MATERIALS AND METHODS

Microorganisms and culture media reagents: Man-Rogosa-Sharpe (MRS) broth was obtained from (Hi-media, India). Nutrient agar and nutrient broth were purchased from Merck, Darmstadt, Germany. Waste substrates such as pineapple, soy-whey, cabbage and molasses were collected from the local market. The other reagents used in the experiment were analytical grade.

The pathogenic microorganisms, E. coli and S. aureus were obtained from General Hospital. Penang, Malaysia. The bacterial species were cultivated and maintained in nutrient agar slants in screw-capped tubes. The agar slants were preserved in a refrigerator at 4°C until use.

Strains of L. acidophilus (FTDC 2804, FTDC 0785, FTDC 8592, FTDC 1295, FTDC 4793, FTDC 4462, FTDC 0582 and FTDC 2916) were obtained from the Culture Collection Center of the School of Industrial Technology, Universiti of Sains Malaysia (Penang Malaysia). The strains were inoculated in sterile MRS broth (dextrose 20.0 g L^-1; meat peptone 10.0 g L^-1; beef extract 10.0 g L^-1; yeast extract 5.0 g L^-1; sodium acetate 5.0 g L^-1; disodium phosphate 2.0 g L^-1; ammonium citrate 2.0 g L^-1; tween 80 1.0 g L^-1; magnesium sulfate 0.1 g L^-1, manganese sulfate 0.05 g L^-1) at 37°C for 36 h. The organisms were activated for three successive times prior to use. The culture centrifuged at 5000 rpm for 15 min and then washed two times by normal saline. The cells re-suspended in four different waste substrates mentioned above (1% v/v inoculum size). All the substrates were incubated at 37°C for 36 h. After incubation the bacterial suspension was centrifuged at 4°C for 15 min at 5000 rpm (Beckman, USA). The metabolites were next evaluated for antimicrobial activity. The entire work was conducted at Department of Pharmaceutical Technology, School of Pharmaceutical Sciences, Univeristi Sains Malaysia, Penang, Malaysia in year of 2010.

Evaluation of antimicrobial activity: This assay was performed using the agar well diffusion method (Fooks and Gibson, 2002; Toure et al., 2003) and is widely used to determine the antimicrobial activity against different types of pathogenic microorganisms.

The antimicrobial activities were determined by measuring the diameter of zone of growth inhibition formed around the wells. To further check whether the pathogens were inhibited or killed, a swab was taken from the growth inhibition zone and then inoculated into nutrient broths and incubated at 37°C for 24 h. The broth tubes were then checked for growth. Presence of growth in the broth was interpreted as an inhibitory activity, while no growth was interpreted as microbicidal activity. Each experiment was conducted in two individual trials, each in triplicate.

Statistical analysis: The results were analyzed statistically using one-way analysis of variance (version 13.0, SPSS, USA). When there was a statistically significant difference, post-hoc Tukey Honestly Significant Difference test was applied. ANOVA data with p<0.001, p<0.01 and p<0.05 were classified as statistically significant.

RESULTS AND DISCUSSION

The selection of suitable probiotics starter culture among the one naturally present in fermented products should be emphasized. Earlier studies showed that the probiotics grown in modified MRS media exerted considerable antagonistic effect against the human pathogens Bacillus cereus, Listeria monocytogenes, MRSA (Methicillin Resistance Staphylococcus aureus) and Pseudomonas aeroginosathe causative agents of food borne disease, Listeriosis, skin infections and lung disease, respectively (Bhilabutra et al., 2007). Yesillik et al. (2011) showed that the antibacterial effects of probiotic yoghurt on viability of Salmonella typhimurium, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus was investigated by using disc diffusion method. In their study it was determined that the most sensitive pathogenic bacteria to the metabolites of probiotics were Salmonella typhimurium where as the least sensitive pathogen was Pseudomonas aeruginosa.

In the present study, the results of the antibacterial activities of probiotic L. acidophilus strains cultivated in pineapple agro waste juice against S. aureus and E. coli is depicted in Fig. 1. The inhibition zones obtained in S. aureus cultures were in the range of 10.08-10.57 mm, whereas, the inhibition zones observed in E. coli cultures were in the range of 9.77-11.13 mm. The highest inhibition was obtained with L. acidophilus FTDC 4462 against S. aureus (10.57 mm) and E. coli (11.13 mm). While, the lowest inhibition effect was obtained with L. acidophilus FTDC 0582 against S. aureus (10.08 mm) and L. acidophilus FTDC 8592 against E. coli (9.77 mm).

Fig. 1:

The antibacterial activities of probiotic Lactobacillus acidophilus strains cultivated in pineapple agro waste juice against Staphylococcus aureus and Escherichia coli. Results are expressed as Mean±SD; each data point is the average of measurement from six independent replicates (n = 6). ***: Significant when compared to the control (p<0.001),**: Significant when compared to the control (p<0.01), *: Significant when compared to the control (p<0.5)

Figure 2 illustrates the antibacterial activities of probiotic L. acidophilus strains cultivated in soy-whey waste juice against S. aureus and E. coli. The inhibition zones measured in S. aureus cultures were in the range of 9.38-10.83 mm, whereas, the inhibition zones found in E. coli cultures were the range 9.67-10.50 mm. The highest inhibition was obtained with L. acidophilus FTDC 0785 towards S. aureus (10.83 mm) and E. coli (10.50 mm), while, the lowest inhibition effect was obtained with L. acidophilus 8592 against S. aureus (9.38 mm ) and L. acidophilus FTDC 2804 in E. coli (10.50 mm).

Figure 3 illustrates the antibacterial activities of probiotic L. acidophilus strains cultivated in cabbage waste against S. aureus and E. coli. The inhibition zones measured in S. aureus cultures were in the range of 9.27-11.12 mm, whereas, the inhibition zones found in E. coli cultures were the range 9.25-10.67 mm. The highest inhibition was obtained with L. acidophilus FTDC 4462 towards S. aureus (11.28 mm) and E. coli (10.67 mm), while, the lowest inhibition effect was obtained with L. acidophilus 2804 against S. aureus (9.27 mm ) and L. acidophilus FTDC 2916 in E. coli (9.25 mm).

Fig. 2:

The antibacterial activities of probiotic Lactobacillus acidophilus strains cultivated in soy-whey waste against Staphylococcus aureus and Escherichia coli. Results are expressed as Mean±SD; each data point is the average of measurement from six independent replicates (n = 6). Comparison between the different strain grown in the same media (p<0.05). **: significant when compared to the control (p<0.001), *: Significant when compared to the control (p<0.01)

Fig. 3:

The antibacterial activities of probiotic Lactobacillus acidophilus strains cultivated in cabbage waste against Staphylococcus aureus and Escherichia coli. Results are expressed as Mean±SD; each data point is the average of measurement from six independent replicates (n = 6). Comparison between the different strain grown in the same media (p<0.05). **: Significant when compared to the control (p<0.01), *: Significant when compared to the control (p<0.5).

Fig. 4:

The antibacterial activities of probiotic Lactobacillus acidophilus strains cultivated in molases waste against Staphylococcus aureus and Escherichia coli, Results are expressed as Mean±SD; each data point is the average of measurement from six independent replicates (n = 6). Comparison between the different strain grown in the same media (p<0.05). ***: Significant when compared to the control (p<0.001)

Figure 4 showed the antibacterial activities of probiotic L. acidophilus strains cultivated in molasses waste against S. aureus and E. coli. The inhibition zones calculated in S. aureus cultures were in the range of 8.48-10.48 mm, whereas, the inhibition zones observed in E. coli cultures were the range 9.18-10.08 mm. The highest inhibition was obtained with L. acidophilus FTDC 2916 towards S. aureus (10.48 mm) and L. acidophilus FTDC 1295 towards E.coli (10.08 mm), while, the lowest inhibition effect was obtained with L. acidophilus 2804 against S. aureus (8.48 mm) and L. acidophilus FTDC 0582 in E. coli (9.18 mm).

All results were expressed as Mean±SD; each data point is the average of measurement from six independent replicates (n = 6).

The results showed that the metabolites of all eight strains exerted considerable antibacterial effect, as the zone of inhibition results were significant when compared to the control. Among the eight strains cultivated in four different growth medium, there was a statistically significant difference in the anti-bacterial activities of the metabolites of S. aureus and E. coli. Some of the strains of L. acidophilus were more effective against S. aureus, whereas, the other strains were more effective against E. coli.

The results indicated that the agar diffusion method was comparatively more superior in the study of antimicrobial activity of L. acidophilus strains. This could be explained by a good diffusion of metabolites from L. acidophilus strains in the well method and thus this may probably be resulted in the growth inhibition of the pathogenic microorganisms. The results revealed that the metabolites from almost all strains tested were microbicidal as there was no growth found in the cultures collected by swabbing from inhibition zones. It is evident from the results of the measurement of the diameters of zone of inhibition that the metabolites are significantly effective. This can be explained from the fact that the metabolites produced by the probiotics include bioactive products such as organic acid, hydrogen peroxide (H₂O₂) and bacteriocins. It was reported that the cell-free supernatant solution from strains of lactic acid bacteria exhibited antimicrobial activity which prevented the growth of different strains of S. aureus and E. coli (Lavermicocca et al., 2000). It is reported that the principal metabolites of probiotics bacteria are acetic acid and lactic acid in ratio 3:2 and these acids are responsible for the consequent drop in pH and may be sufficient to antagonize many pathogenic bacteria belonging to both Gram-positive and Gram-negative bacteria (Cheikhyoussef et al., 2007).

The results showed no significant difference in anti-bacterial activities of L. acidophilus cultivated in pineapple waste substrate, soy-whey substrate and cabbage waste substrate (p>0.05) whereas, it showed significant differences with molasses waste substrate (p<0.05). The results of the inhibition zone diameter of all substrates were considerable and suggested good antimicrobial activity in these waste substrates against pathogenic bacteria. The major advantages of these media are that all the tested media are not toxic, in addition they are suitable for human consumption if they prepared in a suitable formulation and in contrast, MRS medium was toxic to human. The genus Lactobacillus has a long history of safe use and it plays a major role in fermented milk and other food products (Karska-Wysocki et al., 2010). Huttunen et al. (1995) reported that Lactic acid bacterial strains are potentially promising because they generate bactericidal bioactive peptides (bacteriocins) and enzymes that are able to control bio-film formation and the growth of the pathogens. Certain Lactobacillus strains have been reported to be highly antagonistic to S. aureus (Ammor et al., 2006). The current study provides the evidence that the beneficial effects of probiotics are strain specific. According to the World Gastroenterology Organisation (WGO) Practice Guidelines on Probiotics and Prebiotics, “the potential probiotic health benefits can only be attributed to the strain or strains tested and not to the species or the whole group of lactic acid bacteria or other probiotics (WGO, 2008). Therefore, it is obvious that L. acidophilus is effective against E. coli and S. aureus and acted as an bactericidal agent against human pathogenic bacteria. These results were consistent with the findings of other research groups (Choi and Beuchat, 1994; Arihara et al., 1996; Ryan et al., 1996; Aktypis et al., 1998; Jacobsen et al., 1999; Parente and Ricciardi, 1999).

CONCLUSION

The growth of the pathogenic bacteria was successfully inhibited when metabolites of L. acidophilus from all waste substrates were tested. There was a significant inhibition of bacterial growth in well diffusion method as depicted by the zone of inhibition. The effect of metabolites was found to be bactericidal on both pathogenic bacteria, E. coli and S. aureus. It can be concluded that L. acidophilus cultivated in waste substrates produced metabolites with strong bactericidal property and could provide medicinally value added advantages to the human beings.

ACKNOWLEDGMENT

The present study was financially supported by the Universiti Sains Malaysia (USM) Grant No: (1001/PFARMASI/843084) and a USM Graduate Assistance Scheme.