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Isolation and characterization of potential food preservative peptide from Momordica charantia L.
⁎Corresponding author at: Department of Biochemistry, PMAS, Arid Agriculture University Rawalpindi, Murree Road, Rawalpindi 46300, Pakistan. Tel.: +92 51 445 1772; fax: +92 51 442 0894. azrakhanum@uaar.edu.pk (Azra Khanum)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Abstract
Antimicrobial agents (AMAs) also known as preservatives are being used to keep food safe and unspoiled from microorganism. These preservatives, derived from plant sources or synthesized chemically are mostly non-proteinaceous AMAs. Less attention has been given to proteinaceous AMAs which may be used as preservative in food. Thus, there is a need to explore proteinaceous AMAs having antimicrobial activity with potentials to be used as an alternative to the presently used preservatives which are mostly synthetic. Therefore, the present study was carried out systematically to isolate and characterize peptides having antibacterial activity from different parts of bitter melon (Momordica charantia L.). Crude aqueous extracts of seeds, pulp and skin were prepared in phosphate buffer saline (PBS) and antibacterial activity was checked on Luria Bertani (LB) broth agar plates against a number of bacteria such as Escherichia coli, Staphylococcus aureus, Salmonella typhi and Pseudomonas aeruginosa. Since antimicrobial activity was observed only in seeds therefore, seed extract was used for peptide(s) precipitation with 75% ammonium sulfate solution and purification by gel filtration chromatography (GFC) using Sephadex G-100. Antimicrobial activity was also checked in dissolved ammonium sulfate precipitate and GFC peak fractions (1–4). Further, homogeneity and molecular mass of GFC pooled fractions along with crude extract and dissolved ammonium sulfate precipitate were determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). One peptide with molecular mass ∼10 kDa with antimicrobial activity was obtained from peak 3. The purified peptide was stable at 4–50 °C, active between 5–7 pH and inactivated by trypsin and proteinase K. Based on minimum inhibition concentration (MIC) values of purified peptide S. aureus was found to be the most sensitive strain. The use of this strain with minced meat showed significant viable count decreased i.e. from 8.0 to 3.77 log CFU with P < 0.001 at concentration of 40 μg peptide. The results of our study suggest that antibacterial peptide obtained from seeds of M. charantia L. may be used as an alternative natural biopreservative source for minced meat products. However, further studies need to be carried out to check the effect of this peptide on more microorganisms, characterization and its toxicological analysis.
Keywords
Momordica charantia
Antimicrobial agent
Preservatives
Peptides
Bacteria
1 Introduction
From ancient times, a large number of plants and herbs have been used in traditional medicines against bacterial infection (Cook et al., 2000; Martin and Ernst, 2003; Okoli and Iroegbu, 2004). The uses of plant and herb extracts as antimicrobial agents in food and soft drinks have also been reported for centuries (Friedman et al., 2002; Gulmez et al., 2006). Due to potential toxicity of chemical food preservatives, there has been increased demand for food preservatives from natural sources. This has led researchers and food processors to come across natural food additives with a wide range of antimicrobial activities. As a result today plant antimicrobial products have acquired importance in food system to retard bacterial and fungal growth (Souza et al., 2005a,b). Consumer interest is also increasing in consumption of food products having natural ingredients. Several studies have reported the use of natural molecules i.e. organic acids, peptides and essential oils in past several years (Barreteau et al., 2004).
Antimicrobial agents used as preservatives, are chemical compounds that protect food from spoilage by inhibiting growth of pathogenic microorganisms and increases shelf life. Naturally occurring antimicrobial compounds isolated from animal, plant and microbial sources can be used alone or in combination with other approved antimicrobial preservatives (Davidson, 2006). Studies, however, carried out by Akinpelu (2001) and Souza et al. (2005a,b), pointed out that resistant microorganisms are appearing because of extensive and random use of chemical preservatives which can lead to an increase in incidences of food borne diseases.
Besides chemical compounds, in nature, antimicrobial peptides (AMPs) are commonly distributed in all life forms and play an important role in non-specific host defense system. Initially peptides from the skin of frog and lymph of insects were depicted to kill bacteria in vitro. Now more than 800 AMPs have been described in many living organisms including insects, plants, amphibians and mammals (Rydlo et al., 2006). A number of plant proteins have demonstrated activity against bacteria and fungi, which usually play a key role in plant defense, protecting the plant host from phytopathogens. These proteins have been classified into groups and families according to their function and structural similarities. Main proteins include cyclotides, defensins, lipid transfer proteins (LPTs), lectins, pathogenesis-related proteins, chitin-binding proteins, ribosome-inactivating proteins and digestive enzyme inhibitors (Pelegrini et al., 2008).
Momordica charantia L. commonly known as bitter melon, member of the Cucurbitaceae family is a tropical plant currently distributed across the globe and found to have therapeutic properties like anti diabetic, antioxidant, antiviral and antineoplastic activities (Gover and Yadav, 2004; Beloin et al., 2005; Semiz and Sen, 2007; Paul and Raychaudhuri, 2010). Although numerous constituents of bitter melon have been proposed to have therapeutic properties but a polypeptide (polypeptide p) isolated from seeds has been focused more because of its hypoglycemic activity (Khanna et al., 1981). Another protein MAP30 isolated from bitter melon, recombinant MAP 30 and proteolytic fragments of MAP30 was found to exhibit antiviral, anti HIV and antineoplastic activities in several in vivo and in vitro studies (Lee-Haung et al., 1990, 1995a,b; Huang et al., 1999; Basch et al., 2003). The extracts of bitter melon also appear to inhibit the growth of numerous gram-negative and gram-positive bacteria including Escherichia coli, Salmonella typhi, Shigella dysenteriae, Staphylococcus aureus, Pseudomonas aeruginosa, Streptobacillus moniliformis, Streptococcus pneumoniae, and Helicobacter pylori, and parasitic organisms Entamoeba histolytica and Plasmodium falciparum (Anonymous, 2007). Naturally occurring microbial inhibitors have been recovered from a variety of food materials including onions, garlic, fruits, vegetables, cereals and spices. Many of these antimicrobials contribute to the foodstuff’s natural resistance to deteriorations (Kivanc and Kunduhoglu, 1997). A renewed interest in natural preservation appears to be stimulated by present food safety concerns, growing problems with microbial resistance and a rise in production of minimal processed food joined with green image policies of food industries (Suhr and Nielson, 2003). Taking into account, the significance of naturally occurring antimicrobial peptides, the present study was designed to isolate, characterize and determine potentials of peptides having antibacterial activity from different parts of M. charantia L. against common food pathogens.
2 Materials and methods
2.1 Collection of plant and microorganisms
M. charantia was collected from the local markets of Rawalpindi and indentified by a plant taxonomist, Department of Botany, PMAS-Arid Agriculture University Rawalpindi, Pakistan. The vegetable was brought to the laboratory and rinsed with water to remove the soil particles. Cultures of food related bacteria were obtained from the Depository of Biotechnology Laboratory, Department of Biochemistry, PMAS-Arid Agriculture University Rawalpindi to be used for determination of antimicrobial activity. These include E. coli, S. aureus, S. typhi and P. aeruginosa. Luria Bertani (LB) medium was used for culturing the above mentioned bacteria.
2.2 Protein extraction and partial purification
Protein extraction and partial purification were essentially carried out by the method described by Rehman and Khanum (2011) with some modifications. The seeds, skin and pulp of M. charantia; each 20 g were crushed in pestle and then blended with 100 ml phosphate buffer saline (PBS), pH 7.4 in an electric blender. The homogenates were frozen and thawed three times and then centrifuged at 10,000 rpm for 20 min at 4 °C. The supernatants of seeds, skin and pulp were used for determination of antibacterial activity. These supernatants were then precipitated out with 75% ammonium sulfate solution. The precipitated solutions were centrifuged at 10,000 rpm for 30 min at 4 °C. The pellets were dissolved in 1 ml of deionized water by gentle vortexing. The dissolved ammonium sulfate precipitate and supernatant were dialyzed in a dialysis tube MWCO 3500 Da against distilled water with several changes. The protein concentration was estimated in crude extract, dissolved ammonium sulfate precipitate and supernatant.
2.3 Gel filtration chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE)
The samples of both dissolved ammonium sulfate precipitates and supernatant of seeds, pulp and skin were used for estimation of antimicrobial activity. Since activity was observed only in dissolved ammonium sulfate precipitate of seeds, therefore, dissolved ammonium sulfate precipitate was further purified by gel filtration chromatography using column of Sephadex G-100 with 0.02 M sodium acetate buffer (pH 5). The column was eluted with the same buffer at the flow rate of 1 ml/min. The absorbance of collected fractions was monitored at 280 nm. The homogeneity and molecular mass of pooled fractions along with crude extract and dialyzed ammonium sulfate precipitates were run on 15% SDS-gel (Laemmli, 1970). The separating gel was placed in Coomassie blue R-250 staining solution on a shaker for 3 h. Excess dye was removed by destaining with methanol:acetic acid:water (30:60:10 v/v). The approximate molecular mass of fractions having antimicrobial activity was determined by plotting molecular weight of standard proteins versus Rf.
The protein concentration in samples (crude extract, dissolved ammonium sulfate precipitates and in its supernatant, fractions of gel filtration chromatography) was estimated by the method of Bradford (1976) using bovine serum albumin as a standard. These were then saved for further studies.
2.4 Antimicrobial assay
2.4.1 Sub-culturing of bacterial strains
Isolated bacterial colonies grown of Luria Bertani (LB) broth agar plate were picked up to grow in 3 ml LB medium overnight at 37 °C. From overnight grown culture 100 μl culture was taken, transferred to 50 ml of fresh LB medium and grown at 37 °C under shaking (250 rpm/min). The bacterial growth was monitored by measuring optical density at 600 nm. The size of inoculums was calculated from optical density (0.4 OD600nm is equal to 108 colony forming unit (CFU)/ml) (Barreteau et al., 2004).
2.4.2 Disc diffusion method
The antimicrobial activity of extracts of seed of M. charantia (crude, ammonium sulfate precipitated sample, and chromatographic pooled fractions) was determined using the disc diffusion method described by Bauer et al. (1996).
2.4.3 Minimum inhibitory concentration in culture media
Minimum inhibitory concentration (MIC) values of peptide isolated from seed was determined with broth dilution assay as described by Wang (2003). The 108 CFU/ml S. aureus cultures were inoculated into LB broth containing 0–200 μg/ml antimicrobial protein preparation and incubated at 37 °C for 4 h.
2.4.4 Statistical analysis
Mean zone of inhibition in millimeter (mm) and standard deviation of the triplicate incubations were calculated by using Excel statistical software (2007).
2.5 Effect of various temperatures, pH and proteolytic enzymes
The peptide activity was determined at different temperatures; 0 °C, 4 °C, 30 °C, 40 °C, 50 °C, 60 °C ,70 °C, 80 °C and pH; 4, 5, 6, 7 and 8 by heating at different temperatures for 15 min and then bioassayed. Similarly pH effect on isolated peptide was determined by changing pH from 4 to 8. The effect of two enzymes proteinase K (Fermentas® cat #EO0491) and trypsin–EDTA 1X (GIBCO® cat #25300-054) on antimicrobial activity of purified peptide was also checked. Peptide sample of 3–20 μg/10 μl was mixed with 0.02 M Tris–HCl, pH 6.8, 1–2 U/2 μl of enzyme was added into the mixture with total volume of 100 μl and kept for 5 min. Enzyme activity was stopped by heating the solution in a thermo mixer for 5 min. This solution (30 μl) was applied on to the disc to see the effect of different enzymes on bioactivity of the isolated peptides.
2.6 Effect of purified peptide on minced meat inoculated with S. aureus
The antimicrobial activity of purified peptide on minced meat was checked by the method of Wang (2003). Bacterial suspension of S. aureus was centrifuged at 13,000 rpm and pellet was resuspended in 10 mM sodium phosphate buffer, pH 7.4. One gram of minced meat was washed with 70% ethanol, rinsed with deionized water and mixed with 1 ml buffer containing different concentrations of antimicrobial peptide i.e. 0–200 μg/g. Minced meat without antimicrobial peptide was taken as control. Culture of S. aureus (100 μl) was inoculated in this mixture and incubated overnight at 37 °C. After overnight incubation culture was diluted 10-fold serially and streaked on LB agar plates. Cell counts were determined by counting the colonies after plates were incubated overnight at 37 °C.
3 Results and discussion
3.1 Antimicrobial activity
Antimicrobial activity of crude extract, dissolved ammonium sulfate precipitates of seeds, skin and pulp were investigated on LB agar plates against various food pathogens (S. aureus, E. coli, S. typhi, and P. aeruginosa) in triplicate. The results are given in Table 1. Among the tested microorganisms, the crude seed extract showed highest activity against E. coli (6.66 ± 1.15) followed by S. typhi (4.33 ± 0.577) and S. aureus (3.66 ± 0.577) and lowest activity against P. aeruginosa (2.00 ± 0.00). The dissolved ammonium sulfate precipitates of seeds showed highest activity against E .coli (9.33 ± 0.577) followed by S. aureus (8.00 ± 1.00), S. typhi (6.63 ± 0.577) while lowest activity against P. aeruginosa (2.66 ± 0.5). The dissolved ammonium sulfate precipitates have shown better activity as compared to the crude extracts of seeds, thereby indicating that the protein(s)/peptide(s) isolated from the crude extract has been partially purified.
| Section 1.01 | Gram positive | Gram negative | ||
|---|---|---|---|---|
| Protein contents 20 μg/30 μl | S. aureus | E. coli | S. typhi | P. aeruginosa |
| Crude seed extract | 3.66 ± 0.577 | 6.66 ± 1.00 | 4.33 ± 0.577 | 2.00 ± 0.00 |
| Solubilized NH4(SO4)2 precipitate | 8.00 ± 1.00 | 9.33 ± 0.577 | 6.63 ± 0.577 | 2.66 ± 0.50 |
| Fraction (Peak 3) | 11.66 ± 0.577 | 12.00 ± 1.15 | 9.00 ± 0.577 | 3.33 ± 0.577 |
| Cam34 102 μg/30 μl | 25 | 25 | 23 | 20 |
Roopashree et al. (2008) also screened antimicrobial activity of M charantia seeds in aqueous, petroleum ether, methanol and ethanol extracts against S. aureus, E. coli and P. aeruginosa. The results of this study showed that aqueous extract has higher antibacterial activity particularly against S. aureus i.e. 22 ± 0.64 which is comparable to our study. The antimicrobial activity was also checked in skin and pulp of M. charantia L. in this study. However no activity was observed in these parts of vegetable. The results are in contrast to a study carried out by Saeed and Tariq (2005) who observed significant antimicrobial activities in aqueous extracts of skin and pulp of M. charantia against a number of microorganisms like E. coli, P. aeruginosa, S. typhi, K. pneumonia, Salmonella paratyphi A, Salmonella paratyphi B, Proteus vulgaris, S. dysenteriae, Yersinia enterocolitica, Proteus mirabilis, Enterobacter aerogenes with average zone of inhibition of 16.16 mm ± 2.02 mm and 15.88 mm ± 2.24 mm, respectively. The reason could be the use of different varieties of M. charantia in our case.
3.2 Chromatographic profile of dissolved and dialyzed ammonium sulfate precipitates of seeds
The dissolved ammonium sulfate precipitates of seeds were then purified by gel filtration column chromatography using Sephadex G-100. The elution profile of various peptide(s) is shown in Fig. 1. Four peaks were obtained ranging from high molecular mass to low molecular mass peptide(s). All peaks were lyophilized and checked for antimicrobial activity, only peak 3 showed activity against E. coli (12 ± 1.15), S. aureus (11.66 ± 0.577), S. typhi (9 ± 0.577) and P. aeruginosa (4.3 ± 0.577). Chromatographic fraction showed approximately two and one fold increase in activity than crude and dissolved ammonium sulfate precipitates respectively indicating the further purification of peptide. In several other studies using different plant or animal extracts, peptide(s) were isolated and checked for antimicrobial activity. For example, Dahot (1998) obtained three fractions from leaf extracts of M. oleifera on Sephadex G-25 column chromatography. Fraction P1, P2 and P3 showed strong inhibitory activity against E. coli, S. aureus, Bacillus subtilis and Klebsiella aerogenes. Fraction P2 showed significant zone of inhibition against Aspergillus niger, while fractions P1, P2 and P3 were not found effective against the growth of Klebsiella pneumoniae, Aspergillus fumigatus, Aspergillus flavus and Penicillium expansum. Similarly Hou et al. (2007) purified an antibacterial peptide from housefly larvae by using Sephadex G-15, five peaks were obtained but only peak 1 showed antibacterial activity against E. coli and S. aureus. Recently, Rehman and Khanum (2011) isolated two peptides from seed and two peptides from pod of Pisum sativum L. (garden pea) having molecular weight ∼19 kDa, ∼22 kDa, ∼10 kDa and ∼11 kDa, respectively. These peptides have shown strong antimicrobial activity against a number of bacteria.
3.3 SDS–PAGE
The crude extract, dissolved and dialyzed ammonium sulfate precipitates and purified peptide of seeds were subjected to 15% SDS–PAGE along with molecular mass of 14.4–116.0 k Da. The pattern of protein separation on SDS–PAGE was found to be almost same in samples of crude extract and dialyzed ammonium sulfate precipitates while purified peptide obtained by gel filtration chromatography (Peak 3) appeared as a single band with a molecular weight of ∼10 kDa. The result of SDS–PAGE is shown in Fig. 2. Similar to this study, Parkash et al. (2002) isolated antimicrobial peptide with molecular weight ∼9.7 kDa from bitter gourd seeds by affinity chromatography, ion exchange chromatography and gel filtration chromatography. In another study carried out by Diz et al. (2006) they isolated ∼10 kDa antimicrobial peptide from chili pepper seeds while Hou et al. (2007) purified an antimicrobial peptide with molecular weight ∼8.0 kDa. It was obtained as a single band on SDS–PAGE after purification by Sephadex G-15 and HPLC. Xia and Ng (2005) also isolated antimicrobial protein with molecular weight ∼13 kDa from garlic bulbs. All these studies including ours showed that antimicrobial peptides purified by different chromatographic methods and SDS–PAGE have low molecular mass.
3.4 Effect of various temperatures, pH and enzymes on antimicrobial activity of purified peptide
The effect of various temperatures on the antimicrobial activity of purified peptide (Peak 3) of seeds was determined against S. aureus, E. coli and S. typhi. The results are shown in Fig. 3. Peptide exhibited different activities at different temperatures, for example, the activities shown by peptide against S. aureus and E. coli were approximately constant i.e. 10.33 ± 0.577 and 10.66 ± 0.577 respectively at temperatures 4 °C, 30 °C, 40 °C and 50 °C. A decrease of about 40–54% in activities was observed at temperatures 60 °C and 70 °C against S. aureus: 6.66 ± 0.577 and 6.00 ± 0.50 and E. coli: 6.00 ± 0.50 and 4.66 ± 0.10 respectively. In case of S. typhi, the activity was constant i.e. 8.55 ± 0.70 at 4 °C, 30 °C and 40 °C. This activity was then declined at temperatures 50 °C, 60 °C and 70 °C with 40% (6 ± 0.00), 65% (3.66 ± 0.50) and 70% (3 ± 0.70) respectively. The peptide did not show any activity at 0 °C and complete lose of activity was observed at 80 °C against all tested bacteria. These results showed that the peptide was more effective at low and moderate temperatures i.e. 4 °C or 50 °C.
These results support the view that specific conformation of peptide is needed for its function. Any change in conformation leads to the loss of function i.e. antibacterial activity. It is evident from the data that high temperature may have changed the specific configuration of peptide necessary for its antibacterial activity. The data of our study are further supported by Girish et al. (2006) who observed that heating of non-toxic glycoprotein from Withania somnifera up to 60 °C for 10 min did not alter the antimicrobial property, whereas the temperature above 70 °C was found to be inhibitory to antimicrobial activity. Similarly, a study carried out by Ngai and Ng (2004) reported that the functional activity of polypeptide isolated from Chinese white cabbage seeds was stable between 10 and 40 °C.
The effect of pH on the antibacterial activity of purified peptide (Peak 3) of seeds was checked against the above mentioned microorganisms. With pH change, the antibacterial activity of peptide has also been changed. At pH 4 and 8 peptide was inactive against all the microbes tested. Peptide showed a sharp increase in activity against S. aureus with 9 ± 0.00 and E. coli with 9.5 ± 0.70 at pH 5 while a slight increase at pH 6 and pH 7 i.e. 9.66 ± 1.57 and 10 ± 2.82. In case of S. typhi the pattern of antibacterial activity was different. The activity increased at pH 5 i.e. 5 ± 0.00 then pH 6 i.e. 7.5 ± 0.707 thereafter remained constant at pH 7 (Fig. 4). These results show that activity of purified peptides is pH-dependent and varies for different microbes. The results further revealed that purified peptide is stable between narrow ranges of pH 5–7 which is similar to the results obtained by Rehman and Khanum (2011) for peptides purified from P. sativum. However, it is contrast to the broad range antimicrobial substances obtained from Bacillus cereus that has high antimicrobial activity in pH range of 2.0–9.0 (Risoen et al., 2004). Similarly the stability of polypeptide isolated from Chinese white cabbage was retained between pH 4–11 as described by Ngai and Ng (2004). The proteinaceous nature of isolated peptide (Peak 3) was confirmed with use of proteolytic enzymes i.e. proteinase K and trypsin against S. aureus E. coli and S. typhi which proved the evidence that proteolytic enzymes did change the specific structure of peptides necessary for their antimicrobial activity by hydrolyzing it. The study of Wang (2003) supports our results by providing evidence that proteolytic enzymes: pepsin, trypsin, and chymotrypsin drastically decline the bactericidal activities of antimicrobial protein.
3.5 Effect of purified peptide on apparent proliferation of bacteria
The active chromatographic fraction was then used for determination of minimum inhibitory concentration (MIC) in culture media. It was observed that the MIC decreases with the increase of concentration of peptide. Therefore, MIC of S. aureus decreased by 21% at concentration of 40 μg and 57% decrease occurred at 200 μg (Fig. 5a). In case of E. coli bacterial growth dropped down to 25% at 40 μg and 49% at 200 μg (Fig. 5b) while less decrease was observed in case of S. typhi and P. aeruginosa. S. typhi proliferation decreased only by 7% at 40 μg and 29% at 200 μg (Fig. 5c). Similarly P. aeruginosa proliferation decreased 8% at 40 μg and 18% at 200 μg (Fig. 5d). This shows that purified peptide is more effective against S. aureus and E. coli as compared to S. typhi and P. aeruginosa. Effect of various concentrations of antimicrobial protein preparation on the apparent proliferation of S. aureus and E. coli was also studied by Wang (2003). In which it was noted that bactericidal activity of antimicrobial protein preparation on the test cultures increased with concentration. Accordingly test bacteria exposing to the 40 μg/ml antimicrobial protein preparation in culture media caused a decrease in 40–60% MIC. The MIC of test microorganisms was completely inhibited after exposing to 120 μg/ml antimicrobial protein preparation. In another study by Hou et al. (2007), they showed peptide Hf-1 activity against E. coli, S. dysenteriae and S. aureus with MIC ranging from 18 to 72 μg/ml. S. aureus was the most sensitive with the MIC of 18 μg/ml, followed by Salmonella typhimurium and B. subtilis with MIC of 36 and 72 μg/ml, respectively while E. coli Jm109, was inhibited at MIC of 108 μg/ml.
3.6 Effect of purified peptide on viable count formation of S. aureus
In order to check the effectiveness of this peptide as biopreservative agent, minced meat was inoculated with S. aureus which was selected based on MIC results. Thus the effect of purified peptide on viable count formation of S. aureus culture inoculated with minced meat is shown in Fig. 6. Growth pattern indicated that viable count formation decreases with increasing concentration of peptide. Similarly Fig. 6 shows clearly that viable count sharply decreased at concentration of 40 μg i.e. from 8.00 to 3.77 log CFU (P = 0.01). Thereafter a marginal decrease was noted with 160 μg and 200 μg with P values 0.002 and 0.005. When obtained data were compared with other studies it was observed that antimicrobial activity of purified fraction of M. charantia was not very high. However, in a study carried by Wang (2003) they noticed that 20 μg/g antimicrobial protein preparation could decrease in 1.3 log CFU of S. aureus and 160 μg/g antimicrobial protein preparation completely inhibited the growth of test cultures in 24 h. The cause of less effect of purified peptide of seeds of M. charantia L. cannot be explained at this point. More studies are needed to be carried out to address this discrepancy.
4 Conclusion
Based on the results of this study, it is concluded that seeds of M. charantia L. contains a low molecular mass peptide having ∼10 kDa. This peptide may be used as an alternative bio-preservative agent to control the growth of bacteria in fresh minced meat. However, further study is required to enhance the effectiveness of this peptide.
References
- Antimicrobial activity of Anacardium occidentale bark. Fitoterapia. 2001;72:286-287.
- [Google Scholar]
- A rapid method for antimicrobial properties determination of natural molecules. J. Food Prot.. 2004;67:1961-1964.
- [Google Scholar]
- Bitter melon (Momordica charantia): a review of efficacy and safety. Am. J. Health-Syst. Pharm.. 2003;60:356-359.
- [Google Scholar]
- Antibiotic susceptibility testing by standard single disc diffusion method. Am. J. Clin. Pathol.. 1996;45:493-496.
- [Google Scholar]
- Ethnomedicinal uses of Momordica charantia (Cucurbitaceae) in Togo and relation to its phytochemistry and biological activity. J. Ethanopharmacol.. 2005;96:49-55.
- [Google Scholar]
- Rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye-binding. Anal. Biochem.. 1976;72:248-254.
- [Google Scholar]
- Arrowroot as a treatment for diarrhea in irritable bowel syndrome patients. Arq. Gastroenterol.. 2000;37:20-24.
- [Google Scholar]
- Antimicrobial activity of small protein of Moringa oleifera leaves. J. Islamic Acad. Sci.. 1998;11:27-32.
- [Google Scholar]
- Proceedings of Ist international symposium on natural preserve in food systems. Acta Horticulturae. 2006;709:29-33.
- [Google Scholar]
- Antimicrobial peptides from chili pepper seeds cause yeast plasma membrane permeabilization and inhibits the acidification of medium by yeast cells. Biochim. Biophys. Acta. 2006;1760:1323-1332.
- [Google Scholar]
- Bactericidal activities of plant essential oils and some of their isolated constituents against Campylobacter jejuni, Escherichia coli, Listeria monocytogenes and Salmonella enteric. J. Food Prot.. 2002;65:1545-1560.
- [Google Scholar]
- Antimicrobial properties of a non-toxic glycoprotein (WSG) from Withania somnifera (Ashwagandha) J. Basic Microbiol.. 2006;46:365-374.
- [Google Scholar]
- Pharmacological actions and potential uses of Momordica charantia: a review. J. Ethanopharmacol.. 2004;93:123-132.
- [Google Scholar]
- Antibacterial activity of oregano tea and a commercial oregano water against Escherichia coli O157:H7, Listeria monocytogenes 4b, Staphylococcus aureus and Yersinia enterocolitica 03. Int. J. Food Sci.. 2006;8:7-13.
- [Google Scholar]
- Inhibition of food-borne pathogens by Hf-1, a novel antibacterial peptide from the larvae of the housefly (Musca domestica) in medium and orange juice. Food Control. 2007;18:1350-1357.
- [Google Scholar]
- Proteolytic fragments of anti-HIV and anti-tumor proteins MAP30 and GAP31 are biologically active. Biochem. Biophys. Res. Commun.. 1999;262:615-623.
- [Google Scholar]
- Antimicrobial activity of fresh plant juice on the growth of bacteria and yeasts. J. Qafqaz Univ.. 1997;1:27-35.
- [Google Scholar]
- Hypoglycemic activity of polypeptide-p from a plant source. J. Nat. Prod.. 1981;44:648-655.
- [Google Scholar]
- Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680-685.
- [Google Scholar]
- MAP 30: a new inhibitor of HIV-1 infection and replication. FEBS letters.. 1990;272:12-18.
- [Google Scholar]
- Anti-HIV and anti-tumor activities of recombinant MAP30 from bitter melon. Gene.. 1995;161:151-156.
- [Google Scholar]
- Inhibition of the integrase of human immunodeficiency virus (HIV) type 1 by anti-HIV plant proteins MAP30 and GAP31. Proc. Natl. Acad. Sci. USA. 1995;92:8818-8822.
- [Google Scholar]
- Herbal medicines for treatment of bacterial infections: a review of controlled clinical trials. J. Antimicrob. Chemother.. 2003;51:241-246.
- [Google Scholar]
- A napin-like polypeptide from dwarf Chinese white cabbage seeds with translation-inhibitory, trypsin-inhibitory, and antibacterial activities. Peptides. 2004;25:171-176.
- [Google Scholar]
- Evaluation of extracts of Anthocleista djalonesis, Nauclea latifolia and Uvaria afzalii for activity against bacterial isolates from cases of non-gonococcal urethritis. J. Ethanopharmacol.. 2004;92:135-144.
- [Google Scholar]
- Purification and characterization of charantin, a napin-like ribosome-inactivating peptide from bitter gourd (Momordica charantia) seeds. J. Pept. Res.. 2002;59:197-202.
- [Google Scholar]
- Identification of a novel storage glycine-rich peptide from guava (Psidium guajava) seeds with activity against Gram-negative bacteria. Peptides. 2008;29:1271-1279.
- [Google Scholar]
- Medicinal uses and molecular identification of two Momordica charantia varieties-a review. Electro. J. Biol.. 2010;6:43-51.
- [Google Scholar]
- Isolation and characterization of peptide(s) from Pisum sativum having antimicrobial activity against various bacteria. Pak. J. Bot.. 2011;43:2971-2978.
- [Google Scholar]
- Characterization of a broad spectrum range antimicrobial substances from Bacillus cereus. J. Appl. Microbiol.. 2004;96:648-655.
- [Google Scholar]
- Antibacterial activity of antipsoriatic herbs: Cassia tora, Momordica charantia and Calendula officinalis. Int. J. Appl. Res. Nat. Prod.. 2008;1:20-28.
- [Google Scholar]
- Peptide-based antimicrobials: promises and premises in food safety. J. Food Sci.. 2006;71:125-135.
- [Google Scholar]
- Antibacterial activities of Mentha piperita, Pisum sativum and Momordica charantia. Pak. J. Bot.. 2005;37:997-1001.
- [Google Scholar]
- Antioxidant and chemoprotective properties of Momordica charantia L. (bitter melon) fruit extract. Afr. J. Biotechnol.. 2007;6:273-277.
- [Google Scholar]
- Antimicrobial effectiveness of spices: an approach for use in food conservation systems. Braz. Arch. Biol. Technol.. 2005;48:549-558.
- [Google Scholar]
- Inhibition action of some essential oils and phytochemicals on the growth of moulds isolated from foods. Brazil. Arch. Biol. Technol.. 2005;2:245-250.
- [Google Scholar]
- Antifungal activity of essential oils evaluated by two different application techniques against rye bread spoilage fungi. J. Appl. Microbiol.. 2003;94:665-675.
- [Google Scholar]
- Effect of antimicrobial proteins from porcine leukocytes on Staphylococcus aureus and Escherichia coli in comminuted meats. J. Meat Sci.. 2003;65:615-621. www.spcforexcel.com
- [Google Scholar]
- Isolation of alliumin, a novel protein with antimicrobial and anti-proliferative activities from multiple-cloved garlic bulbs. Peptides. 2005;26:177-183.
- [Google Scholar]