Identification of sources of Pediococcus species

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RESULTS AND DISCUSSION

5.1 Identification of sources of Pediococcus sps.

In the present study the various sources chosen for isolating Pediococcus sps. were cow milk, sheep milk, fresh cream, buffalo milk, curd and idli batter.

It was observed that in a study of Characterization of Lactic Acid Bacteria from Raw Milk Samples of Cow, Goat, Sheep, Camel and Buffalo with Special Elucidation to Lactic Acid Production, various lactic acid bacterial strains isolated from these sources were found to produce numerous colonies (Sharma et al., 2013).

Similarly another study was conducted in Nigeria relating to Screening of Lactic Acid Bacteria Strains Isolated from Some Nigerian Fermented Foods for EPS Production showed about One hundred and thirteen lactic acid bacterial isolates that were obtained from seven varieties of fermented foods (Bukola C. Adebayo-tayo and Abiodun A. Onilud., 2008). Lactic acid bacteria are most commonly found to occur in milk and milk products and also from locally produced yoghurt (Azadina et al., 2009; Forouhandeh et al., 2010).

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5.1.1 Collection transportation and processing of samples

The samples were collected in sterile containers. Aseptic conditions were maintained while collecting the samples to ensure no contamination. These samples were transported to the laboratory. The samples were processed accordingly for the isolation of Pediococcus sps. Recent studies have shown that the samples like milk, curd, butter and idly batter have to be processed to isolate Pediococcus from them.

5.2 Isolation and enumeration of Pediococcus sps. from selected samples

5.2.2 Use of Selective media

The Pediococcus sps. were successfully isolated using the Pediococcus selective media (PSM). We observed that the colonies of Pediococcus sps.were present within the agar due to its microaerophilic nature.

From a study for Enumeration and identification of pediococci in powder-based products using selective media and rapid PFGE, it has been proved that PSM is the selective medium for the growth of Pediococcus sps. It supports only the growth of Pediococcus sps. and not any other organism (Simpson et al., 2006). Similarly another study for the Enumeration of probiotic pediococci in animal feed: interlaboratory study, showed that this media supports the growth of Pediococcus sps. even if this species is present at a much lower concentration than other species that can be normally produced on the MRS agars (Leuschner et al., 2003). Previous studies have revealed that isolates when subjected to growth on selective MRS agar media produced round shape, off-white to cream coloured colonies, similar to colonies of Lactobacillus sps. produced on MRS agar media (Chowdhury et al., 2012).

5.3 Morphological studies of isolated Pediococcus sps.

The isolated samples of Pediococcus were streaked to study the colony morphology. It was seen that Pediococcus sps. forms a convex shaped colony with regular margins. Colonies developed show opaque nature. Very small cream colored colonies were observed which are of small size and numerously scattered.

Gram staining for the isolated colonies of Pediococcus sps. was performed to provide more information on the morphological characteristics. It was observed from our study that the colonies obtained were gram positive cocci arranged in tetrads. They were found to be very small in size. Samples of cow milk and idly batter contained gram positive cocci. They were violet colored non motile colonies that are circular and arranged in tetrads both scattered and numerous in number. Samples of goat milk and curd contained gram positive bacilli. They were Violet colored, rod shaped, non-motile, bacilli in chains and clusters present in numerous numbers. Hence this supports in the partial identification of the isolated bacteria as Pediococcus sps. (Cai et al., 1999).

A study conducted on Isolation, Identification and Characterization of Lactic Acid Bacteria from Dairy Sludge Sample showed the Cultural and morphological characteristics which showed different types of lactic acid bacteria, majority of them being Gram positive rods and cocci shaped bacteria (Choksi Nikita and Desai Hemangi, 2012). Similarly another study for Screening of Lactobacillus spp. from Buffalo Yoghurt for Probiotic and Antibacterial Activity showed that the isolated bacteria from yoghurt were rod shaped convex colonies that are circular and gram positive being facultative anaerobic belonging to the Lactobacillus sps. Most isolates when subjected to Gram staining were found to be rod shaped or cocci, positive in Gram reaction being the typical characteristics of Lactobacillus sps. (Chowdhury et al., 2012).

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5.4 Biochemical characterization of isolated Pediococcus sps.

Various biochemical tests were performed to determine whether the isolated bacteria were Pediococcus sps. The biochemical tests that were performed were indole production test, methyl red and Voges-Proskauer test, citrate utilization test, triple sugar iron test, mannitol motility nitrate reduction test, catalase,urease and starch hydrolysis.

Pediococcus sps. showed positive results for mannitol motility nitrate reduction test and urease test. Since Pediococcus is a lactic acid producing bacteria it metabolizes the mannitol in mannitol motility test. Nitrate provided in the mannitol motility medium was reduced. By production of urease, urea was split and thus degraded. It showed negative results for IMVic test, catalase test, starch hydrolysis test and triple sugar iron test.

In a study conducted for Screening of Lactobacillus spp. from Buffalo Yoghurt for Probiotic and Antibacterial Activity the biochemical characterization showed catalase, oxidase, indole, MR, VP and citrate tests as negative for most isolates of lactic acid bacteria. However most of the samples were positive for carbohydrate utilisation (Chowdhury et al., 2012). In a study of Effect of Natural Foods Used in Traditional South Indian Cuisine that Contain Prebiotics on Probiotic Lactobacillus Species, most of the Lactobacillus sps. such as L.plantarum, L.acidophilus and L.rhamnosus showed positive results for methyl red test. An exemption of L.casei showed a negative result (Joel et al., 2015).

5.5 Differential medium

In our study it was observed that Mannitol salt agar media and Mac Conkey media showed no growth. Since MSA showed no growth we concluded that the organism was Gram positive. No growth in Mac Conkey media proved that the organism was non Staphylococcus.

In the study of Antibacterial activity ofLactobacillus acidophilusandLactobacillus caseiagainst methicillin-resistantStaphylococcus aureus(MRSA) it was observed that only MRSA could grow on Mannitol salt agar medium, but Lactic acid bacterial strains used in this study were unable to grow on the MSA agar plates.(Karska-Wysocki et al., 2009)

5.5.1 Blood agar

In our current study we observed that there was no growth of bacterial colonies seen on the blood agar plate. This confirmed that there was no hemolytic activity found on the blood agar plates hence confirming that the isolated bacteria were non pathogenic.

This is supported by a study conducted by Estifanos Hawaz on the isolation and identification of probiotic lactic acid bacteria from curd and in vitro evaluation of its growth inhibition activities against pathogenic bacteria. From this study it is evident that when the selected strains were grown in when grown in Columbia human blood agar, they did not exhibit beta hemolytic activity. Whereas, seven other strains did not show any hemolytic activity which confirms non pathogenecity.

5.6 Study of extension of the shelf life of selected food samples using Pediococcus sps.

The study of extension of the shelf life of certain set of food samples was done by spraying the culture of Pediococcus sps. The changes occurring in the physical features of food samples were continuously monitored and recorded. The various samples like tomatoes, fish, strawberries, mutton were sprayed with different volumes of Pediococcus sps. cultures such as 5ml, 10ml and 15ml. During the initial two days the samples developed a characteristic odor but however no changes in the physical features were observed. On the third day the fish sample containing 15ml of the culture was found to be spoilt. On the same day all samples of mutton containing different volumes of culture were rotten with green coloration. It was also noticed that strawberries with 10ml and 15ml of culture had lost their firmness but however the sample containing 5ml and all tomato samples remained fresh. On the fourth day it was seen that all strawberries and fish samples were spoilt and tomato still retained their freshness. On the fourteenth day the tomato sample sprayed with 5ml sample culture continued to maintain its freshness and therefore proved to show the best result. The remaining samples of tomatoes were found to be spoilt. From this continuous monitoring of food samples we could analyze that our samples sprayed with the Pediococcus sps. culture showed to have extended shelf life when compared with the control. Hence we can state that our study clearly indicates the biopreservative efficiency of Pediococcus sps. and it can thus successfully be used as a biopreservative.

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A research based on inhibition of Listeria monocytogenes in cottage cheese manufactured with a lacticin 3147-producing starter culture, proved the effect of utility of lacticin which is a bacteriocin like substance that has shown to inhibit various strains of Listeria monocytogenes and hence improved the shelf life significantly (Mcauliffe et al., 1999)

5.7 Extraction of bacteriocin

Chloroform extraction method was used to extract the bacteriocin produced by the Pediococcus sps. which yielded three layered complex namely, supernatant layer, interfacial layer and the bottom layer. The interfacial layer which was assumed to be the extracted bacteriocin was produced in very small amounts.

A study on the molecular profiling and antimicrobial activity of bacteriocin from Bacillus subtilis revealed that the interfacial layer which when subjected to HPLC confirmed that the extract was bacteriocin (Berlina Dhas S and J.Vimalin Hena, 2012).

5.8 Inhibitory effect of bacteriocin- Well diffusion technique

The presence of the extracted bacteriocin was tested using traditional well diffusion technique. The presence of zone of inhibition was recorded. Intracellular product that was inoculated on the swabbed media of Shigella culture showed positive results and negative results for extracellular product. The zone of inhibition for intracellular product was measured to be 0.8cm. Furthermore, E. coli showed positive results for both extracellular and intracellular products and their zone of inhibitions were found to be 1cm and 0.5cm respectively. However, both the intracellular and extracellular products that were inoculated on the swabbed media of Streptococcus culture showed negative results. Hence, it was observed that a larger zone of inhibition was observed by extracellular product. Thus, the inhibitory effect of the bacteriocin on the clinical isolates was confirmed.

Our results are in good agreement with the research conducted by (Nivedita Sharma and Neha Gautam, 2007) which showed that the zone of inhibition to be 5mm and 3mm when culture supernatant of B. mycoides was inoculated into the wells thus inhibiting L. mesenteroides.

5.9. Protein detection

5.9.1. Biuret test

The presence of protein in the bacteriocin extracted was detected by using the Biuret assay in the supernatant layer that was obtained. Purple colored solution was obtained when the Biuret reagent was added confirming the protein nature of the bacteriocin.

Biuret test is the simplest test that can be conducted for the detection of the protein. The principle behind Biuret reagent is the binding of the copper(II) ions to the peptide bonds in the protein in an alkaline solution. Thus a purple colored complex is obtained. Hence the intensity of the purple color obtained would be proportional to the amount of the peptide bonds present in the sample. As a result of this the protein can be detected with ease (Rose, 1833).

5.9.2. Millon’s test

The presence of protein in the bacteriocin extracted was also detected by using the Millon’s test in the supernatant layer that was obtained. Red colored solution was obtained in the when the Millon’s reagent was added determining the protein nature of the bacteriocin.

Millon's test candetectphenolic compounds, such astyrosine. When a reagentcontaining mercuric nitrate dissolved in nitric acid is added to the test solution and in presence of heating, a pink to dark-red solution or precipitate is formed. This is an indication thattyrosineis present. As a result, any compound containing a phenolic hydroxyl group is confirmed to be positive. Sincetyrosineis anamino acid that is present in most of theproteins, Millon's test can be used for detectingproteins (O’Farrell, 1961).

5.10. Ammonium sulphate precipitation

The protein thus detected was subjected to ammonium sulphate precipitation to purify the protein. The ammonium sulphate precipitation was carried out at 40% saturation. Furthermore the supernatant and pellet thus obtained was tested for estimation of protein concentration and antimicrobial potential.

Earlier, the inhibitory activity of bacteriocin isolated from malted barley was precipitated from cell free supernatant using 40 % ammonium sulphate saturation, and resuspended in 2 mmol sodium phosphate buffer, pH=6.0 and purified using chromatography (Vaughan et al., 2001).

Lactacin F, a bacteriocin produced by Lactobacillus acidophilus 11088 (NCK88), was purified by ammonium sulphate precipitation at 40% saturation successfully and thus resulted in a 474-fold increase in specific activity of lactacin F (Muriana PM and Klaenhammer TR, 1991).

5.11. Lowry’s test

The protein that was subjected to ammonium sulphate precipitation was estimated by Lowry’s test. Lowry’s test was separately performed for supernatant and pellet after centrifugation. Following the incubation durations O.D readings at 660 nm was recorded. By plotting a graph of O.D vs amount of protein it was possible to estimate the amount of protein present in supernatant and pellet respectively. After extrapolation the amount of protein present in supernatant was found to be 87ug and amount of protein present in pellet was found to be 18ug respectively.

By this method, total level of protein was determined. Under alkaline conditions the divalent copper ion reacts with peptide bonds to form a complex that is in turn reduced to obtain a monovalent ion. Then a reaction between monovalent copper ion and the radical groups of tyrosine, tryptophan, and cysteine occurs with Folin reagent to produce an unstable product. Next this gets reduced to molybdenum/tungsten blue. The main basis being the reduction of the Folin–Ciocalteu reagent and oxidation ofaromaticresidues such astryptophan andtyrosine. The total concentration of protein in the sample was deduced from the concentration of Trp and Tyr residues that reduce the Folin–Ciocalteu reagent (Lowry et al., 1951).

5.12. Determination of bacteriocin-intracellular or extracellular

To identify if the bacteriocin is intracellular or extracellular standard antimicrobial activity was performed. The antimicrobial potential of supernatant and pellet was determined by using the traditional well diffusion technique. Supernatant and pellet were added to the wells and were observed for the zone of inhibition results. Positive results were obtained for supernatant with zone of inhibition of 0.8mm and for pellet with zone of inhibition of 0.6mm. Clearly zone of inhibition was identified for supernatant as well as for pellet. However, supernatant was found to have larger zone of inhibition with better antimicrobial activity. Hence it is evident that ammonium sulphate precipitation was unsuccessful as purified protein (pellet) is supposed to show better antimicrobial activity than supernatant solution.

In a study of Purification, Stability and Efficacy of Bacteriocin from Isolates of Natural Lactic Acid Fermentation of Vegetables the fraction with the highest bacteriocin activity was precipitated with 20–30 % (by mass per volume) ammonium sulphate. The antimicrobial activity (in terms of inhibition zone diameter) increased from 12 to 23 mm. There was 1.91-fold increase in the partially purified bacteriocin activity than that of crude bacteriocin (Vinod Kumar Joshi et al., 2006).

5.13 Comparative studies of preservation efficiency against chemical preservatives

The tomato samples were sprayed with the chemical preservative sodium sulphite and their efficiency of preservations was compared along with the biopreservative efficiency of lactic acid bacteria. The changes in the physical features of the food samples were recorded. Various changes include freshness, firmness, colour, damage etc. Different conditions like 30ºC, 4ºC, light and dark conditions were set. It was observed that at 30ºC the tomato sample sprayed with extracellular product remained fresh for longest period of 8 days whereas control and intracellular sample were spoilt on day 3 and sodium sulphite sample was spoilt on day 6.

In dark condition it was observed that the tomato sample sprayed with extracellular product remained fresh for the longest period of 8 days whereas control and intracellular sample were spoilt on day 4 sodium sulphite sample was spoilt on day 6.

It was observed that at 4ºC the tomato sample sprayed with extracellular product remained fresh for longest period of 20 days whereas tomato samples sprayed with control and intracellular samples were spoilt on day 8, crude culture was found fresh only till day 9. The tomato samples sprayed with sodium sulphite was found to be fresh only till day 16. Sodium chloride is another chemical preservative that was chosen and this lasted only for about 15 days.

Therefore, all these data confirm that extracellular product has the highest antimicrobial activity and is much better than the chosen chemical preservatives.

In a study of Application of purified bacteriocin produced by Lactococcus lactis AP2 as food biopreservative in acidic foods it was noticed that among biopreservatives and chemical preservatives used to enhance the shelf life of orange juice, purified bacteriocin confirmed to be better a preservative having less mean value 7.65 log CFU/ml as compared to sodium benzoate with indicated a higher mean value 7.97 log CFU/ml at 4ºC (Amit Pratush et al., 2012).

5.14 SDS Page

The protein was purified by SDS Page method. A single band was observed in supernatant lane and no bands were seen in pellet lane. Molecular weight was hence determined corresponding to the marker with respect to the band. It was observed that the supernatant band corresponded to 30kDa. Thus the result obtained by SDS Page was recorded and hence approximated.

With respect to a study conducted on Purification and characterization of a new bacteriocin isolated from a Camobacterium sps. produced a singular indicating molecular weight of approximately 4,500 to 5,000kDa (Stoffels et al., 1992).

Additionally in another study of partial purification and characterization of a bacteriocin produced by Enterococcus faecium 130 isolated from mozzarella cheese, the bacteriocin of E. faecium 130 which was resolved by SDS-PAGE. It was seen that the molecular weight of the bacteriocin was identified between 3.5 and 14.3 kDa with silver staining (Tulini et al., 2011).