Introduction

The problem of food spoilage has plagued humans since ancient times. Spices and herbs are used in foods mainly for their flavour and aroma but it is recognized that they may fulfill more than one function in foods to which they are added. In addition to imparting flavour, certain spices prolong the storage life of food by preventing rancidity through their bacteriostatic or bactericidal activity (Beuchat and Golden, 1989). Being natural foodstuff, they appeal to consumers who tend to question the safety of synthetic food additives. Many natural substances of plant origin may play a fundamental role in the host-pathogen relationship, and products from different plant genera are reported to be biologically active, endowed with antimicrobial, allelopathic and antioxidant properties.

Amchur (dried pulp of unripe Mangifera indica) is used in Indian spices as a souring agent to provide the desired acidity in the various food recipes.

Mango is considered as a king of fruits in Indian delicacy. The roots and bark of mango Mangifera indica (Anacardiaceae) are astringent, acrid, anti-inflammatory, and constipating. The leaves and flowers are refrigerant, styptic, vulnerary and constipating. Amchur (dried or dehydrated product of unripe mango flesh in the form of peeled slices or powder) is used as an acidulant or a souring agent for curries. Amchur is rich in citric acid.

Very limited literature is available on the antimicrobial activity of amchur extract. In the present study, we have investigated the antibacterial activity & phytochemical study of dried pulp of unripe Mangifera indica for the first time.

Materials And Methods

Materials: All chemicals used were of analytical-reagent grade and obtained from E. Merck (Mumbai, India). Amchur (Mangifera indica) was collected from local market of Meerut (Uttar Pradesh, India). Dr. C.M Govil, Botany Department, C.C.S University, Meerut, India confirmed the species.

Bacterial & fungal strains: Ten bacterial strains (7 Gram positive and 3 Gram negative), mostly food borne including pathogens, were selected for the study. Gram positives were Bacillus cereus, Bacillus mycoides, Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus, Staphylococcus epidermidis and Listeria monocytogenes while Gram negatives were Escherichia coli, Enterobacter aerogenes and Pseudomonas aeruginosa. The fungal species used in the present study were Alternaria sp., Aspergillus fumigatus, Aspergillus niger, Aspergillus sp., Penicillium sp., Rhizopus sp. & Rhizomucor sp. The bacterial & fungal stock cultures were obtained from the Department of Microbiology of this University. The viability tests for each isolate were carried out by resuscitating the organism in nutrient agar medium & Sabouraud’s dextrose agar (SDA) medium respectively. The stock on nutrient agar medium (Hi Media, Mumbai, India) & potato dextrose agar medium was incubated for 24h at 37°C (bacteria) & 28°C for 3 days (fungi) respectively following refrigeration storage at 4°C until required for sensitivity testing.

Extraction: The pulp of unripe mango (Mangifera indica) was dried and powdered in milling machine (Inalsa Mixer Grinder) to obtain fine dry powder called amchur. The powder was weighed using single pan electronic weighing balance (Ohaus model). The herbal extract was prepared at the rate of 1g/5ml of solvent (50% ethanol) in a 250mL Erlenmeyer flasks. The flasks were closed with cotton plug and aluminium foil. The spice powder was soaked in 50% ethanol for 48h at room temperature with intermittent shaking. The mixture was centrifuged at 3500xg for 20min and finally filtered through Whatmann filter paper No.1 (Azoro, 2000). The pellet was discarded and the supernatant was collected and concentrated under reduced pressure in a rotary vacuum evaporator (Buchi Type) until semisolid substance was obtained. This was dried inside the crucible under a controlled temperature (45ºC) to obtain solid powder (Jonathan and Fasidi, 2003). The process of extraction was repeated until the weight of 500mg was obtained.

The powder was weighed and reconstituted in dimethyl sulfoxide (DMSO). These were stored in the refrigerator at 4ºC for testing antimicrobial sensitivity. Once the extracts are dissolved in pure DMSO, these are also sterilized, and thus, a very costly and time consuming step of membrane filtration sterilization was omitted (Zgoda and Porter, 2001).

Antibacterial assay: The antimicrobial activity of amchur extract was determined by agar well diffusion method (Okeke et al., 2001). Pure isolate of each bacterium was first subcultured in nutrient broth at 37ºC for 24h. One hundred microlitres (100μL) of standardized inoculum (106CFU/mL; 0.5 Mac-Farland) of each test bacterium was spread with the help of sterile spreader on to a sterile Muller-Hinton Agar plate (Hi Media, Mumbai, India) so as to achieve a confluent growth. The plates were allowed to dry and a sterile cork borer (6.0mm diameter) was used to bore wells in the agar. Subsequently, a 50μL volume of the extract was introduced in triplicate wells of the agar plates. Sterile DMSO & sodium propionate (standard food preservative) served as negative & positive control respectively. The plates were allowed to stand for 1h or more for diffusion to take place and then incubated at 37ºC for 24h. The zone of inhibition was recorded to the nearest size in mm (Norrel and Messely, 1997).

Figure 1

Table1: Zone of inhibition (mm) of ethanolic extract of amchur () on selected bacteria that cause food spoilage

Incubation temperature: 37ºC; Incubation period: 24h

Positive control- Sodium propionate; Negative control- Dimethyl sulfoxide

Each value is the average of three independent replicates.

Antifungal assay: For determining the antifungal activity of the oil, the fungal isolates were subcultured on SDA at 28ºC for 3-4 days. Sterilized Sabouraud’s Dextrose Agar plates were taken and a sterile cork borer (6-mm diameter) was used to bore wells in the agar. A 50L volume of the oil was introduced into each of the peripheral wells while a fungal disc was inoculated into the central well. A negative control (sterilized DMSO) was also included in one of the peripheral wells to compare the activity. The plates were then incubated at 28ºC. The evaluations were carried out by means of daily measurement of colony diameter, starting 24h after the experiment began and finishing when 2/3rd the plate surface of the control treatment was covered by the fungus (Fiori et al., 2000). The appearance of zones of inhibition was regarded as the presence of antimicrobial action in the test substance.

The results were expressed in terms of the diameter of the inhibition zone: <9mm, inactive; 9-12mm, partially active; 13-18mm, active; >18mm, very active (Junior and Zanil, 2000).

Figure 2

Table 2: Zone of inhibition (mm) of ethanolic extract of amchur () against common food spoilage fungi on SDA medium

Positive control- Sodium propionate; Negative control- Dimethyl sulfoxide

Each value is the average of three independent replicates.

Determination of Minimum Inhibitory Concentration (MIC) of amchur extract: The method of Thongson et al., 2004 was applied. The MIC for the crude extract was determined by agar-well diffusion method. A two-fold serial dilution of the test extracts was prepared by first reconstituting it in DMSO. It was then diluted in sterile DMSO to achieve a decreasing concentration range of 1000- 31.25mg/mL. A 50L volume of each dilution was added aseptically into Mueller Hinton agar plates that were already seeded with the standardized inoculum (106CFU/mL) of the test bacterial cells. Sodium propionate only served as positive control. All the experiments were performed in triplicate. The same procedure was used for fungi, except that SDA plates were used and the plates were incubated at 28ºC. The lowest concentration of amchur extract showing a clear zone of inhibition was considered as the MIC.

Figure 3

Table 3: The MIC (Minimum inhibitory concentration) values of amchur extract (mg/mL) against different bacteria on Mueller- Hinton Agar Medium

Incubation temperature: 37ºC; Incubation period: 24h

Negative control- Dimethyl sulfoxide

Each value is the average of three independent replicates.

Preliminary phytochemical analysis of amchur extract: The ethanolic extract of the amchur powder was subjected to phytochemical tests for the presence of tannin, alkaloid, saponin, cardiac glycoside, steroid, flavanoid &.terpenoid.

Tannins- (200 mg plant material in 10 ml distilled water, filtered); a 2 ml filtrate + 2 ml FeCl3, blue-black precipitate indicated the presence of Tannins.

Alkaloids- (200 mg plant material in 10 ml methanol, filtered); a 2ml filtrate + 1% HCl + steam, 1 ml filtrate + 6 drops of Mayor’s reagents/Wagner’s reagent/ Dragendroff reagent, creamish precipitate/brownish-red precipitate/orange precipitate indicated the presence of respective alkaloids.

Saponin- (frothing test: 0.5 ml filtrate + 5 ml distilled water); frothing persistence indicated presence of saponins.

Cardiac glycosides- (Keller-Kiliani test) 2 ml filtrate + 1 ml glacial acetic acid + FeCl₃ + conc. H₂SO₄); green-blue color indicated the presence of cardiac glycosides.

Steroids- (Liebermann-Burchard reaction) 200 mg plant material in 10 ml chloroform, filtered); a 2 ml filtrate + 2 ml acetic anhydride + conc. H₂SO₄. Blue-green ring indicated the presence of terpenoids.

Flavonoids- (200 mg plant material in 10 ml ethanol, filtered); a 2 ml filtrate + conc. HCl + magnesium ribbon pink-tomato red color indicated the presence of flavonoids

Terpenoid- 2mL of chloroform & concentrated sulphuric acid was added to 1mg of extract & observed for reddish brown colour (Harbone, 1973).

Figure 4

Table 4: Results of phytochemical tests for amchur extract

Results

Following the extraction of the dried unripe pulp of Mangifera indica (Amchur) using 50% ethanol by maceration method, the antimicrobial activity of the extract was determined. Table 1 shows the antimicrobial activity of the amchur extract on the selected food borne bacteria that cause spoilage. The extract was effective against both Gram positive and Gram negative bacteria. However the ethanolic extract was most effective against Staphylococcus aureus with an inhibition zone diameter (IZD) of 26mm followed by Staphylococcus epidermidis with an IZD of 19mm (both Gram positive bacteria). Amongst the Gram negative bacteria, the extract showed highest activity against Pseudomonas aeruginosa with an IZD of 17mm followed by E. coli & Enterobacter aerogenes (IZD= 15, 14mm respectively).

Antifungal effects of the amchur extract against some fungi have also been investigated as shown in Table 2. The extract was only partially active against Alternaria sp. & Penicillium sp. while the other test fungi were resistant to it. The MIC for the fungal species was therefore not determined. In contrast, sodium propionate which is used as a standard food preservative inhibited all the test fungal species. The highest zone of inhibition was observed in Alternaria sp. & Rhizopus sp. with an IZD each of 14mm.

Table 3 shows the minimum inhibitory concentration (MIC) of the ethanolic extract of amchur. It ranged from 500 to 62.5 mg/mL. Bacillus mycoides was found to be highly sensitive to the extract exhibiting lowest MIC of 62.5 mg/mL followed by Staphylococcus aureus & Pseudomonas aeruginosa with MIC of 125 mg/mL each while Micrococcus luteus was found to be resistant to it (MIC= 500 mg/mL).

The phytochemical analysis of the ethanolic extract of amchur powder was also carried out (Table 4). The study revealed the presence of tannins & terpenes in the amchur powder.

Discussion

From this investigation, it was observed that amchur is effective against both groups of bacteria. It is due to the change in pH of the medium due to amchur which cause the pH to bring down in acidic range. pH is known to control the growth, development and sporulation of all microbes including bacteria (Frazier, 1958). Amchur contains citric acid related compounds which is responsible for its sour taste.

The antimicrobial activity of amchur extract is also due to presence of tannins & terpenes. It has been well documented that several terpenes (ocimene, myrcene, limonene), aldehydes and esters occur in dried unripe mango fruit. (Gernot Katzer’s Spice Pages- An encyclopedia of Spices). Moreover, investigations into the effect of terpenoids upon isolated bacterial membrane have suggested that their activity is a function of the lipophilic properties, the potency of their functional groups and their aqueous solubility (Knobloch et al., 1989, Elgayyar et al., 2001). Their site of action is at the phospholipid bilayer, caused by biochemical mechanisms catalysed by the phospholipid bilayers of the cell. These processes include the inhibition of electron transport, protein translocation, phosphorylation steps and other enzyme – dependent reactions (Knobloch et al., 1989).

Conclusion: In conclusion, amchur extract was found to be a much better antagonistic agent, exhibiting broad range of antibacterial activity against common bacteria than sodium propionate. It is therefore conceivable that it represents an inexpensive source of food preserving agents.

Acknowledgements