International Journal of Pharma and Bio Sciences ISOLATION AND CHARACTERIZATION OF BACTERIOCINS FROM FERMENTED FOODS AND PROBIOTICS

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1 International Journal of Pharma and Bio Sciences ISOLATION AND CHARACTERIZATION OF BACTERIOCINS FROM FERMENTED FOODS AND PROBIOTICS RAJA NARENDER.B* 1, RAVI.P 1, A. SHYAM SUNDER 2 and V. MALLIKARJUN 1. 1 Department of Microbiology &, SR College Of Pharmacy, Warangal, , India. 2 Department of Microbiology, Kakatiya University, Warangal, , India. *Corresponding Author rajanarenderbongoni@gmail.com ABSTRACT Isolation and characterization of bacteriocins from fermented food by using the various food isolates tested against different test organisms. Several steps of isolation of bacteriocins is carried out by Agar Well Diffusion Assay Test and preparing the cell free extract by the centrifugation of overnight broths and pouring the cell free extract and the overnight broth of the food isolates tested against the test organisms by a process called overlaying on nutrient agar plates. Standardization procedure was carried out so as to make the test organisms to spread on the Petriplates. Tween20 is used to know whether the bacteriocins are attached to the cell. Microscale Optical Density Assay was carried out on the 96 wells Petri plate. Then the extraction of protein was done by the process of dialysis taking the dialysis membrane to know whether the proteins passes through the membrane or the membrane with holds the protein. KEY WORDS Bacteriocins, Lactic acid bacteria, Fermented foods, Probiotics. INTRODUCTION Lactic acid bacteria (LAB) occur naturally in several raw materials like milk, meat and flour used to produce foods 1. Gram negative and Gram positive bacteria produce bacteriocins. Bacteriocins are proteinaceous antibacterial compounds, which constitute a heterologous subgroup of ribosomal synthesized antimicrobial peptides 2. Some investigations have reported on the ability of bacteriocins to also inhibit Gram-negative bacteria 3. Bacteriocins and the organisms that produce them have potential in the food and feed industry as a source of probiotics, as well as in the pharmaceutical industry as a source of probiotics. Bacteriocins are also useful in food and feed industry because of their antibacterial characteristics; moreover, bacteriocins can be used as bio preservatives in fermented foods. On a sound scientific basis three defined classes of bacteriocins have been established 4. Class I, the lantibiotics; class II, the small heat stable non lantibiotics; and class III, large heat labile bacteriocins. A fourth class of bacteriocins is 1

2 composed of an undefined mixture proteins, lipids and carbohydrates. The existence of the fourth class was supported mainly by the observation that some bacteriocin activities obtained in cell free supernatant 5. The aim of the study was to Isolate Bacteriocins producing Bacteria from fermented foods (Probiotic Drink, yogurt and Meat) by centrifugation, filtration and to characterize Bacteriocins by Agar diffusion method, MODA Test and Dialysis. In this study, we report the detection, isolation, and partial characterization of a broad-range bacteriocin from cell free optical density assay(moda), agar well diffusion assay (AWDA), Dialysis, Standardization method for its ability to inhibit a wide range of Grampositive and Gram-negative bacteria. The majority of bacteriocin producing is natural food isolates; they are suited to food applications. An increasing health conscious public seeks to avoid foods with chemical preservatives which have undergone expensive processing or which contains chemical preservatives. Bacteriocins are used as a tool in the food industry in controlling the undesirable bacteria in a food-grade and natural manner. MATERIALS AND METHODS Cultures used as test organisms were Staphylococcus aureus, Pseudomonas, E.coli, Bacillus subtilis, Klebsiella pneuomoniae and Listeria monocytogenes procured form department of microbiology, Kakatiya University. The food samples used are Yogurt, Meat and Probiotic Drink, collected from reliance super market, Warangal. Media used are Nutrient Agar and Nutrient Broth, procured from Hi- Media Laboratories, Pvt. Ltd, Mumbai. (i) Gram Staining: A colony was fixed onto a slide passing through a Bunsen flame then stained with crystal violet for one minute. Then the stain was poured off by holding the slide at an angle over the sink water was used to wash off excess crystal violet. The slide is returned to the rack and was covered with the fresh Lugol's iodine for one minute. Then the slide was washed with tap water and drained and treated with alcohol for ten seconds as the alcohol removes the excess gram complex and also decolorizes gram negative cells. The slide was counterstained with safranin for two minutes, then washed with tap water and blotted with a tissue to remove excess water. The slide was looked at under the microscope (100 x objectives with oil) 6. (ii) Preparation of Cell Free Extract: Nutrient broth was prepared (100ml) and inoculated with bacteria and incubated for 18-24hr at 37 C. The culture was centrifuged in AUTOSPIN (400) at 8500rpm for 10min the cells were discarded and the cell free extract was filtered using a syringe with 0.2µm filter. The cell free extract was gently filtered into sterilized test tubes with 0.2µm acetate cellulose filter 7. (iii) Screening for Antagonistic Activity: An agar well diffusion assay (AWDA) 8 was carried out for detection of antagonistic activity. Nutrient agar plates were prepared. The test organism and the food isolates were grown in Nutrient broth for over night at 37 c in agitator incubator and checked for the growth. The food isolates were then centrifuged to obtain cell free extract. Small tubes of nutrient agar (15ml) were made autoclaved, cooled to 50 c and were used for overlaying. 25µl of the overnight grown test organisms was added to the overlay, mixed well were overlaid on to the Petri plates with agar. Wells were made in the solid overlay. Six wells in each plate with a sterile well maker of 5mm. Cell free extract (15µl) of the isolates was added to three wells and to the other wells is added the broth with isolates. The plates were then incubated at 37 c overnight and were examined for the zones of inhibition. Inhibition was recorded as negative if no zone of inhibition was observed around the agar well. The wells were measured in mm. (iv) Standardizing of Test Organisms: A loop full of test organism was diluted in sterile water and checked for absorbance of 0.1 at A ml of the diluted was taken to ix with the overlaying. Wells were made in the solid overlaid. Cell free extract and the broth were added and incubated at 30 C overnight and were examined for the zone of inhibition. (v) Comparison by Using Tween 20: 5mls of the overnight Nutrient broth was taken and centrifuged the cells down. Supernatant was taken off and resuspended (vortex) the cells in 2mls of sterile water with 0.005% of Tween 20 for 10mis at 7000rpm. The cells were centrifuged again and the supernatant was taken to a tube and the cells are 2

3 resuspended into 1ml of sterile water. The supernatant and the cells were tested on Petri plates 9. (vi) Microscale Optical Density Assay (MODA) 10 Test: Cultures of test organism grown in Nutrient broth were mixed with serial dilutions with sterile ringer s solution to give 1:10,000 (used 9ml of ringer s in glass tube and add 1 ml of broth to give 1:10, mixed well, took 1ml added 9ml of ringer s to give 1:100 etc. down to 1:10,000). Isolates grown in Nutrient broth and 0.005% Tween 20 were centrifuged and took 5ml of Cell free extract. Then took sterile 96 well plate and tried combinations of cell free extract against 1:10,000 diluted test organism. Duplication have been done for each test by taking 100 µl of diluted test organism plus 15µl of Nutrient broth for well 1 and to well 2 100µl of diluted test organism plus 15µl of Nutrient broth plus 15µl of cell free extract. The 96 wells plate was then incubated overnight at 37C and read absorbance at 570nm on plate reader. (vii) Extraction of Protein: From the reports observed from the Microscale Optical Density Assay Test, is done the Dialysis for the extraction of protein 11. The best food isolate and the test organisms were grown in Nutrient Broth at 30C in shaking incubator. Then was centrifuged for cell free extract to which Ammonium sulphate was added (60grs/100ml) and cooled in cold room for day for precipitation. Dialysis membrane (mol wt , diameter of 15.9mm) was made ready by boiling for 10mins in a large volume of 2% (w/v) sodium bicarbonate and 1mM EDTA (ph.8.0), washed the tubing thoroughly in distilled water. Then was again boiled for 10mins in 1mM EDTA and was cooled and had dropped the sample with a sterile pipette. And kept the tubes in sterile water in cold room for 6hrs hours by changing the water for every 2hrs.test organisms overlaid Petri plates are made well and was added with the Undialyzed and dialyzed sample and incubated overnight at 30C, and recorded the results. RESULTS AND DISCUSSION After mashing the food isolates, gram staining was carried out and the table 1 showing bacteria stained with respective stains and shapes. The food isolates were grown on the Nutrient Agar. All the isolates were grown at 30 0 c overnight. Table shows the food isolates containing bacteria of different bacteria got stained differently, if considered the table 1 the bacteria from the meat got stained in pink make a conclusion that the bacteria present in it is the gram negative and shaped like cocci. Table: 1 Staining of food samples and test organisms Food/ Test organisms Stain Shape Medium Temperature Meat Pink Cocci Nutrient Agar 30 0 c Probiotic drink Purple Rod Nutrient Agar 30 0 c Yogurt Purple Rod Nutrient Agar 30 0 c Staphylococcus aureus Pink Cocci Nutrient Agar 37 0 c E.coli Pink Rod Nutrient Agar 37 0 c Pseudomonas Pink Cocci Nutrient Agar 37 0 c Klebsiella Purple Rod Nutrient agar 37 0 c Listeria Purple Rod Nutrient agar 37 0 c Bacillus subtilis Purple Rod Nutrient agar 37 0 c Gram positive bacteria were found in probiotic drink with rod shaped and stained in purple color. Gram staining was also carried out for the test organisms and from table-1, it was known that the test organisms used contains both the gram positive and gram negative bacteria and grow better at 3

4 37 0 c. klebsiella, Listeria monocytogenes and. Bacillus subtilis were stained purple as they are gram positive bacteria and shaped rods, Staphylococcus aureus, E. coli, Pseudomonas were concluded to be gram negative bacteria as they have stained in pink with spherical shaped bacteria. Table 2 showing about the bacteriocins from bacteria are tightly attached to the bacteria as the zone of inhibition was found in both the Cell Free Extract and Broth. All the bacteria were showing the presence of bacteriocins that are bound to the cell especially in Probiotic drink and Meat when they are grown against all the six test organisms. It can be concluded from the table 3, that the bacteriocins are a bit tightly attached to the cells and so showing the zones of inhibition even in cell free extract. The zone of inhibition was quiet more in broth in both Meat and Probiotic drink. Table 2 Showing the zone of inhibition when isolates against the test organisms Meat Probiotic drink Yogurt Test organism Broth CFE Broth CFE Broth CFE cm cm cm cm cm cm Bacillus subtilis E.coli Staphylococcus aureus Klebsiella Diameter of well: 0.5cm Table: 3 showing the zone of inhibition by Agar Well Diffusion Assay (AWDA) when isolate against the test organisms on standardization. The method of standardization was calculating the absorbance for the test organism at 540nm. The standardization was carried out to get the same amount of test organisms in all the plates. Standardization was calculated for the absorbance needed divided by the obtained absorbance multiplied with the amount of mixture of water and cells that is used for adding to the media to be overlaid is made to the final volume by adding either water or cells as per required, and after carrying out the procedure and on overnight incubating, it was shown form the table 4 that all the test organisms have shown results against Probiotic drink and Pseudomonas showing the in both the meat and Probiotic drink. And even shows that the bacteriocins are well attached to cells. The broth showing higher zone of inhibitions than the cell fee extract. Table : 3 Showing the zone of inhibition when isolates against the test organisms on standardization Meat Probiotic drink Yogurt Test organism Broth CFE Broth CFE Broth CFE cm cm cm cm Cm cm Pseudomanas E.coli Klebsiella Staphylococcus Bacillus Listeria Diameter of well: 0.5 cm 4

5 Table: 4 The MODA (Microscale Optical Density Assay) Test was carried out to know the percentage reduction in growth due to bacteriocins. It was carried out on sterile 96 wells plate on serial dilution (1:10,000) of test organisms grown in overnight Nutrient Broth mixed with sterile Ringers solution. After adding different samples(100µl) of test organisms with 15µl of Nutrient Broth and 10µl of diluted test organisms plus 15µl of Nutrient Broth plus 15µl of cell free extract to two different wells. Well 1 acted as the negative control and showing how much growth the test organisms after incubation at 37 0 C and well 2 showing the effect on growth of the bacteriocins in the cell free extract. 96 wells plate was incubated overnight and took the absorbance at 570nm on plate reader. The anti bacterial activity is calculated by taking the difference in absorbance between the control (well 1) and well 2 and dividing this difference by the control (well 1) absorbance and multiplied by 100 gave the percentage reduction in growth due to the bacteriocin. % reduction in growth due to bacteriocins = abs difference (well 1 abs - well2 abs) 100 Well 1 abs Table 4 showing % reduction in growth due to bacteriocins, as per the reading got as shown in table indicates the Bacillus subtilis, Klebsiella pneumoniae, showing the better results indicating the presence of Bacteriocins with Meat of food isolate and remaining test organisms showing low results indicating growth in cell free extract. MODA (Microscale Optical Density Assay) TEST Table : 4 Showing % reduction in growth due to bacteriocins Test Organism Meat Probiotic Drink Yogurt Bacillus 45.12% 08.33% 18.90% Listeria 19.14% 11.80% 10.92% Psuedomanas 13.78% 04.38% 12.82% E.Coli 18.44% % 15.50% Klebsiella 34.40% 07.54% 00.39% Staphylococus 10.56% 07.83% 05.68% Table: 5 showing the recorded measurements of wells of dialysis method. Meat was taken from the Microscale Optical Density Assay Test which was shown to be as an active isolate producing the bacteriocins and carried out Dialysis for the protein extraction for which Agar Well Diffusion Assay (AWDA) was carried out to know the molecular weight and diameter of protein by using membrane of ( Daltons) molecular weight and diameter of 15.9mm. And from table 6 it was known that the proteins were more size that of the membranes as so could not pass through the membrane when kept in sterile water on stirring on cold temperature for six hours. Table: 5 Showing the zone of inhibition with Undialysed and dialysed product Test organism Meat Undialysed Dialysed Bacillus subtilis E.coli 0.8cm 0.7cm 0.7cm 0.9cm Diameter of well: 0.5 cm 5

6 For the future on going of the experiment, the proteins would be extracted and experimented to know the molecular weight of the protein by performing gel electrophoresis REFERENCES 1. Rodriguez E, Gonzalis B, Gaya P, Nunez M, Medina M: Diversity of bacteriocins prod y lactic acid bacteria isolated from raw milk. Intl Dairy J, 10: 7-15, (2000). 2. De Vugst L, Vandamme E J: Bacteriocins of lactic acid bacteria. Microbiol, Genet Appl. London: Blackie Acad and professional. ISBND , (1994). 3. Ennahar S, Sashihara T, Sonomoto K, Ishzaki A: Class IIa bacteriocins: biosynthesis, structure and activity. FEMS Microbiol.Rev 24: , (2000). 4. Nettles CG, Barefoot SF: Biochemical and genet characteristics of bacteriocins of food associated lactic acid bacteria. J. Food Prot., 56: , (1993). 5. Jimenez-Diaz R, Rios-Sanchez RM, Desmazeaud M, Ruiz-Barba JL, Piard JC: Plantaricin S and T, two new bacteriocins produced by Lactobacillus plantarum LPCO 10 isolated from a green olive fermentation. Applied Environ. Microbiol, 59, , (1993). 6. Sridhar Rao PN: Department of Microbiology, JJMMC, DAVANGERE. / 7. Mitsunobu Sakajoh, Nadine A. Solomon and Arnold L. Demain: Cell-free synthesis of the dipeptide antibiotic bacilysin. Journal of Industrial Microbiology, 2: , (1987) 8. Jin-Woo Kim and S.N. Rajagopal: Antibacterial Activities of Lactobacillus crispatus ATCC and Lactobacillus gasseri ATCC Journal of Industrial Microbiology, 4: , (1988). 9. Nakamura T., Yamazaki N., Taniguchi H. and Fujimura S: Production, purification, and properties of a bacteriocin from Staphylococcus aureus isolated form saliva. Infection and Immunity, 39(2): , (1983). 10. Bradley W. Lash, Tami H. Mysliwiec and Hassan Gourama: Food Microbiology, 22 (2-3): , (2005). 11. Fanny Guzmán, Sonia Barberis, Andrés Illanes: Peptide synthesis: chemical or enzymatic. Electronic Journal of, 10 (2): (2007). 6