Isolation of bacteriocin producing bacteria from mango pulp and its antimicrobial activity

Transcription

1 Journal of Microbiology and Biotechnology Research Scholars Research Library J. Microbiol. Biotech. Res., 2011, 1 (2): ( ISSN : CODEN (USA) : JMBRB4 Isolation of bacteriocin producing bacteria from mango pulp and its antimicrobial activity V. Ravi 1*, M.Prabhu 2, D.Subramanyam 3 1. Department of Environmental Science, Sri Venkateswara University, Tirupati, A.P, India 2. Department of Microbiology, Sri Venkateswara University, Tirupati, A.P, India 3. Department of Biotechnology, Dravidian University, Kuppam, A.P, India ABSTRACT The objective of the present study was to isolate, characterize, and identify the bacteriocin producing lactic acid bacteria (LAB) from mango pulp. Antimicrobial activity of the isolated strain was tested against with spoilage pathogens. The extracted antimicrobial compound was also tested for bactericin assay and antimicrobial properties against to pulp spoilage bacteria. The present work was also focused on effect of some physiological conditions like detergents, heat and ph, and enzymes. The antimicrobial compound was also tested for maintaining the decreased CFU during the pulp storage along with the chemical preservative Potassium Meta bisulphate (PMS). Total six bacterial isolates were isolated, out of all the six isolated strains one was selected for further work, which shows the probiotic characters like bacteriocin assay and antimicrobial properties. The extracted compound shows the decreased CFU used as preservative during the pulp storage when compared to a without any preservative and also to the chemical preservative. The proteinase K, pepsin shows the negative effect on the activity of extracted compound from Lactic acid bacteria. Although, enzymes like trypsin and α- chymotrypsin has no effect on bacteriocin, and there is no effect of surfactants on bactreiocin.the effect of ph on the culture supernatant was studied in range from 2 to 12. They are not changed under a wide ph range from 3 to 9, increased ph after 9 decreases the activity of the compound. The results obtained from this study the isolated lactic acid bacteria show the potent bacteriocin and antimicrobial properties that exerts in the usage of this compound as a preservative in mango or fruit pulp industry. Key words: Lactobacilli, Bacteriocin, Mango pulp, Antimicrobial activity, biopreservative. 54

2 INTRODUCTION Mango (Mangifera indica L.), as an emerging tropical export crop is produced in about 90 countries in the world with a production of over 25.1 million tones. Asia is the main producer with 76.9% of the total world production, followed by America with 13.38%, Africa with 9% and less than 1% each for Europe and Oceania. Pakistan is the 5th largest mango producer with production of tones per year contribution with a share of 7.6% in the world market [1]. Mango is a short seasoned fruit and being highly perishable does not withstand even in cold storage. Therefore, most of the fruit processing industry in Pakistan preserves mango pulp for the manufacture of mango products around the year. Mango pulp is not generally consumed directly rather used as fillings for pastries, jams, sauces, fruit juices and drinks [2]. The conventional method to prevent the spoilage of mango pulp is the applying a heat treatment to the fruit juices during manufacturing and by the natural low ph of the juices [3,4]. Spoilage of fruit juices and fruit juice products by thermophilic bacteria has become a major concern as spoilage cases of acidic vegetables, fruit juices and fruit juice products Alicyclobacillus spp. has been reported [5]. Most of these spoilage bacteria has the fatty acids as the major components of the cellular membrane [6], and they can be grown at C [7]. Apart from this spoilage bacterial group some of the other microbial populations were also present in the mango fruit juices, and they must be protecting the fruit juices from the spoilage microbial population. Being the next cheapest method of preservation, chemical preservation of mango pulp is the most common and widely used in Pakistan. The chemical preservatives are used to prevent the food spoilage due to microbial attack and thus are effectively used in combinations for better preservation. No single preservative is completely effective against all microorganisms [8]. Sodium benzoate (SB) and potassium metabisulphite (PMS) are commonly used as preservatives for long term storage of fruit pulp because of their better antimicrobial activity [9,10,11]. The effect of SB on the growth and survival of some yeast strains, food poisoning and spoilage organisms has been widely reported [12, 13]. Among the chemical preservative tested in single doses of ppm PMS and 500-ppm SB showed no sign of micro flora up to 90 days. Besides having an inhibitory effect on micro flora in the stored pulp, addition of these chemicals may adversely influence its physico-chemical and sensory profile. It has been reported earlier that preservatives have influence on physico-chemical characteristics of mango pulp as storage caused an increase in acidity, brix, reducing sugars and decrease in sucrose [2]. Lactobacilli are important organisms recognized for their fermentative ability as well as their health and nutritional benefits [14]. They produce various compounds such as organic acids, diacetyl, hydrogen peroxide, and bacteriocin or bactericidal proteins during lactic fermentations [15]. Antagonistic effects produced by lactic acid bacteria toward other organisms may play an important role in maintaining a proper microbial balance in the intestinal tract and preserving certain foods. Some bacteria produce bacteriocins which are protein or protein related complexes that inhibit other bacteria that are usually closely related. A number of lactobacilli including strains of Lactobacilus acidophilus produce bacteriocins. Most bacteriocins are heat stable and 55

3 sensitive to certain proteolytic enzymes. Lactic acid bacteria are widely used as starter cultures and play an important role in food preservation, microbiological stability and production of aroma compounds. Lactobacillus lactis and Lactobacillus plantarum were potent producers of bacteriocins. Bacteriocin produced by these Lactobacillus species had a large spectrum of inhibition against food spoilage microorganisms and various related strains of lactic acid bacteria. The bacteriocin inhibited the growth of Escherichia coli but it showed least activity against Candida albicans. The antibacterial activity appeared to be pronounced between late logarithmic phase and early stationary phase. The present work was carried out in order to investigate the inhibitory effect of bacteriocin- a compound extracted from lactic acid bacteria isolated from mango pulp itself and play a major role in the inhibition of spoiled bacteria along with PMS used as preservative for controlling the growth and survival of micro flora of mango pulp. MATERIALS AND METHODS Sample collection Mango fruit pulp was collected from Mango pulp industry in Chittoor, Andhra Pradesh, India. The entire collected sample was stored at 4 0 C until the microbiological experiments in the laboratory. Isolation of bacteriocin producing bacteria from mango pulp Homogenized mango pulp was serially diluted and inoculated on a selective MRS agar media (HI MEDIA) for Lactobacillus and the plates were allowed to incubate at 37 0 C in a candle jar for 48 h. The lactobacilli were identified on the basis of growth on selective MRS agar (ph 5.2), cell morphology, gram staining, and catalase activity. Further identification of the species of these lactobacilli was performed according to carbohydrate fermentation patterns, and growth on MRS broth (HI MEDIA), as described in Bergey s Manual of Systematic Bacteriology [16]. The isolated lactobacilli were sub cultured and the purified cultures were maintained at C in MRS broth with 10% glycerol. Screening for Bacteriocin producing strain Screening activity of pure isolated strain was determined by agar disc method [17] against the standard laboratory test organisms like E.coli, Staphylococcus, Bacillus, pseudomonas, Proteus and Klebsiella. Morphological and Biochemical identification of isolated strain The potent bacterial strain were characterized by morphological and biochemical methods. The biochemical tests includes Indole test, Methyl red- Voges prosker test, Citrate utilization test, catalase test, Starch, Gelatin and Casein hydrolysis tests and carbohydrate fermentation test were performed. Antimicrobial activity of isolated strain by Disc diffusion method 56

4 The antimicrobial activity of culture supernatants of bacteriocin producing strain on various gram positive, gram negative organisms and C. albicans were tested by the disc diffusion. In brief, 0.5 mm-diameter containing Whatmann Number 1 filter paper discs were prepared and placed on preinoculated agar medium and press gently and the discs are filled with bacteriocin extract at different concentrations. The plates were incubated at 37 0 C for h; inhibition zones around the wells were measured and recorded. Preparation of culture supernatants The bacteriocin-producing strain was grown in MRS broth (ph 5.5) at 37 0 C for h. The isolated lactobacilli culture was centrifuged at 10,000 rpm for 5 min, and then the supernatant was adjusted to ph with 1N NaOH [18]. Bacteriocin assay The antimicrobial activity of Bacteriocin was detected by the agar-spot test 18. The test was performed as follows: 200 µl of each isolated bacterial culture at the early exponential growth phase (OD 600 of ) in MRS broth was mixed with 4 ml of MRS soft agar (0.6% agar, prewarmed to 48 0 C) and poured on an MRS agar plate. Then, 3 µl of each culture supernatant was dropped onto the solidified soft agar. The plates were incubated for 48h unaerobically in a candle jar. Bacteriocin inhibition was indicated by a clear zone in the soft agar layer. Effect of bcateriocin on mango pulp preservation by the estimation of CFU in pulp The determination of the total microbial contamination of the pulp samples was performed after 30 days by the addition of Bacteriocin compound extracted from lactic acid bacteria until three months by the method (with some modification) outlined in compendium of methods for the microbiological examination of foods [19]. Nutrient agar, (HI MEDIA) was used for periodical determination of total bacterial count (TBC) in the stored mango pulp sample. One-gram sample was serially diluted and enumerated onto Nutrient agar. Plates were subsequently incubated for 24-48h at 37 C and TBC was calculated using colony counter. The experiment was repeated twice and reported data represent mean values (CFU/g) of these measurements Effect of enzymes, ph, detergents, and heat treatment The sensitivity of the active substance to proteolytic and other enzymes was tested on cell-free supernatant (ph 6.0) of 24 h cultures incubated at 37 0 C. Samples of 100µl were treated for 2 h with 0.1 mg/ml -1 and 1.0 mg/ml -1 final concentration of the following enzymes: proteinase K, pepsin, trypsin, α-chymotrypsin. The surfactants tested were Tritone X-100, sodium dodecyl sulphate (SDS), Tween 80, EDTA, and urea at final concentration 0.1, 1, 1.5, 2, 3 and 5%. Controls consisted of either active supernatant or detergents used. All samples and controls were incubated at 37 0 C for 5 h and tested for activity. The sensitivity of the active substance to different ph was estimated by adjusting the ph of supernatant samples to ph 2, 4, 6, 8, 10, and 12 with NaOH or HCl and testing against the indicator strain after 30 min and 2 h incubation. The sensitivity to heat was tested by heating cell free supernatant samples to 30, 50, 70, 90, 100, and C and testing the residual activity after 30 min by the agar dilution assay. Optimum conditions for bacteriocin Production All the optimum conditions are tested in specific production medium which can replaces the different types of carbon and nitrogen sources. Production medium contains Peptone10g; Carbon 57

5 source 20; Potassium phosphate 2g; Sodium acetate 5g; Magnesium sulphate 0.2g; Manganese sulphate 0.05g; with 1L distilled water. Effect of Carbon and Nitrogen source The study of bacteriocin production with various carbon sources in the production medium supplemented with different types of carbon sources like lactose, starch, maltose and sucrose and nitrogen sources like peptone, tryptone, yeast extract. The sterilized medium flasks were inoculated with 0.1% inoculum and incubated on rotary shaker at 120rpm for 2days and after those microbial cells were removed by centrifugation at 3000rpm for 15min. Anti-microbial activity in the supernatant was estimated by disc method shown in the above procedure. RESULTS Isolation and Screening of Bacteriocin producing bacteria form mango fruit pulp Total six similar (color and appearance) bacterial strains were isolated form mango pulp and all the six isolated strains were tested to produce the bacteriocin. The morphological, physiological, biochemical characteristics and anti-microbial properties were observed; finally one isolate is selected for optimization based on the above characteristics. Morphological and Biochemical characteristics of LB strain Growth and Colony characteristics The morphological features of bacterial colonies were observed on culture medium. On the culture agar plate, colonies were white in color, circular in shape and convex elevated with flat and rough textured. Table: 1 Morphological Characteristics and Biochemical tests for isolated strain S. No Tests LB Strain 1 Media used for inoculums MRS medium 2 Colony character Round, yellow-whitish 3 Colony on Nutrient media White 4 Grams staining +ve rods 5 Biochemical tests a) Indole tests - b) Methyl red tests - c) VP tests - d) Citrate utilization tests - e) Oxidase tests - f) Nitrate tests - g) Catalase tests - h) Gelatin - i) casienase + Antimicrobial activity The culture supernatants obtained from isolated strain was tested for antimicrobial activity against the laboratory test organisms and some fruit spoilage bacteria like E.coli and Staphylococcus aureus and Pseudomonas. aeruginosa and Candida albicans. The isolated strain 58

6 V. Ravi et al J. Microbiol. Biotech. Res., 2011, 1 (2):54-63 inhibits the growth of the E.coli, Pseudomonas. aeruginosa and Staphylococcus aureus. However, there is no inhibitory activity on Candida albicans A) B) Fig: 1 Antimicrobial activity of isolated strain against to A) E.coli B) Staphylococcus Effect of bcateriocin on mango pulp preservation by the estimation of CFU in pulp The present result indicates the bacterial growth pattern at various time intervals and the systematic increase in CFU/g of the mango samples, corresponding to the storage time. The growth was found to be highest at the termination of the storage period which may be attributed to the variability in the temperature and the chemical changes, specifically alteration in ph of the system that would take place resulting from the presence of the chemical preservatives in the samples CFU/gr Effect of Storage on microbial growth of mango pulp preserved with LAB and PMS Control LAB PMS Time in Days Fig: 2 Effect of storage on microbial growth of mango pulp preserved with bacteriocin and PMS. The values are means of at least two experiments (SD±) Effect of enzymes, ph, detergents, and heat treatment Result The inhibitory activity of the extracted compound in supernatant was tested under conditions which eliminate the possible effect of organic acids by adjusting the ph of the cell free supernatant to ph 6.5 and of hydrogen peroxide by catalase treatment. In the both cases the activity was not affected. The effect of various enzymes on the inhibitory agent was studied. Complete inactivation or significant reduction in the activity was observed after treatment of the cell free supernatant or purified inhibitory substance with proteinase K, pepsin which indicated 59

7 of the proteinaceous nature of the active agent. At the same time trypsin and chymotrypsin had no effect. In the present investigations the tested enzymes like amylase and lipase, did not cause inactivation. This concerned that carbohydrate and lipid moieties were not required for the inhibitory activity (Table 2). The exposure to surfactants showed they did not affect the activity of the bacteriocin in the concentration used. The antimicrobial substance in neutralized active culture supernatant appeared to be relatively heat labile as it was inactivated partially at 80 0 C for 10 min and completely at C for 10 min (Table 2). The effect of ph on the culture supernatant was studied in range from 2 to 12. They are not changed under a wide ph range from 3 to 9 increased ph after 9 decreases the activity of the compound. Table: 2 Effect of enzymes, ph, detergents and heat treatment on inhibitory activity of LB strain cell-free supernatant: S. No Treatment Activity (AU ml -1 ) 1 Untreated Enzymes α-chymotripsin + 3 trypsin + 4 pepsin - 5 proteinase K - 6 α-amylase + 7 lipase + ph Heat Agent 20 Tween 20 (1%) + 21 Tween 80 (1%) + 22 Urea (1%) + 23 SDS (1%) + 24 Triton X-100 (1%) + 25 EDTA (0.1%) + 26 EDTA (2%) + Effect of Carbon and Nitrogen sources Different carbon sources are used for the Bacteriocin production, when maltose was used as carbon source it shows (Fig: 3) maximum antimicrobial activity compared to remaining carbohydrates such as sucrose, lactose and starch respectively. In the case of nitrogen sources the maximum growth was observed (Fig: 4) in yeast extract compared to remaining sources. 60

8 Effect of carbon soure on Bacteriocin production 12 Inhibition zone in mm E.coli Staphylococcus Pseudomonas 0 Glucose Sucrose Lactose Maltose Starch Carbon sources Fig: 3 Effect of Carbon Sources on bacteriocin production Effect of Nitrogen source on Bacteriocin Production Inhibition zone in mm Tryptone Yeast Extract Peptone Nitrogen Sources E.coli Staphylococcus Pseudomonas Fig: 4 Effect of Nitrogen Sources on bacteriocin production DISCUSSION Because viable bacterial strain isolated from mango can inhibit pulp spoilage bacteria and enteric pathogenic microorganisms by producing lactic acid and other antimicrobial substances. In this study, of six isolated strains, lactobacilli culture supernatant was examined for acid and bacteriocin production and had inhibitory effects on sensitive bacteria including E.coli Staphylococcus aureus and Pseudomonas aeruginosa. Mango pulp is source of micro-organisms associated with mango pulp based beverages. Despite the overall significance of lactic acid bacteria in mango beverages, there are only occasional reports of their isolation from fruit pulp. Lactic acid bacteria are particularly important because of its role in preventing the spoilage bacteria from fruit pulp but, to our knowledge, it has never been isolated from mango pulp. Its ecological origin in wine production remains a mystery but, like the principal fruit pulp contains yeast and some pathogenic especially spoilage bacteria. We have had little success in isolating lactic acid bacteria from mango pulp by MRS agar plate culture. Instead, we found a predominance of Lactic acid bacteria by this method, and this 61

9 correlated with its widespread use as a biopreservative during the storage of fruit pulp, in pulp industry. Consequently, in this study, we focused on enrichment culture as an approach to determine the antimicrobial activity of the compound- bacteriocin of lactic acid bacteria. The extracted compound shows antimicrobial activity against to the spoilage bacteria like E.coli, and Bacillus and Pseudomonas. And it is also tested for the ability to produce optimum bacteriocin at maltose and yeast extracts as a carbon and nitrogen sources respectively. The present study also focused on the suppression of spoilage bacteria by counting the CFU before and after the addition of bacteriocina as preservative and compared with a chemical preservative PMS and the results indicated that the decreased CFU in bacteriocin containing sample that very similar to the sample containing PMS, hence it is important to note that the addition of biopreservative is very useful in preservation of fruit pulp when compared with chemicals as a preservative The extracted compound was also tested for their activity in the presence of certain physiological and enzymatic conditions. Around the ph and low temperature shows its activity, increased or decreased that condition may leads to decreased activity. In the case of detergents, almost all the detergents had no effect on its activity at certain concentrations after that may leads to negative effect. Finally the proteolytic enzymes like pepsin and Proteinase-K has a negative effect, due to the protenacous nature of antimicrobial compound, and there is no effect of α-amylase, lipase, α- chymotrypsin and trypsin. Their potential significance in protecting the fruit pulp, it is possible that the growth of these Lactic acid bacteria during enrichment and suppressed the growth other spoilage bacteria, but this requires further study. According to Rattanachaikunsopon and Phumkhachorn [20] the Lactic acid bacteria which produce antimicrobial compounds like bacteriocin most widely used in food production. It may be necessary to design more highly selective enrichment media to detect the high concentration of antimicrobial compound-bacteriocin of lactic acid bacteria that occur mango pulp. CONCLUSION These results demonstrated that the inhibitory effects of isolated strain on microbial growth in the mango pulp. The usage of chemical preservatives had significantly affected the physicochemically profile of the pulp samples during storage. However, a variety of other bacterial members may be present at this habitat, and suppress the growth of other spoilage bacteria. Bacteriocin, a biological compound extracted from lactic acid bacteria shows a pronounced increase in acidity and corresponding decrease in ph during storage for 3-6 months so the product remained acceptable after three months storage. The results obtained from this study, the isolated lactic acid bacteria show the potent bacteriocin and antimicrobial properties that exerts in the usage of this compound as a preservative in mango or fruit pulp industry. 62

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