INTRODUCTION:

The emergence of outbreaks of foodborne illness associated with fresh fruits and vegetables has revived interest among public health agencies and sparked a new wave of research on food safety issues related to microbial contamination of fresh produce. Several studies have demonstrated the presence of foodborne human pathogenic bacteria on crops grown in soil to which were manured with animal dung. Animal feces may serve as potential source of inoculum of these human enteric pathogens (HEPs). Irrigation with water contaminated with faeces is also a primary source of inoculum in the field. Air-borne contamination of food crops is also a potent possibility.¹ Establishment of HEPs on fresh farm produce is a matter of concern and development of resistance in them against several synthetic drugs indicate the need to search for efficient alternative, natural and eco-friendly means of controlling those.² Tomato is one such crop which is extensively included in day to day diet of humans, mostly served raw in the form of salad, which if contaminated can be a chief cause of spread of pathogenic organisms in the consumers.³

Recent investigations have shown that extracts from higher plants contain substances which inhibit the growth of several harmful pathogens and could provide suitable biological control options.

Banana is one of the medically acclaimed plants, every part of which possesses antimicrobial properties. Banana plants are rich in phenolic compounds and flavonoids which are antioxidant in nature. Antifungal and antibiotic principles are found in the peel and pulp of fully ripe banana.^4,5 Consumption of banana flower also helps to treat intestinal and stomach upsets.⁶ The antibacterial properties in banana seeds, peel and pulp have been reported earlier.⁷ The banana pseudostem is also known to possess antimicrobial properties.⁸

Aegle marmelos (Bael) is another medically important plant. It has a reserve of a bioactive molecule with broad range antimicrobial properties.⁹ Fruit and leaves are of great importance in particular for the control of different stomach and intestine dwelling pathogens.¹⁰ Pandey and Mishra have evaluated the antibacterial properties of Aegle marmelos leaves, fruits and peels against various human enteric pathogens.¹¹

The present study thus aimed at formulating of a potent biocide from banana pseudostem and leaves of Aegle marmelos, to reduce the population of HEPs on leaves of tomato plants so as to ensure that the fruits borne subsequently will have lesser load of HEPs.

MATERIALS AND METHODS:

Raising of plants:

Surface sterilized and aseptically dried tomato seeds (local variety) were sown in sterilized soilrite in plastic trays (35cm×25cm×6 cm; L×W×H). The plants were raised in a sterile culture room maintained at 25±1ºC with a relative humidity (RH) of 70% and a photoperiod of 12 h of light and dark. Trays were watered daily with sterile distilled water and once a week with sterilized Hoagland’s solution. Six weeks old plants were selected for the study.

Isolation of HEPs from phyllopane of tomato:

Naturally grown tomato leaves were procured from fields near Amity University, Noida. The leaves were imprinted onto nutrient agar media plates and incubated at 37˚C for 24 hours. Two HEPs i.e E. coli and Salmonella sp. were isolated from the resulting colonies on EMB agar and nutrient agar respectively. 16S rRNA sequencing was performed for their identification. 16S rRNA sequencing was performed as follows:

DNA extraction:

The DNA of the isolated microbe was extracted from its pure cultures using InstaGene^TM Matrix Genomic DNA isolation kit (BIORAD). Single colony was selected and resuspended in 1ml of autoclaved distilled water in a sterile microcentrifuge tube followed by centrifugation at 12000 rpm for 1 minute. To the pellet, 200µl of InstaGene^TM matrix was added and incubated at 56˚C for 30 minutes. The tube contents were mixed by vortexing at high speed for 10 sec and kept in boiling water bath for 8 minutes. This was followed by vortexing at high speed for 10 seconds and centrifugation at 12000 rpm for 3 min. The supernatant obtained was stored at -20˚C and used for further amplification by PCR.

16S rRNA amplification by PCR:

Amplification was done on Mastercycler® Pro (Eppendorf). Universal 16S rRNA primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTTACGACTT-3′) were used for amplification of complete 16S rRNA gene. The amplification cycle included 2 minutes of initial denaturation at 94˚C followed by 35 cycles, each of which had 45 seconds denaturation at 94˚C, 1 minute annealing at 56˚C, 1 minute extension at 72˚C followed by 2 minutes final extension at 72˚C.

16S rRNA Sequencing:

Chain termination by dideoxynucleoside triphosphates during in vitro DNA synthesis was used for bacterial DNA sequencing. Primers 518F (5′-CCAGCAGCCGCGGTAATACG-3′) and 800R (5′-TACCAGGGTATCTAATCC-3′) were used. Sequencing was done at Medox biotech. private limited, India. The data was analyzed using Basic Local Alignment Search Tool (BLAST) at National Centre for Biotechnology Information (NCBI).

Extraction of Aqueous Banana pseudostem extract:

Two different concentrations of aqueous Banana pseudostem extract were selected for the experiments i.e 20 % (w/v) and 50 % (w/v). In accordance with the concentrations, banana pseudostem was weighed, surface sterilised and macerated in 100 ml sterilized distilled water in a pre chilled mortar and pestle under aseptic conditions. The extract thus obtained was filtered twice through four layers of muslin cloth. The filterate was again filtered through 0.22 µm syringe-driven filters and used as the Aqueous Banana pseudostem extract.

Extraction of Aqueous Aegle marmelos leaf extract:

Two different concentrations of aqueous Aegle marmelos leaf extract were selected for the experiments i.e 20 % and 50 %. In accordance with the concentrations, Aegle marmelos leaves were weighed, surface sterilised and macerated in 100 ml sterile distilled water in a pre chilled mortar and pestle under aseptic conditions. The extract thus obtained was filtered twice through four layers of muslin cloth. The filterate was again filtered through 0.22 µm syringe-driven filters and used as the Aqueous Aegle marmelos leaf extract.

In vitro microbial growth inhibition measurement:

The in vitro bacterial growth inhibition activity of the aqueous extracts from banana pseudostem and A. marmelos leaves was calculated by measuring the diameter of the culture of the two isolated HEPs on culture media supplemented with extracts of banana pseudostem and Aegle leaves either singly or in combination as described below. 1 ml sterilized plant extract was added to the media before pouring. After solidification, a 5 mm agar plug was removed using a sterile cork borer. A similar plug was removed from the plate containing either E. coli or Salmonella. This was placed in the centre of the control plate or that supplemented with plant extracts. The plates were incubated at 37˚C for 48 hours. Thereafter, the diameter of growth of the HEP on the plate was measured. The plate devoid of any plant extract was considered as control. All the experiments were performed in triplicates. 19 different combinations of the Banana pseudostem and Aegle leaf extract were used for the experiments as follows:

C = Control (without plant extract)

1 T1 = 20 % banana pseudostem extract

2 T2 = 10 times diluted 20 % banana pseudostem extract

3 T3 = 100 times diluted 20 % banana pseudostem extract

4 T4 = 20 % Aegle marmelos leaf extract

5 T5 = 10 times diluted 20 % Aegle marmelos leaf extract

6 T6 = 100 times diluted 20 % Aegle marmelos leaf extract

7 T7 = 50 % banana pseudostem extract

8 T8 = 10 times diluted 50 % banana pseudostem extract

9 T9 = 100 times diluted 50 % banana pseudostem extract

10 T10 = 50 % Aegle marmelos leaf extract

11 T11 = 10 times diluted 50 % Aegle marmelos leaf extract

12 T12 = 100 times diluted 50 % Aegle marmelos leaf extract

13 T13 = 1:1 combination of 20 % banana pseudostem and 20 % Aegle marmelos leaf extract

14 T14 = 1:1 combination of 10 times diluted 20 % banana pseudostem and 20 % Aegle marmelos leaf extract

15 T15 = 1:1 combination of 100 times diluted 20 % banana pseudostem and 20 % Aegle marmelos leaf extract

16 T16 = 1:1 combination of 50 % banana pseudostem and 50 % Aegle marmelos leaf extract

17 T17 = 1:1 combination of 10 times diluted 50 % banana pseudostem and 50 % Aegle marmelos leaf extract

18 T18 = 1:1 combination of 100 times diluted 50 % banana pseudostem and 50 % Aegle marmelos leaf extract

Inoculum preparation:

The pure cultures of E. coli and Salmonella sp. were sub cultured separately on nutrient broth and after 24 hours of inoculation, it was used for spraying on the plants.

Treatment of plants:

The tomato plants were divided into three groups. One was sprayed with E. coli, the second with Salmonella sp. and the last one was treated as control (without any microbial spray). After 48 hours of incubation, these plants were sprayed with 50 ml of Banana pseudostem and Aegle leaf extract either singly or in different combinations as follows:

1 C = Control (autoclaved sterile distilled water)

2 T1 = 20 % banana pseudostem extract

3 T2 = 10 times diluted 20 % banana pseudostem extract

4 T3 = 100 times diluted 20 % banana pseudostem extract

5 T4 = 20 % Aegle marmelos leaf extract

6 T5 = 10 times diluted 20 % Aegle marmelos leaf extract

7 T6 = 100 times diluted 20 % Aegle marmelos leaf extract

8 T7 = 50 % banana pseudostem extract

9 T8 = 10 times diluted 50 % banana pseudostem extract

10 T9 = 100 times diluted 50 % banana pseudostem extract

11 T10 = 50 % Aegle marmelos leaf extract

12 T11 = 10 times diluted 50 % Aegle marmelos leaf extract

13 T12 = 100 times diluted 50 % Aegle marmelos leaf extract

14 T13 = 1:1 combination of 20 % banana pseudostem and 20 % Aegle marmelos leaf extract

15 T14 = 1:1 combination of 10 times diluted 20 % banana pseudostem and 20 % Aegle marmelos leaf extract

16 T15 = 1:1 combination of 100 times diluted 20 % banana pseudostem and 20 % Aegle marmelos leaf extract

17 T16 = 1:1 combination of 50 % banana pseudostem and 50 % Aegle marmelos leaf extract

18 T17 = 1:1 combination of 10 times diluted 50 % banana pseudostem and 50 % Aegle marmelos leaf extract

19 T18 = 1:1 combination of 100 times diluted 50 % banana pseudostem and 50 % Aegle marmelos leaf extract

The leaf samples were collected after 24 h of treatment. One gram of leaves was washed in 5 ml autoclaved sterile distilled water. The leaf wash thus obtained was diluted 10³, 10⁴ and 10⁵ times respectively. One ml of the obtained leaf wash from each dilution was cultured on media plates by spread plate technique. After 24 h of incubation at 37˚C, the colony forming units (cfu’s) of the HEPs were counted. All the experiments were conducted in triplicates.

Statistical analysis:

The data were statistically analyzed by analysis of variance (ANOVA) using the general linear model procedure and the least squares means test of the statistical software SAS (version 9.2 developed by SAS institute Inc., Cary, NC). Multiple pairwise-comparison tests using least-square means were performed for post-hoc comparisons after two-way ANOVA with treatment and time as two factors with replications. The corrections used for multiple comparisons were Tukey’s honest significantly differences (HSD) test, Dunnett and Bonferroni procedure.

RESULTS:

Identification of isolated microbes:

Figure 1. 16S rRNA sequence of Escherichia coli. Figure 2. 16S rRNA sequence of Salmonella typhiii.

The 16S rRNA sequencing results (Figures 1 and 2) of the two microbes isolated from tomato phylloplane confirmed their identities as Escherichia coli and Salmonella typhi.

The most notable reduction in in vitro growth of Salmonella was observed when 50% Aegle marmelos leaf extract was incorporated in the culture media. Upto 40% inhibition was observed at this concentration. The 1:10 and 1:100 dilutions also had significant antibacterial activity against Salmonella. The effects of A. marmelos leaf extract at lower concentration appear to be more pronounced on Salmonella than on E. coli.

The combination of 20% banana pseudostem and Aegle marmelos leaves extracts showed remarkable antibacterial activity against Salmonella. The mixture was able to suppress the in vitro growth of Salmonella by about 38%. The 1:10 and 1:100 dilutions of this combination were also significantly effective against the pathogen. The combination of 50 % banana pseudostem and 50 % Aegle marmelos leaf extracts was most effective against Salmonella than E. coli. The formulation could reduce the pathogen growth by up to 48% as compared to control. The 1:10 and 1:100 dilutions of this combination had the highest antibacterial effect against Salmonella as compared to other concentrations (Table 2).

Table 2. In vitro bacterial growth inhibition measurement of different combinations of extracts from banana pseudostem and Aegle leaves against Salmonella.

Treatment	Growth Diameter (in mm)
C	50
T1	37
T2	39
T3	41
T4	33
T5	36
T6	38
T7	31
T8	34
T9	37
T10	30
T11	34
T12	36
T13	32
T14	34
T15	35
T16	26
T17	29
T18	36

C = Control (autoclaved sterile Type I water); T1 = 20 % banana pseudostem extract; T2 = 10 times diluted 20 % banana pseudostem extract; T3 = 100 times diluted 20 % banana pseudostem extract; T4 = 20 % Aegle marmelos leaf extract; T5 = 10 times diluted 20 % Aegle marmelos leaf extract; T6 = 100 times diluted 20 % Aegle marmelos leaf extract; T7 = 50 % banana pseudostem extract; T8 = 10 times diluted 50 % banana pseudostem extract; T9 = 100 times diluted 50 % banana pseudostem extract; T10 = 50 % Aegle marmelos leaf extract; T11 = 10 times diluted 50 % Aegle marmelos leaf extract; T12 = 100 times diluted 50 % Aegle marmelos leaf extract; T13 = 1:1 combination of 20 % banana pseudostem and 20 % Aegle marmelos leaf extract; T14 = 1:1 combination of 10 times diluted 20 % banana pseudostem and 20 % Aegle marmelos leaf extract; T15 = 1:1 combination of 100 times diluted 20 % banana pseudostem and 20 % Aegle marmelos leaf extract; T16 = 1:1 combination of 50 % banana pseudostem and 50 % Aegle marmelos leaf extract; T17 = 1:1 combination of 10 times diluted 50 % banana pseudostem and 50 % Aegle marmelos leaf extract; T18 = 1:1 combination of 100 times diluted 50 % banana pseudostem and 50 % Aegle marmelos leaf extract

The results of cfu calculations of HEPs from the leaf wash of the treated plants are in agreement with the in vitro bacterial growth inhibition measurements. The observations demonstrate the efficacy of extracts from banana pseudostem and A. marmelos leaves in reducing the populations of E. coli and Salmonella on the phylloplane of tomato.

Both the extracts were significantly (P ≤ 0.05) inhibitory to E. coli at 20% and 50% concentrations when applied singly as well in combinations. The 20% banana pseudostem extract when applied singly, reduced the E. coli cfu by about 36%. 50% extracts of banana pseudostem was most effective and could reduce E. coli population to half as compared to control when applied singly or in combination with leaf extracts of Aegle marmelos. The combination of 20% extracts of banana pseudostem and Aegle leaves also had an equivalent inhibitory effect on the pathogen population. The 10 times diluted extracts from both plants were also significantly (P ≤ 0.05) inhibitory to the enteric pathogen and banana pseudostem extract was most significantly effective against E. coli amongst all the combinations tested at this dilution. The 100 times diluted extracts from banana pseudostem and A. marmelos leaves also inhibited the growth of E. coli but not significantly (P ≤ 0.05) enough, either singly or in combination. Thus, the efficacy of the formulated extract in inhibiting the pathogen decreased with the increase in its dilution. With the increase in the dilution of the leaf wash, the inhibitory effect of the botanical formulation was observed to be elevated (Figures 5a, 5b and 5c).

The inhibitory ability of extracts of banana pseudostem and Aegle leaves had similar behavior against Salmonella on the phylloplane of tomato plants. The botanical formulation was significantly (P ≤ 0.05) effective in reducing the count of the colony forming units of the lethal pathogen. As in E. coli, 50% extracts of banana pseudostem and Aegle leaves were most effective and could reduce the pathogen population by up to 40% on tomato leaves, when sprayed either singly or in combination. Dilution of the plant extracts was found to be inversely proportional to their ability in inhibiting pathogen growth. 20% extracts of banana pseudostem and Aegle leaves inhibited Salmonella growth. However, unlike their action against E. coli, the inhibition at this concentration was not significant (P ≤ 0.05) enough, when sprayed singly or in combinations (Figures 6a, 6b and 6c)

Figure 5. Effect of banana pseudostem and Aegle leaf extract on the colony forming units (cfu/ml) of E. coli at (a) 10³ dilution, (b) 10⁴ dilution and (c) 10⁵ dilution of leaf wash.

Figure 6. Effect of banana pseudostem and Aegle leaf extract on the colony forming units (cfu/ml) of Salmonella at (a) 10³ dilution, (b) 10⁴ dilution and (c) 10⁵ dilution of leaf wash.

DISCUSSION:

The findings of the present study clearly demonstrate the antibacterial ability of aqueous extracts of banana pseudostem and Aegle marmelos leaves against lethal human enteric pathogens E. coli and Salmonella sp. 50 % concentration of the two extracts depicted the best anti-HEP activity both in vitro as well as on the phylloplane of the growing plant, amongst the two concentrations tested. Banana pseudostem extract was more active against E. coli and A. marmelos leaf extract was more active against Salmonella. The combinations of these two extracts had significant inhibitory effects upon the growth of both pathogens. The dilutions of extracts from the two plants could significantly suppress the pathogens.

Mohamed et al. demonstrated the antibacterial activity of unripe banana against wide range of gram negative and gram positive bacteria including E. coli and Salmonella typhii.¹² Similar E. coli growth suppressing effect of the compounds isolated from the banana fruit was also observed.¹³ Mokbel and Hashinaga and Fagbemi et al. demonstrated significant antioxidant and antibacterial abilities of ethyl acetate and aqueous fractions of fresh green peel of banana and tannins present in the banana pericarp against S. aureus, B. subtilis, B. cereus, S. enteritidis, E. coli, P. aeruginosa, S. paratyphi, S. flexneri and K. pneumonia.^4,14 Li et al. elaborated the antibacterial properties of banana bericarp against several microbes including bacteria.¹⁵ Notable observations of growth reducing activity of water extracted fraction of banana against food borne pathogens like Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes and Vibrio parahaemolyticus have been made, hence concluding that Musa paradisiaca water fraction could be potential source of natural antimicrobials to be used in foods before being served to the consumers.¹⁶ In their review, Mohapatra et al. have mentioned about the antimicrobial abilities of all the parts of banana plant especially emphasizing on the pseudostem which contains pathogenesis proteins possessing these properties of killing microbes.⁸ The methanolic extracts of peel, pulp, flower and seeds of banana had lytic activity against several bacteria including E. coli and Salmonella.^6,7,17 Hossain et al. elaborated the anti-HEPs potentiality of banana seeds from his observations made on the lytic capabilities of their extracts against Staphylococcus aureus, Escherichia coli, Pseudomonus aeruginosa, Salmonella typhi, Shigella boydii, Shigella flexneri and Shigella dysenteriae.¹⁸ Proteus vulgaris and Klebsiella pneumonia were shown to be susceptible to extract from banana peel.¹⁹ From their findings on the potential bacteriolytic nature of banana inflorescence, Padam et al. stated that it can be exploited as a natural antibacterial.²⁰ It has been reported that banana peel extracts possess significant antibacterial activity against several human pathogens without affecting the normal flora of human body and hence can be implicated in reducing human-pathogenic bacteria populations on the surface of diet-important crops and vegetables.²¹

Several investigations have evidenced the remarkable role of water and organic extracts from various parts of Aegle in controlling the growth and population of lethal human enteric pathogens including E. coli and Salmonella.^11,22 Broad range biological activity of crude extracts from different parts (leaf, fruit, stem and root) of Aegle marmelos have been discussed earlier.^23-28 Several other studies involving in vitro antibacterial effects of aqueous and ethanolic extracts of Aegle leaves and other parts on several human pathogenic bacteria concluded that the extracts possess promising antibiotic properties.^29-31 Tambekar and Dahikar have mentioned the bactericidal properties of Aegle marmelos root extract against E. coli.^2,32 The broad spectrum lytic properties of the organic extracts of Aegle leaves against human pathogenic bacteria and fungi have been observed and hence they recommended it as a potential source for development of novel bioactive antimicrobial agents.³³ In various reviews, the antimicrobial properties of Aegle marmelos extracts have been enumerated, discussing in details the multi ranged effect of the extracts from this plant over a large array of microbes that cause diseases in humans.^9,34-36

CONCLUSIONS:

The investigation here reported thus emphasizes on the ability of aqueous extracts of banana pseudostem and Aegle marmelos leaves to reduce loads of HEPs on the surface of the diet-necessary crops. The formulation from these two extracts could significantly control the growth of two common enteric pathogens i.e E. coli and Salmonella both in vitro as well as on the surface of growing plants. The extract could effectively be used to treat fruits and vegetables after harvest thereby significantly reducing HEPs load on them and also possibly reducing post-harvest decay by inhibiting post-harvest microbial pathogens.

ACKNOWLEDGEMENT:

The authors thank Amity University Uttar Pradesh for providing necessary infrastructural facilities for carrying out the research work. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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Received on 22.03.2018 Modified on 18.04.2018

Research J. Pharm. and Tech 2018; 11(6): 2408-2417.

DOI: 10.5958/0974-360X.2018.00445.6