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Detection and Biochemical Characterization of Microorganisms in Milk and Cocoa powder samples by FTIR and subsequent production of Bacteriocin from Lactobacillus

Ramalingam C*, Harshita Jain, Kirti Vatsa, Naushin Akhtar, Bhaskar Mitra, D. Vishnudas, Sharad Yadav, Kunal Garg, Alok Prakash, Amit Rai
  1. Industrial Biotechnology Division, School of Biosciences and Technology, VIT University, Vellore
Corresponding Author: SHARMA VIVEK, E-mail: vivek03sharma@rediffmail.com
Received:23 January 2013 Accepted: 28 February 2013
Citation: Ramalingam C, Harshita Jain, Kirti Vatsa, Naushin Akhtar, Bhaskar Mitra, D. Vishnudas, Sharad Yadav, Kunal Garg, Alok Prakash, Amit Rai “Detection and Biochemical Characterization of Microorganisms in Milk and Cocoa powder samples by FTIR and subsequent production of Bacteriocin from Lactobacillus” Int. J. Drug Dev. & Res., January-March 2013, 5(1): 310-320.
Copyright: © 2013 IJDDR, Ramalingam C et al. This is an open access paper distributed under the copyright agreement with Serials Publication, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Abstract

Cocoa and milk powder samples were taken from a confectionery and tested for presence of microbes (harmful and pathogenic).Biochemical characterization of isolated microbes was carried out for confirmation. Lactobacillus was isolated from milk powder. When a culture of Lactobacillus sp. was inoculated into milk and incubated at room temperature, it multiplies and converts lactose to lactic acid. Fourier transform infrared spectroscopy was used to study the variation of functional group peaks in milk by the action of Lactobacillus sp. The spectral changes were also observed. Our main aim of this project is the production of bacteriocin from isolated lactobacillus species; it showed broad range of antibacterial activity against some food borne pathogens like staphyloccus, Ecoli, streptococcus, Enterococcus etc. The bacteriocin is purified by ammonium sulfate precipitate and dialysis. Biochemically it was pure protein moiety. Maximum bacteriocin concentration was found after dialysis. Project revealed the possibility of using bacteriocin as food preservative.

Key words:

Fourier Transform Infrared Spectroscopy, Lactobacillus, antibacterial activity, bacteriocin

INTRODUCTION

Lactobacillus have been used as a flavoring and texturizing agent as well as a preservative in food for centuries [1] and are now added as starters in food. LAB such as lactobacilli, inhibit food spoilage [2] and pathogenic bacteria and preserve the nutritive qualities of raw food material [3] for an extended shelf life. Recently, the use of metabolites of Lactobacillus as biological preservatives in food packaging materials [4] has been discussed. The antimicrobial effect of Lactobacillus is mainly due to their lactic and organic acid production [5], causing the pH of the growth environment to decrease [6]. Low pH induces organic acids to become lipid soluble and diffuse through the cell membrane into the cytoplasm [7].
Over the last three decades, a variety of lactic acid bacteria that are generally-recognized-as-safe (GRAS) [8] for use in foods have been found to produce bacteriocins that kill [9] or inhibit the growth of other bacteria [10]. Because of potential food-related applications [11], many bacteriocins have been identified and characterized, both biochemically [12] and genetically [13].
Consumers have been concerned about possible adverse health effects [14] from the presence of chemical additives in their foods. As a result, consumers are often drawn to natural and “fresher” foods with no chemical preservatives added [15]. This perception, coupled with the increasing demand for minimally-processed foods [16] with long shelf life [17] and convenience [18], has stimulated research interest in finding natural but effective preservatives. Bacteriocins, produced by LAB, may be considered natural preservatives [19] or biopreservatives [20] that can possibly fulfill these requirements. Biopreservation refers to the use of antagonistic microorganisms or their metabolic products to inhibit or destroy undesired microorganisms in foods to enhance food safety and extend shelf life [21]. Nisin, the best-known LAB bacteriocin, has been repeatedly shown to be safe and effective [22] for use in foods over the past 30 years. Pediocin [23] is another well-studied bacteriocin that will likely be the second LAB bacteriocin to be widely used in the food industry .A large number of other LAB bacteriocins has been identified and the list [24] is still growing.
Research on LAB bacteriocin Lactic acid bacteria produce a variety of antibacterial compounds such as organic acids, diacetyl, hydrogen peroxide and bacteriocin or bactericidal proteins during lactic fermentations [25] .Most of bacteriocins produced by grampositive bacteria are from lactic acid bacteria. The isolated bacteriocin was then tested for its antibacterial activity against various pathogenic microbes like staphylococcus aureus, enterococcus, pseudomonas, vibrio cholera, E coli, bacillus cereus etc.

MATERIALS AND METHODS:

Collection of samples:

Three sample units were collected from a food industry located in Chitoor. Each collected sample unit was placed in a separate sterilized container.

Serial dilution

1 g of sample was taken in 1st test tube to which 9 ml of distilled water was added. 1 ml was pipetted out of the first test tube and added to the second test tube containing 9 ml distilled water to make the total volume remain 10 ml. This was continued for 6 eppendrofs totally to reach a dilution factor of 10- 6.100 μl each was spread plated with an L-rod onto autoclaved MRS agar, nutrient agar, YPD, EMB agar plates. The plates were left overnight in the incubator at 37ºC. The plates were observed for presence of mixed colonies and each morphologically varying colony was tested by Gram staining and Biochemical tests to identify and confirm the different colonies. Once identified, the different colonies were subcultured and streaked onto an autoclaved plate of respective agar to obtain pure colonies.

Biochemical Characterization:

Voges Proskauer Test

100 ml of MR VP medium was prepared and autoclaved. 15 ml of medium was poured into 6 test tubes. Each test tube was inoculated with a single colony of Lactobacillus isolated from Aavin curd, Ranipet Curd, Benny Buttermilk, Bifilac, Darolac and Lactobacillus MTCC 2997 and left overnight on the shaker.
The next day, 1 ml of Barritt’s Reagent A and 1 ml Barritt’s Reagent B was pipetted and added to each test tube and the tubes left at room temperature for 15-30 min and color change observed.

Methyl Red Test

100 ml of MR VP medium was prepared and autoclaved.15 ml of medium was poured into 6 test tubes. Each test tube was inoculated with a single colony of Lactobacillus isolated from Aavin curd, Ranipet Curd, Benny Buttermilk, Bifilac, Darolac and Lactobacillus MTCC 2997 and left overnight on the shaker.
The next day, 1 ml methyl red indicator was pipetted and added to each boiling tube and the tubes left at room temperature for 15-20 min and color change observed.

Citrate Utilization Test

200 ml Simmons Citrate Agar was prepared and autoclaved. 15 ml agar each was poured into 6 boiling tubes. Each tube was inoculated with a colony by means of stab and streak inoculation. The boiling tubes were incubated at 35ºC for 48 hours and color change observed.

Catalase Test

A single colony was touched with a loop and smeared onto clean glass slide. Two drops of hydrogen peroxide was added to the smear. Presence or absence of free oxygen gas bubbles was checked for.

FTIR analysis

At the 0th hour, just after the side arm flasks containing milk were inoculated and kept in the shaker, 200 μl of sample from each of the side arm flasks except control was pipetted into eppendorfs. These eppendorfs were labeled, N0 - N7, covered tightly with paraffin wax and kept at -20ºC. Steps 1 and 2 were repeated after every hour for a period of 6 hours. Thus samples were collected for FT-IR analysis (Fig 15, 16, 17, 18).

Lyophilization of samples

The samples that were kept at -20ºC needed to be lyophilized before analysis by FT-IR.

Preparation of samples to obtain pure spectra for the 6 Lactobacillus strains by FTIR

6 flasks of MRS broth were prepared and autoclaved. A single colony from each pure culture plate of isolated Lactobacillus was picked and inoculated in MRS broth. The flasks were incubated overnight on the shaker. When the broths were found to be turbid, they were transferred to centrifuge tubes and centrifuged at 10,000 rpm for 10 min. The supernatants obtained were discarded and to the pellets 1 ml of phosphate buffer added. The contents were transferred to eppendorfs and centrifuged again at 10,000 rpm for 10 min. The pellets were washed with phosphate buffer once again. These pure pellets were first kept at -20º C overnight and then kept for lyophilization. The lyophilized pellets were then processed for FTIR and pure spectra for the 6 isolated Lactobacillus strains obtained.

Preparation of samples to obtain pure spectra for milk by FTIR

Pure pasteurized Aavin milk was bought from the retail outlet in Vellore. 100 μl of milk was poured on the sides of an eppendorf and immediately kept at - 20º C overnight. The eppendorf was then kept in the sample chamber of the lyophilizer and lyophilization carried out for 5-6 hours till solid flakes are obtained. The solid milk powder obtained after lyophilization were processed for FT-IR. A pure spectrum of milk was obtained.

Statistical Analysis from FTIR spectra data

The FTIR data obtained was used for statistical analysis using SPSS version 17.0 and Microsoft Excel 2007. Spectral changes were observed. Changes in peak area and height for characteristic lactic acid groups were done using Microsoft Excel 2007.
Using the software SPSS version 17.0, a number of analyses were done. Similarity matrices cluster analysis and correlation plots were made to compare how similar 2 strains of isolated Lactobacillus sp. are over the entire FTIR range of wave numbers are. ANOVA tables and curve estimation was done to compare each isolated strain with MTCC 2997. Distance correlation was also done to confirm results obtained by curve estimation. Hierarchical cluster analysis was also done to determine the most closely related strains.

Preparation of Crude bacteriocin

To prepare crude bacteriocin from Lactobacillus the isolates have to be grown in TGE broth and incubated at 37 degrees for 12 to 16 hours and then the culture from TGE broth is immobilized

TGE Broth

TGE Broth is a nonselective nutrient medium for the determination of bacterial counts by the membrane filtration method. This broth has the same formulation as Tryptone Glucose Extract Agar, except the agar has been omitted and ingredients are at twice the concentration.
Composition of TGE broth (Table 2)

Immobilization Test

Sodium alginate 6% and 4% of CaCl2 solution are autoclaved separately. Bacterial culture is mixed with 6% sodium alginate solution in the ratio of 25:40.Then the mixer is introduced as drops into the 4% CaCl2 solution with the help of a sterile syringe. Sodium alginate reacts with CaCl2 to produce beads in which bacterial cells are entrapped. The beads are washed in sterile water. After washing, the beads are inoculated into the production medium and incubated at 30 degree Celsius. For aeration the flask is kept in a shaker

Production Media

One percentage level of immobilized culture is transferred to the modified dairy based medium and incubated at 30 degree Celsius for 24 hours.

Kirby-Bauer Antimicrobial Susceptibility Test

Obtain a plate culture of one of the organisms to be tested. Using a sterile loop, emulsify a colony from the plate in the sterile saline solution. Mix thoroughly making sure that no solid material from the colony is visible.
Inoculation of Test Plates Optimally, within 15 minutes after adjusting the turbidity of the inoculum suspension, a sterile cotton swab is dipped into the adjusted suspension. The swab should be rotated several times and pressed firmly on the inside wall of the tube above the fluid level. This will remove excess inoculum from the swab. The dried surface of a Müeller-Hinton agar plate is inoculated by streaking the swab over the entire sterile agar surface. This procedure is repeated by streaking two more times, rotating the plate approximately 60° each time to ensure an even distribution of inoculum. As a final step, the rim of the agar is swabbed. The lid may be left ajar for 3 to 5 minutes, but no more than 15 minutes, to allow for any excess surface moisture to be absorbed before applying the drug impregnated disks.

Ammonium Sulfate Precipitation

This is a classic first step to fractionate proteins by causing perturbations in the solvent with respect to ionic strength. Historically, separation methods were limited and as a result precipitation methods were highly used with very fine cuts in precipitated conditions [26]. As more choices of inexpensive and quality resins are commercially available precipitation steps are typically limited to one or two initial cuts in the beginning of purification or simply used to concentrate the proteins. The major advantage to (NH4)2SO4 precipitation is that it easily causes the reversible precipitation of the protein and is non-denaturing [27] to the protein structure.

Protein Estimation by Lowry s Method

The phenolic group of tyrosine and trytophan residues in a protein will produce a blue purple color complex with maximum absorption in the region of 660 nm wavelength, with Folin- Ciocalteau reagent which consists of sodium tungstate molybdate and phosphate. Thus the intensity of color depends on the amount of these aromatic amino acids present and will thus vary for different proteins. Most proteins estimation techniques use Bovin Serum Albumin (BSA) universally as a standard protein, because of its low cost, high purity and ready availability. The method is sensitive down to about 10 μg/ml and is probably the most widely used protein assay despite its being only a relative method, subject to interference from Tris buffer, EDTA, nonionic and cationic detergents, carbohydrate, lipids and some salts. The incubation time is very critical for a reproducible assay. The reaction is also dependent on pH and a working range of pH 9 to10.5 is essential. Different dilutions of BSA solutions are prepared by mixing stock BSA solution (1 mg/ ml) and water in the test tube as given in the table. From these different dilutions, pipette out protein solution to different test tubes and add 500 ul of alkaline copper sulphate reagent (analytical reagent). Mix the solutions well.This solution is incubated at room temperature for 10 mins. Then add 150ul of reagent Folin Ciocalteau solution (reagent solutions) to each tube and incubate for 15min. Zero the colorimeter with blank and take the optical density (measure the absorbance) at 660 nm.Plot the absorbance against protein concentration to get a standard calibration curve. Check the absorbance of unknown sample and determine the concentration of the unknown sample using the standard curve plotted above.

Bradford protein assay

The Bradford assay is very fast and uses about the same amount of protein as the Lowry assay. It is fairly accurate and samples that are out of range can be retested within minutes. The Bradford is recommended for general use, especially for determining protein content of cell fractions [28] and assessing protein concentrations for gel electrophoresis [29].Assay materials including colour reagent, protein standard, and instruction booklet are available from Bio-Rad Corporation [30]. The method described below is for a 100 μl sample volume using 5 ml color reagent. It is sensitive to about 5 to 200 micrograms protein, depending on the dye quality. In assays using 5 ml color reagent prepared in lab, the sensitive range is closer to 5 to 100 μg protein. Scale down the volume for the "microassay procedure," which uses 1 ml cuvettes. Protocols, including use of microtiter plates are described in the flyer that comes with the Bio-Rad kit [31].
The assay is based on the observation that the absorbance maximum for an acidic solution of Coomassie Brilliant Blue G-250 shifts from 465 nm to 595 nm when binding to protein occurs [32]. Both hydrophobic and ionic interactions stabilize the anionic form of the dye, causing a visible color change. The assay is useful since the extinction coefficient of a dye-albumin complex solution is constant over a 10-fold concentration range [33].

Results and discussions:

Isolated Staphylococcus aureus colonies in Milk powder (Figure 1) and cocoa powder

Voges- Proskauer Test

Development of a deep rose colour in the culture; 15 minutes following the addition of Barritt’s reagent is indicative of the presence of acetylmethyl carbinol and represents a positive result.

Methyl Red Test

The pH indicator methyl red detects the presence of large concentrations of acid end products. The methyl red indicator in the pH range of 4 will turn red which is indicative of a positive test (Fig. 11).

Citrate Utilization Test

Citrate-positive cultures are identified by the presence of growth on the surface of the slant, which is accompanied by blue coloration (Fig.12).

Oxidase Test

The enzyme in the presence of atmospheric O2 oxidises colourless substrate tetramethyl- p – phenylenediamine dihydrochloride to form a dark purple patch (Fig.13).

Catalase Test

Catalase production can be determined by adding the substrate hydrogen peroxide to a smear of the culture on slide [34]. If catalase is present, bubbles of free oxygen gas is produced (Fig. 14). This is a positive catalase test and the absence of bubble formation is a negative catalase test.
Organism A may be Staphylococcus aureus. Organism B may be Bacillus cereus. Colonies were seen in MRS media .It showed positive results for methyl red and voges proskauer Test and negative result for catalase test. It showed gram positive result. Morphology was long slender rods which formed chains. Hence we infer that, colonies found in MRS media were of lactobacillus.

Result of FTIR analysis:

The prominent groups of lactic acid are the carboxylic group (OH-C=O) which lies in the range of wave numbers 1760 – 1670 cm-1 and the alkyl group (-CH3) which lies in the range of wave numbers 2960 – 2850 cm-1. The detection and increase of these peaks in the spectra obtained by FTIR indicate the positive production of lactic acid in both milk powder and cocoa powder. An increase in peak height is directly proportional to an increase in quantity [35]. Various other functional groups were also found in cocoa powder and milk powder as indicated in the table.
After the FTIR analysis, it was found that the functional groups present in cocoa powder and milk powder is the almost the same as the functional groups present in cocoa powder and milk powder after the addition of microorganisms.

Results of Lowry estimation:

Crude bacteriocin
Stock Solution= 1 ug/ul
Crude bacteriocin formed zone of inhibition of 18mm against enteroccoccus and 15mm against staphyloccus aureus. As bacteriocin was purified by ammonium sulfate precipitate and dialysis; diameter of zone of inhibition was increased respectively and highest zone of inhibition was found after dialysis. It was found that the bacteriocin obtained was effective against enterococcus and staphylococcus aureus as it showed the zone of inhibition. From the Kirby bauer chart, it was concluded that enterococcus was susceptible to bacteriocin and staphylococcus aureus showed intermediate effect. Cocoa powder and milk powder were tested for the presence of bacteria and it was found that it contains Staphylococcus aureus, Bacillus cereus,E Coli and lactobacillus. FTIR results showed the presence of functional groups. Lactobacillus was used for the production of bacteriocin. The present investigation highlights the isolation, characterization and activity of bacteriocin produced by Lactobacillus. Culture supernatant of Lactobacillus was tested for antibacterial activity. Protein (bacteriocin )after 30% ammonium sulfate precipitate 70%ammonium sulfate precipitate and dialysis was also tested for antibacterial activity against gram-positive and gram-negative bacteria such as staphylococcus aureus, enterococcus, pseudomonas, vibrio cholera, E coli, Bacillus cereus. Crude bacteriocin formed zone of inhibition of 18mm against enteroccoccus and 15mm against staphyloccus aureus. As bacteriocin was purified by ammonium sulfate precipitate and dialysis; diameter of zone of inhibition was increased respectively and highest zone of inhibition was found after dialysis. From the Kirby bauer chart [36], it was concluded that enterococcus was susceptible to bacteriocin and staphylococcus aureus showed intermediate effect. Protein concentration of bacteriocin was estimated after centrifugation, 30%ammonium sulfate precipitate, 70%ammonium sulfate and dialysis. Estimation of protein concentration was done by lowry’s method.63.7ug/ml protein concentration was found in crude bacteriocin protein concentration was increased with more purification and results showed maximum protein concentration of 80.1ug/ml after dialysis. Possession of bacteriocin by Lactobacillus is an indication that the bacteria can be used as probiotic and as biopreservative. Various physicochemical factors seemed to affect bacteriocin production as well as its activity.

CONCLUSION:

Cocoa powder and milk powder were tested for the presence of bacteria and it was found that it contains Staphylococcus aureus, Bacillus cereus, E.Coli and lactobacillus. FTIR results showed the presence of functional groups. The bacteriocin produced by Lactobacillus was assayed by agar well diffusion method and bacteriocin concentration was measured. The zone of inhibition was measured and it was found to be the maximum after dialysis. Mode of action of bacteriocin produced by Lactobacillus was tested and the behavior of the bacteriocin produced by isolated strain was considered as bactericidal.

Tables at a glance

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