Trakia Journal of Sciences, No 2, pp 110-117, 2013 Copyright 2013 Trakia University Available online at: http://www.uni-sz.bg ISSN 1313-7050 (print) ISSN 1313-3551 (online) Original Contribution HEAT STABILITY AND OPTIMIZATION OF INVITRO ANTIMICROBIAL ACTIVITY OF METABOLITES PRODUCED BY RHIZOPUS OLIGOSPORUS NRRL 2710 AGAINST SOME PATHOGENIC BACTERIA I. F. Fadahunsi*, S. T. Ogunbanwo, D. T. Ogundana Department of Microbiology, University of Ibadan, Ibadan, Nigeria ABSTRACT The antimicrobial activity of Rhizopus oligosporus as a food grade microorganism is highly important to the food Industry in assessing the microbial safety, and shelf life of finished fermented food product. Studies were carried out to investigate the heat stability, optimization of antimicrobial activity, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the growth filtrate produced by Rhizopus oligosporus NRRL 2710 using the agar well diffusion method. When the filtrate was heated at 20 o C, 40 o C and 60 o C for 20-60 min, it was inhibitory, but when the filtrate was heated at 40 o C, 60 o C, 80 o C and 100 o C for 80-100min the antimicrobial activity was not detected. The highest zone of inhibition (9.0mm) was observed against Escherichia coli and Salmonella typhi followed by Pseudomonas aureginosa showing an inhibition zone of 8.0mm while Staphyloccouss aureus, Corynebacteria diphtheriae and Klebsiella pneumoniae recorded an inhibition zone of 7.0mm. The least zone of inhibition (6.5mm) was detected against Proteus mirabilis. (control experiment). Optimization showed that the best ph and temperature were 4 and 37 o C respectively. The C: N ratio investigation indicated that the highest zone of inhibition of 15.0mm was observed against Salmonella typhi at ratio 3:5. The MIC and MCB were 2.5mg/ml for Proteus mirabilis and 5.0mg/ml for Corynebacteria diphtheria respectively. From this study, antimicrobial activities can be improved. Key words: Rhizospus oligosporus; Metabolite; Screening; Antagonistic Pathogenic bacteria INTRODUCTION Rhizopus oligosporus is popularly known as Rhizopus microsporus var. oligosporus. It also has a binomial name of Rhizopus oligosporus saito (1). In 1895, the Dutch scientist Prinsen Geerlings first identified the tempeh mold (2). It has a rapid growth between 30-42 o C (mesophile) and grows best at ph 4, does not ferment sucrose; possess high lipolytic activities and low amylase activity with no detectable pectinase activity. It is a good producer of strong antioxidants. Within the R. microsporus group, Rhizopus oligosporus has the greatest number of large sized irregular spores and a proportion of more than 10% irregular spores, can be used as a *Correspondence to: I. F. Fadahunsi, Department of Microbiology, University of Ibadan, Ibadan, Nigeria, e-mail: sanmifadahunsi @yahoo.com Trakia Journal of Sciences, Vol. 11, 2, 2013 marker to distinguish it from other strains within the Rhizopus oligosporus group (3). However, the strain of Rhizopus oligosporus NRRL 2710 are the most acceptable starter culture used in tempeh production and mostly preferred in tempeh fermentation (2). It has been observed to be one of the best producers of tempeh and is still the most widely used tempeh culture strain in the USA (2) There is available documented information about the use of Rhizopus sp. in the reduction of cassava toxicity by heap fermentation (5). It is also reported to possess the ability to grow successfully in solid substrate system using fruit residues as substrate and enhanced its total phenolic compounds. Great viability of biological effects is attributed to phenolic compounds including anti-inflammatory, anti- 110
microbial and anti-oxidant activities (6). This organism has recently received attention in other fields of applied microbiology such as sufu production (7). Agro industrial solid waste treatment (8) production of phenolic compounds from agricultural wastes (9) and bioremediation of heavy metals(10, 11, 12, 13) Other species of Rhizopus had been found to be useful in the commercial preparation of some anesthetics, birth control agents, industrial alcohol, meat tenderization and yellow coloring used in margarine and butter substitute (14). There are documented reports on the ability of Rhizopus oligosporus NRRL 2710 to inhibit some pathogens. The antimicrobial activity of Rhizopus oligosporus as a food grade microorganism is highly important to the food Industry in assessing the microbial safety and shelf life of finished fermented food product. Therefore there is need to investigate the thermal stability and optimize the antimicrobial activity of this microorganism. This work is intended to provide information on different physicochemical and physiological parameters that could be used to optimize the activity of the antimicrobial compounds produced by Rhizopus oligosporus NRRL 2710 MATERIALS AND METHODS Collection of sample Rhizopus oligosporus NRRL 2710 was obtained from Professor Esther Aderibigbe of the Department of Microbiology, Ekiti State University, and Ado- Ekiti (EKSU) Nigeria. The strain was subcultured, maintained on Potato Dextrose Agar (PDA) slant and stored in a refrigerator at 4 o C. The test organisms used for this research work were obtained from the University College Hospital (UCH) Ibadan Nigeria. The test organisms used include Escherichia coli, Salmonella typhi, Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Proteus mirabilis and Corynebacteria diphtheriae. They were subcultured to obtain pure cultures which were stored on nutrient agar slant and kept in a refrigerator at 4 o C. Preparation of cell-free filtrate Five - day culture of Rhizopus oligosporus was inoculated into 25ml of sterilized Potato Dextrose Agar broth in 150 ml Erlenmeyer flask using a sterile cork borer (5mm diameter). Incubation was carried out at 28 o C for 8days and, the broth was filtered thrice using Whatman filter paper no.1 in order to obtain a cell-free filtrate. The filtrate was used for further work in this study. Preparation of Inoculum Twenty hours old cultures of the test organisms were used. A serial dilution technique was used for the inoculum preparation; the turbidity was adjusted to that of McFarland Standard. The inoculum size was verified by measuring the absorbance of the suspension spectrophotometrically. The absorbance was in the same range as that of the McFarland Standard 0.5 (OD625 nm 0.08 0.13) (15). Preparation of Basal Medium for supplementation studies The basal medium used for this study consisted of Yeast Extract Powder (2.5g); Potassium dihydrogen phosphate-kh 2 P0 4 (0.05g); Magnesium Sulphate Hepta-hydrate-MgS0 4. 7H 2 0 (0.05g) and potassium nitrate (1.5g) all dissolved in 1litre of distilled water and sterilized at 121 o C for 15mins (16). Thermal Stability of the antimicrobial metabolite of Rhizopus oligosporus NRRL 2710 One hundred milliliters of culture filtrate of Rhizopus oligosporus NRRL 2710 was transferred into 150mls Erlenmeyer flask and heated in a water bath set at 20 o C, for 20, 40, 60, 80 and 100 min respectively. This procedure was repeated for other temperatures (40, 60, 80 and100 o C), the antimicrobial activity of the heated filtrate was determined using the Agar well diffusion method of (17). Determination of Antagonistic activity of Rhizopus oligosporus NRRL 2710 Antimicrobial activity of Rhizopus oligosporus NRRL 2710 against the indicator organisms was carried out using agar well diffusion assay as described above. Inhibition zones around the wells were measured at 24h and 48h respectively (18). This serves as the control experiment. Effect of different ph on the Antimicrobial Activity of Rhizopus oligosporus NRRL 2710 111 Trakia Journal of Sciences, Vol. 11, 2, 2013
This was carried out according to the method described by (16) Effect of different temperatures on the Antimicrobial Activity of Rhizopus oligosporus NRRL 2710 This was carried out according to the method described by (16) Effect of C: N ratio on the Antimicrobial Activity of Rhizopus oligosporus NRRL 2710 The best carbon and nitrogen sources were combined in various ratios and introduced into the basal medium to determine which ratio would support the optimum antimicrobial activity of the filtrate, using the method employed by (16). For the control experiment the organism was inoculated into the basal medium with no nitrogen source. Determination of Minimum inhibitory concentration (MIC) MIC was determined using broth dilution technique of (19). The minimum inhibitory concentration (MIC) of the filtrate was estimated for each of the test organisms in duplicates. Determination of Minimum bactericidal concentration (MBC) The MBC was determined using a loopful of broth cultures collected from tubes which did not show any growth during MIC and inoculated on a sterile nutrient agar by streaking. This was incubated at 37 0 C for 24h (19). RESULTS Table 1 shows the effect of heat treatment on antimicrobial activity while Table 2 shows zones of inhibition against some test organisms. Table 3 shows the effect of different ph on the antimicrobial activity. Table 4 shows effect of different temperature on the antimicrobial activity; Table 5 shows effect of different C: N ratio on the antimicrobial activity. Table 6 shows the result MIC and MBC of Rhizopus oligosporus NRRL 2710 against the test organisms. Table 1. Effect of Heat Treatment on antimicrobial metabolites produced by Rhizopus oligosporus NRRL 2710 Organism 20 0 C 40 0 C 20min 40min 60min 80min 100min 20min 40min 60min 80min 100min E. coil 8.0±0.493 7.5±0.239 7.5±0.00 7.0±0.365 6.5±0.282 7.0±0.476 6.5±0.000 6.0±0.141 - - S. typhi 9.0±0.365 8.0±0.416 7.0±0.476 6.5±0.00 6.0±0.216 8.0±0.577 6.5±0.424 6.0±0.000 - - S. aureaus 7.5±0.408 7.0±0.282 7.0±0.355 6.5±0.200 7.0±0.182 8.0±0416 7.0±0.365 6.0±0.163 6.0±0.1-15 B.subtilis 9.0±0.577 8.0±0.408 8.0±0.577 7.0±0.424 6.5±0.230 7.0±0.400 6.0±0.594 6.0±0.182 - - P. aeruginosa 8.0±0.476 7.0±0.424 7.5±0.428 7.0±0.465 6.5±0.000 7.0±0.522 6.0±0.163 6.5±0.496 - - C. diphtheriae 7.0±0.522 7.0±0.469 7.0±0.469 6.5±0.408 6.0±0.496 6.0±0.653 6.0±0.216 - - - K. pneumoniae 7.0±0.365 7.0±0.282 6.5±0.577 6.0±0.571 6.0±0.141 6.0±0.496 6.0±0.182 6.0±0.141 - - P. mirablis 7.0±0.400 7.0±0.465 6.5±0.570 6.0±0.594 6.0±0.547 7.0±0.282 6.5±0.000 6.0±0.115 - - Organism 60 0 C 80 0 C 20min 40min 60min 80min 100min 20min 40min 60min 80min 100min E. coil 7.0±0.355 6.0±0.476 6.0±0.216 - - 7.0±0.408 7.0±0.476 6.5±0.424 6.0±0.5-47 S. typhi 7.0±0.469 6.0±0.000 6.0±0.163 - - - - - - - S. aureaus 8.0±0.400 8.0±0.424 6.0±0.115 6.0± 0.141-6.0±0.496 - - - - B.subtilis 8.0±0.408 6.0±0.216 6.0±0.163 - - - - - - - P. aeruginosa 7.5±0.408 6.0±0.571 6.0±0.547 - - - - - - - C. diphtheriae 6.5±0.300 6.0±0.115 - - - 6.0±0.182 - - - - K. pneumoniae 6.0±0.216 6.0±0.00 6.0±0.182 - - 6.0±0.216 - - - - P. mirablis 8.0±0.476 6.5±0.282 6.5±0.000 - - 6.5±0.282 6.0±0594 - - - Trakia Journal of Sciences, Vol. 11, 2, 2013 112
Test Organisms Escherichia coli Salmonella typhi Staphylococcus aureus Pseudomonas aeruginosa Bacillus subtilis Corynebacteria diphtheriae Klebsiella pneumonia P. mirabilis K pneumonia C. diphtheria P.aeruginosa B. subtilis S. aureus S. typhi E. coli ph P. mirabilis K. pneumonia C. diphtheriae P. aeruginosa B. subtilis S. aureus S. typhi E. coli Temperature Proteus mirabilis Table 2. Zones of Inhibition of Rhizopus oligosporus NRRL 2710 against some Test Organisms Zone of Inhibition (mm) 8.0 0.408 8.0 0.678 7.0 0.522 8.0 0.439 8.0 0.000 7.0 0.365 7.0 0.400 6.5 0.577 Table 3. Effect of different ph on the antimicrobial activity of metabolites produced by Rhizopus oligosporus NRRL 2710 4 9.0±0.230 11.0±0.365 8.0±0.282 9.5±0.816 9.0±0.577 8.0±0.816 9.5±0.408 9.0±0.40 5 9.0±0.416 9.0±0.439 8.0±0.414 8.0±0.282 8.0±0.000 7.0±0.493 8.5±0.522 9.0±0.408 6 9.0±0.816 7.5±0.577 7.5±0.000 6.0±0.577 8.0±0.541 6.0±0.230 6.5±0.230 8.5±0.577 7 7.5±0.408 8.0±0.163 7.0±0.816 8.5±0.416 9.0±0.678 7.5± 0.000 7.5±0.577 7.0±0.191 8 8.0±0.163 8.0±0.476 6.8±0.000 10.0±0.77 9.0±0.707 6.5±0.416 6.5±0.416 8.0±0.439 Control 8.0 0.408 8.0 0.678 7.0 0.522 8.0 0.439 8.0 0.000 7.0 0.365 8.0 0.400 6.5 0.577 Table 4. Effect of different temperature on the antimicrobial activity of metabolites produce by Rhizopus oligosporus NRRL 2710 4 0 C 7.5±0.408 7.0±0.400-6.0±0.365 6.0±0.416 7.0±0.326 6.0±0.658-15 0 C 7.0±0.577 7.0±0.678 9.0±0.416 6.5±0.326 6.0±0.365 - - 6.0±0.282 27 0 C 8.0±0.416 9.0±0.522 8.0±0.408 9.0±0.678 8.0±0.439 8.5±0.476 7.0±0.730 6.0±0.476 37 0 C 9.5±0.770 11.0±0.493 8.0±0.326 8.5±0.658 9.0±0.577 7.5±0.365 8.0±0.493 6.0±0.478 42 0 C - - - - - - - - Control 8.0 0.408 8.0 0.678 7.0 0.522 8.0 0.439 8.0 0.000 7.0 0.365 7.0 0.400 6.5 0.577 113 Trakia Journal of Sciences, Vol. 11, 2, 2013
P. mirabilis K. pneumonia C. diphtheriae P. aeruginosa B. subtilis S. aureaus S. typhi E. coli C:N Table 5. Effect of different C: N Ratio on the antimicrobial activity of metabolites produced by Rhizopus oligosporus NRRL 2710 Zone of Inhibition (mm) 1:2 7.0±0.4.00 7.0±0.326 7.5±0.424 7.0±0.577 7.5±0.365 7.0±0.528 7.0±0.577 7.0±0.678 1:3 7.0±0.476 8.0±0.540 8.0±0.408 9.0±0.522 7.5±0.493 8.5±0.770 8.0±0.439 8.0±0.416 1:4 7.0±0.400 9.0±0.678 8.0±0.476 8.0±0.577 7.5±0.540 8.0±0.486 10.0±0.730 9.0±0.628 1:5 8.0±0.664 9.0±0.500 9.5±0.512 7.5±0.424 8.0±0.439 8.5±0.602 8.0±0.522 8.0±0.493 2:1 7.5±0.540 10.0±0.476 9.0±0.408 8.5±0.678 8.0±0.414 9.0±0.522 7.0±0.658 7.5±0.577 2:3 8.0±0.282 7.0±0.365 7.5±0.424 8.0±0.416 8.5±0.408 7.0±0.365 9.0±0.400 8.0±0.476 3:1 8.0± 0.577 8.5±0.424 10.0±0.400 8.5±0.282 8.0±0.000 7.0±0.408 8.0±0.577 7.0±0.408 3:2 8.0±0.682 7.5±0.00 7.5±0.424 7.5±0.577 9.0±0.493 8.5±0.548 9.5±0.577 7.0±0.439 3:5 8.5±0.493 15.0±0.77 13.0±0.707 9.0±0.486 10.5±0.512 8.5±0.424 14.0±0.678 11.0±0.476 4:1 4.5±0.408 12.5±0.577 11.0±0.400 8.5±0.00 8.5±0.476 9.0±0.365 13.5±0.522 8.0±0.400 4:3 8.5±0.416 9.5±0.577 11.0±0.365 8.0±0.678 9.5±0.424 8.5±0.577 14.0±0.00 8.0±0.00 8.0 0.408 8.0 0.678 7.0 0.522 8.0 0.439 8.0 0.000 7.0 0.365 7.0 0.400 6.5 0.577 Control Table 6. Minimum inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of Rhizopus oligosporus NRRL 2710 against the test organisms Test Organisms MIC (mg/ml)/ MBC (mg/ml) E.coli 2.5 5.0 S.typhi 2.5 5.0 S.aureus 5.0 10.0 P.aeruginosa 2.5 5.0 B.subtilis 2.5 5.0 C.diphtheriae 5.0 10.0 K.pneumoniae 5.0 10.0 P.mirabilis 10.0 20.0 DISCUSSION Determination of the thermal stability of metabolites, effects of different ph, temperatures, C: N ratios on the antimicrobial activity, MIC and MBC of the metabolites produced by Rhizopus oligosporus NRRL 2710 were carried out. Table 1 shows the thermal stability of Rhizopus oligosporus NRRL 2710, when the filtrate was heated at 20 0 C, 40 0 C and Trakia Journal of Sciences, Vol. 11, 2, 2013 60 0 C for 20-60 min, it exhibited antimicrobial activity and at 80-100mins for 20-60mins antimicrobial compounds was no more inhibitory. The heat treatment showed that the filtrate was effective at lower temperature for longer period, while it was not inhibitory at higher temperature for shorter periods. This result agrees with that of (20) who obtained similar gradual decrease in the antimicrobial 114
effectiveness of the extract of L. edodes as the temperature was elevated and also reported loss of activity of L. plantarum filtrate after heat treatment at 121ºC for 15 min (21). This observation may be due to the denaturation of the antimicrobial compounds which may be glycoprotein in nature. Table 2 shows zones of inhibition of Rhizopus oligosporus NRRL 2710 against some test organisms. The result revealed that Escherichia coli, Salmonella typhi, Pseudomonas aeruginosa, Bacillus subtilis, recorded the highest zones of inhibition (8mm) while the lowest (6.5mm) was recorded by Proteus mirabilis (control experiment). The ability of Rhizopus oligosporus metabolite to inhibit the test pathogens had earlier been reported by (22, 23, 1, 24, 9, 6). Rhizopus oligosporus NRRL 2710 has been reported to produce phenolic compounds that inhibit the growth of pathogenic bacteria such as Helicobacter pylori. Kier et al (25) also reported that Rhizopus oligosporus NRRL 2710 can also produce certain compounds that interfere with the adhesion of E. coli to small intestinal brushborder membrane. Table 3 represents the result of the effect of different ph on activity of the metabolite produced by Rhizospus oligosporus. The best ph for optimization was 4 and the zone of inhibition increased by 33.3%, 100%, 150%, 33.3%, 33.3%, 50.0%, 125%, 166% for E. coli S. typhi, S. aureus, B subtilis, P. aeruginosa, C. diptheriae, K. pneumoniae and P. mirabilis and respectively when compared to the control. Such occurrence has earlier been documented by Richard and Dale (26). This may be due to the fact that at ph 4, there was an increase in the permeability of the cell membrane which aids rapid diffusion of the nutrients in the cell thereby leading to increase in the production of antimicrobial compounds. The effect of different temperature shows that 37 o C was the best for optimization and the zone of inhibitions increased by 50%, 100%, 50%,16.7%, 33.3%, 25%, 50% and 33.3% for E. coli, S. typhi, S. aureus, B subtilis, P. aeruginosa, C. diptheriae, K. pneumoniae and P. mirabilis respectively when compared to the control (Table 4). Such finding is in conformity with Sparringa and Owens (27). Reasons such as optimum growth of Rhizopus oligosporus NRRL 2710 at 37 o C may be responsible for the highest zone of inhibition that was observed. The effect of C:N ratios shows that the optimum was 3:5mm showing an increase in inhibition zone of 50%,250%,300%,33.3%,66.7%,125%,350% and 300 % against E. coli, S. typhi, S. aureus, B subtilis, P. aeruginosa, C. diptheriae, K. pneumoniae and P. mirabilis respectively when compared to the control (Table 5). This ratio may probably be the best carbon to nitrogen ratio based on the nutritional requirement of the organism that would stimulate the optimum antimicrobial potency of the metabolites produced by the organism. Also the relatively higher resistance of some test organisms such as P. aeruginosa, and others might be caused by the tendency of these microorganisms to adapt and circumvent the inhibitory effect of antimicrobial agent and hence becomes resistance to this agent (28). The results of (MIC) and (MBC) of the metabolites are shown in Table 6, the highest (MIC) of 2.5mg/ml was shown by E coli, S typhi, P. aeruginosa and B. subtilis. The lowest value of 10mg/ml was recorded by P mirabilis. The result of (MBC) showed that Escherichia coli, Salmonella typhi, Bacillus subtilis and Pseudomonas aeruginosa showed highest value of 5.0mg/ml while Proteus mirabilis showed the lowest value of 20.0mg/ml. The MIC and MBC concentrations vary from one test organism to the other. This may be due to the cell wall composition (peptidoglycan, techoic acid) of the organisms which confers on the organism resistance to antimicrobial substances (28) However, in this study it was observed that the Gm-ve bacteria were more susceptible to the antimicrobial agent, than the Gm+ve bacteria, because the bacteria cell wall is the site of action of several antimicrobial agents and specifically the cell wall of the Gm+ve is thicker with 20-80nm homogenous layer of Peptidoglycan laden with techoic acid thus protecting antimicrobial agent inhibition. On the other hand the Gm-ve organisms consist of 2 to 7nm thick layer of peptidoglycan without techoic acid, and this structural arrangement makes the Gm-ve organisms will prone to antimicrobial attack (29). 115 Trakia Journal of Sciences, Vol. 11, 2, 2013
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