.7 mmol NaCl) and 1 ml of 1% (w/v) starch. The contents of the tube (sample) were mixed and incubated in a water bath at 37°C for 15 min. In the second control tube, all the ingredients were added as in the test tube and the tube was placed in ice until the incubation of the test tube was completed. 1 ml of the contents of both tubes were taken and 1 ml of dinitrosalicyl reagent (5.3 M K-Natartrate and 96 mmol of 3,5-dinitrosalicylic acid) was added. The test tubes were incubated at 100°C for 15 min. The tubes were cooled to room temperature and then the absorbance was spectrophotometrically read at 540 nm.
In order to construct a standard curve, a 0.2% maltose solution (2 mg/ml solution of D -(+) -maltose standard (Sigma Aldrich)) should be made. 2 ml of the final concentration of standard were added to each of the eight tubes: 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, and 0.80 mg/ml. 2 ml of distilled water should be taken as a blank instead of the standard. Then, 1 ml of the dinitrosalicylic reagent was added to each tube. The contents of the tubes were carefully mixed and incubated at 100°C for 15 min. The tubes were cooled at room temperature and the absorbance was read at 540 nm. Based on the obtained absorbance and known concentration of standards, the standard curve was constructed and the amount of reducing sugar in the sample was determined from it. The enzyme activity unit determines the following formula: Unit of enzymatic activity: where 3 -total sample volume (ml), 15-incubation time of the sample (min), 0.5 -volume of enzyme extract (ml), 1-volume of the sample taken for colorimetric detection (ml).

Determination of total protein amount
The total amount of protein in the fermentation liquid was determined using the Bradford method (BRADFORD, 1976). The method is based on the creation of a complex between the color of Brilliant Blue G. and the protein in solution. The color intensity is proportional to the amount of protein in the solution. In order to determine the amount of protein in the sample, a standard curve for the BSA initial concentration of 1 mg/ml should be prepared. In each of the ten tubes, 0.1 ml of the final concentration standards: 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, and 1.00 mg/ml should be pipetted. The blank tube contains 0.1 ml of distilled water instead of standard. 3 ml of Bradford's reagent were added to all tubes and then mixed slightly. The tubes were left to incubate at room temperature for 30 min. After the incubation was finished, the absorbance was read at 595 nm using a spectrophotometer. Based on the values of the standards of known concentrations and the resulting absorbances, the standard curve was constructed.

Data analysis
Paired samples T-test was used for comparing the amount of enzyme in TSB and MH, while Spearman correlation coefficient was used for comparing the amount of proteins and proteases at two broths, via IBM SPSS Statistics 20.

The total amount of protein
The total amount of protein in the substrates was measured. The higher concentration of proteins was measured in TSB than in MH. The highest absorbance was measured in the TSB where the isolates E. coli KGPMF 1). A statistically significant correlation between the amount of protein and protease activity in TSB (Spearman correlation coefficient -P <0.05) was obtained, while in MH it was not noticed.

The acid and alkaline invertase activity
The acid invertase activity was higher in MH for most of the tested bacteria (E. coli KGPMF 14, E. coli KGPMF 21, E. coli KGPMF 24, S. odorifera KGPMF 18, K. oxitoca KGPMF 1, K. oxitoca KGPMF 2, K. pneumoniae KGPMF 10, K. pneumoniae KGPMF 11). For other isolates, the acid invertase activity was higher in TSB, except for K. pneumoniae KGPMF 12 where the acid invertase activity was equal in both broths. The alkaline invertase activity of the tested bacteria was not detected (Tab. 2). 129  Table 3. Amylase activity and alkaline phosphatase activity.

The alkaline phosphatase activity
The alkaline phosphatase activity was higher in TSB for all tested isolate except E. coli KGPMF 14, where alkaline phosphatase was active in MH. Alkaline phosphatase of K. In this paper, the activity of extracellular enzymes of bacteria isolated from cheese made in a traditional way was measured for the first time. By measuring extracellular enzymes, using specific methods, it could be concluded that the activity of extracellular enzymes depended on the type of broth (statistically significant difference in alkaline phosphatase activity in TSB than MH, P<0.05) and the type of bacteria. Based on the screening method, isolates from cheese belonging to the collection of microbiology laboratory of the Faculty of Science, University of Kragujevac (KGPMF), from the genus Escherichia and Klebsiella, except K. oxytoca KGPMF 1, did not possess proteolytic and lipolytic activities. S. odorifera KGPMF 18 and S. marcescens biogp 1 KGPMF 19 exhibited lipolytic activity (MLADENOVIĆ  et al., 2018a). Bacteria from the spring collection, belonging to the genus Escherichia demonstrated no proteolytic or lipolytic activity. Bacteria isolated from Sokobanja cheese mostly did not manifest proteolytic and lipolytic activity. Their activity was presumably controlled or inhibited since lactic acid bacteria could develop a low pH, which was responsible for the sour taste of cheese. The results of the study from MURUZOVIC et al. The cheese investigated in our study was produced in countryside households around Sokobanja (Southeastern Serbia) from fresh, processed, and unpasteurized cow's milk by using a traditional method. Bacteria penetrated the cheese, presumably, during the production or storage. It was assumed that poor hygiene and unpasteurized cow's milk caused the appearance of enterobacteria. Detection of enzyme activity of the isolates was crucial since the bacteria in a changing natural environment demonstrated the potential of becoming a good producer of extracellular enzymes. In our research, it was shown that the substrate, in our case two different broths, affected differently the production of enzymes of isolates.

Species
Microbial enzymes have contributed to the development and growth of the food industry, especially to the optimization process and efficiency. They have also contributed to the improvement of organoleptic characteristics, nutritive value, quality, conservation, and shelf life of the product. The important role of enzymes in monitoring and quality control of the process and products is significant in various sectors of the food industry such as dairy, meat, baking, etc. (AVENDAÑO et al., 2016).
Bacteria from the fam. Enterobacteriaceae syntheses proteolytic and lipolytic enzymes which are responsible for the deterioration of milk and dairy products (ZAJÁC et al., 2015;MASIELLO et al., 2016). In the production of cheese, these enzymes destabilize casein and may modify or even prevent milk coagulation, which could directly affect the formation of the product (CALDERA et al., 2015). Another major problem is that these bacteria can significantly affect color, odor, taste, and texture ( ), coliform detection in cheese was associated with specific cheese characteristics, but there was no connection to pathogen detection.
Bacteria from the fam. Enterobacteriaceae were detected in cheese from Sokobanja and demonstrated enzyme activity. The presence of enterobacteria was not desirable, from the aspect of the sanitary safety of the product. The cheese is safe for consumption since it contained a higher concentration of salt which was added during cheese production. Salt was a good preservative and prevented the growth and development of conditionally pathogenic bacteria. Consequently, the number and all the activities, such as enzyme activity of the bacteria, were controlled. Only in the case when bacteria were found out of their natural habitat (cheese with high salt concentration), they can develop enzyme activity (production of extracellular enzymes).