Preservatives are usually added to food products to ensure longer shelf life and prevent decomposition process and microbial growth. However, synthetic food preservatives can also give negative side effect to health and are harmful to human and animal physiology. Based on the potential of herbs and spices as antimicrobial agent, the purpose of this study is to identify antibacterial activity from extracts of some local herbs and spices: Phaeomeria speciosa ( P. speciosa), <i>Aquilaria subintegra </i> ( <i>A. subintegra </i>), <i>Polygonum minus </i> ( <i>P. minus </i>), <i>Syzygium aromaticum </i> ( <i>S. aromaticum </i>), <i>Cinnamomum verum </i> ( <i>C. verum </i>) and <i>Piper nigrum </i> ( <i>P. nigrum </i>) against food bacteria using disc diffusion method. Results revealed that dichloromethane extracts of <i>C. verum </i>, hexane extracts of <i>S. aromaticum </i> and <i>P. minus </i> showed the most active antibacterial against tested bacteria. The Minimal Inhibitory Concentrations (MIC) ranged from 25 to 75 mg/ml for dichloromethane extract of <i>C. verum </i>, hexane extract of <i>S. aromaticum </i> and <i>P. minus </i>. Therefore further research should be pursued to identify the chemical structure of antibacterial agents from the active extracts as an alternative source of natural preservatives.
Preservatives are crucial in food manufacturing, cosmetics, medicine and pharmaceutical products to extend their shelf life and avoid the growth of bacteria. Besides that, preservation also plays an important role in the taste, colour and texture as well as improves nutrition value of the foods. The source of preservatives can be natural or synthetic. Preservatives from natural sources such as herbs and spices have long been practiced in traditional food and medicinal preparations, and recent studies have shown their potential as antibacterial, antifungal, antiviral and antioxidant agents [
On the other hand, synthetic preservatives synthesized through chemical reactions produce cheaper alternative and now are becoming more reliable and in favour. Previous studies have shown that without proper management, synthetic food preservatives could give negative effect not only to human and animal health but also to the physiology of other living organisms, damage the biology system and pollute the environment [
Solvent used for plant extraction will determine the compound extracted and its bioactivity. The solvent can be grouped into different polarities which are low, medium and high polarity [
P. speciosa, A. subintegra, and P. minus were collected around Tanjung Malim, Perak while S. aromaticum, C. verum and P. nigrum were purchased from the local market in Behrang, Perak in 2013. Plant species and their parts used in this study were summarized in
Powdered samples were sequentially extracted with hexane, dichloromethane and methanol by maceration tech-
Plant species | Parts used | Remarks |
---|---|---|
Phaeomeria speciosa | Flowers | Only widely open petals were chosen |
Aquilaria subintegra | Young leaves | Healthy young leaves were chosen |
Polygonum minus Huds. | Leaves | Only healthy and green leaves were chosen |
Syzygium aromaticum | Buds | Brownish and dry buds were chosen |
Cinnamomum verum | Stem barks | Light reddish brown barks were chosen |
Piper nigrum | Drupes | Only dried and black drupes were chosen |
nique at room temperature for 72 hours and then extract with water by reflux technique for two hours to avoid deterioration of the chemical compounds. The organic extracts (18 extracts) were evaporated in vacuum by using rotary evaporator at 40˚C while the water extracts (6 extracts) were freeze-dried. The dried extracts were stored in glass bottle at 4˚C for further analysis.
Bacteria cultures of Escherichia coli, Bacillus cereus, Pseudomonas aeruginosa, Staphylococcus aureus and Salmonella typhi were obtained from culture collection centre, Department of Biology, Universiti Pendidikan Sultan Idris, Perak, used for antibacterial test microorganisms. All bacteria strains were maintained and grown according to standard procedures, unless otherwise stated.
After overnight incubation in nutrient broth at 37˚C, a sterile cotton swab was dipped into each nutrient broth suspension containing the tested bacteria; E. coli, B. cereus, P. aeruginosa, S. aureus and S. typhi, were swab on the agar surface. Then the 5 mm-diameter sterile filter paper discs were impregnated with different concentration of extracts and a control. The antibacterial test was done at concentration 25, 50, 75 and 100 mg/ml. Methanol and distilled water that used to dilute the extract were choose as the control. Then, they were placed on the NA plate with suitable distance from each other. The discs were gently pressed down by using sterile forcep to ensure complete contact with agar surface. The plates were then sealed with parafilm and incubated at 37˚C overnight in inverted to prevent the moisture build up on the lid from dripping onto the bacteria. Observations were recorded after 24 hours incubation. Antibacterial activities were interpreted from the diameters of inhibition zone around the disc and done in triplicate [
The minimal inhibitory concentration was performed on extracts which showed zone of inhibition in the preliminary screening by using dilution method according to Nascimento et al. [
The disc diffusion method was used to evaluate the antibacterial activity. In this study, 18 organic extracts and 6 water extracts from P. speciosa, A. subintegra, P. minus, S. aromaticum, P. nigrum and C. verum were tested against Gram negative bacteria, E. coli, S. typhi, P. aeruginosa and Gram positive bacteria, B. cereus and S. aureus which commonly found in food such as chilli sauce. Four out of six herbs and spices tested in this study have shown antibacterial activity by using disc diffusion method (
A study carried out on pure piperine isolated from P. nigrum’s drupes showed zone of inhibition against E. coli, P. aeruginosa and S. aureus [
Other than environmental condition, genetic factor, solvent used for extraction also affects the degree of antibacterial activity [
Plants | Extracts | Test bacteria | Conc. (mg/ml)/Zone of inhibition (mm ± s.d) | ||||
---|---|---|---|---|---|---|---|
0 | 25 | 50 | 75 | 100 | |||
A. subintegra | MeOH | E. coli | - | - | - | - | - |
S. aureus | - | - | - | - | - | ||
S. typhi | - | - | - | - | - | ||
B. cereus | - | - | - | - | - | ||
P. aeruginosa | - | - | - | 6.67 ± 0.58 | 7.00 ± 0.00 | ||
P. minus Huds. | Hex | E. coli | - | - | - | - | - |
S. aureus | - | 6.67 ± 0.58 | 7.33 ± 0.58 | 7.67 ± 0.58 | 7.67 ± 0.58 | ||
S. typhi | - | - | - | - | - | ||
B. cereus | - | 7.00 ± 0.00 | 8.00 ± 0.00 | 8.67 ± 0.58 | 9.33 ± 0.58 | ||
P. aeruginosa | - | 7.67 ± 0.58 | 8.67 ± 0.58 | 9.00 ± 1.00 | 9.33 ± 0.58 | ||
DCM | E. coli | - | - | - | - | - | |
S. aureus | - | - | - | - | - | ||
S. typhi | - | - | - | - | - | ||
B. cereus | - | - | - | 6.67 ± 0.58 | 6.67 ± 0.58 | ||
P. aeruginosa | - | - | 6.33 ± 0.58 | 6.67 ± 0.58 | 7.67 ± 0.58 | ||
MeOH | E. coli | - | - | - | - | - | |
S. aureus | - | - | - | 6.67 ± 0.58 | 7.33 ± 1.15 | ||
S. typhi | - | - | - | - | - | ||
B. cereus | - | - | - | 6.33 ± 0.58 | 6.67 ± 0.58 | ||
P. aeruginosa | - | - | 6.67 ± 0.58 | 6.67 ± 0.58 | 7.00 ± 0.00 | ||
S. aromaticum | Hex | E. coli | - | 7.33 ± 0.58 | 7.00 ± 1.00 | 7.60 ± 0.58 | 8.00 ± 0.00 |
S. aureus | - | 7.00 ± 0.00 | 8.67 ± 0.58 | 9.00 ± 1.00 | 9.00 ± 1.00 | ||
S. typhi | - | 6.33 ± 0.58 | 7.00 ± 0.00 | 7.67 ± 1.15 | 9.00 ± 1.00 | ||
B. cereus | - | 7.00 ± 0.00 | 8.33 ± 0.58 | 8.33 ± 0.58 | 9.00 ± 0.00 | ||
P. aeruginosa | - | 6.33 ± 0.58 | 7.00 ± 0.00 | 7.60 ± 0.58 | 8.00 ± 0.00 | ||
DCM | E. coli | - | - | - | - | 7.00 ± 0.00 | |
S. aureus | - | - | - | - | 7.00 ± 1.00 | ||
S. typhi | - | - | - | - | - | ||
B. cereus | - | - | - | - | - | ||
P. aeruginosa | - | - | 7.00 ± 1.73 | 6.67 ± 1.15 | 7.67 ± 0.58 | ||
C. verum | Hex | E. coli | - | - | - | 7.00 ± 1.00 | 8.00 ± 0.00 |
S. aureus | - | 7.67 ± 0.58 | 7.67 ± 0.58 | 7.67 ± 1.15 | 8.00 ± 1.00 | ||
S. typhi | - | 7.00 ±0.00 | 7.33 ± 0.58 | 8.33 ± 0.58 | 9.00 ± 1.00 | ||
B. cereus | - | 7.00 ± 0.00 | 7.67 ± 0.58 | 11.00 ± 0.00 | 10.00 ± 0.00 | ||
P. aeruginosa | - | 7.00 ± 0.00 | 8.67 ± 0.58 | 11.00 ± 0.00 | 10.67 ± 0.58 | ||
DCM | E. coli | - | - | - | - | - | |
S. aureus | - | - | 7.00 ± 0.00 | 8.33 ± 0.58 | 9.00 ± 1.00 |
C. verum | S. typhi | - | - | - | 6.67 ± 0.58 | 7.67 ± 0.58 | |
---|---|---|---|---|---|---|---|
B. cereus | - | 8.00 ± 1.00 | 10.67 ± 0.58 | 14.00 ±1.73 | 13.67 ± 1.15 | ||
P. aeruginosa | - | 9.00 ± 0.00 | 11.33 ± 0.58 | 13.33 ± 2.52 | 14.33 ± 1.53 | ||
MeOH | E. coli | - | - | - | - | - | |
S. aureus | - | 6.00 ± 0.00 | 7.00 ± 1.00 | 7.33 ± 0.58 | 7.00 ± 0.00 | ||
S. typhi | - | - | - | - | - | ||
B. cereus | - | 6.33 ± 0.58 | 7.00 ± 1.00 | 7.67 ± 0.58 | 8.67 ± 0.58 | ||
P. aeruginosa | - | 7.33 ± 0.58 | 9.00 ± 1.00 | 10.33 ± 0.58 | 10.33 ± 0.58 | ||
Aqueous | E. coli | - | - | - | - | - | |
S. aureus | - | - | 7.00 ± 1.00 | 8.33 ± 0.58 | 8.67 ± 0.58 | ||
S. typhi | - | - | - | - | - | ||
B. cereus | - | 8.00 ± 0.00 | 8.67 ± 0.58 | 9.67 ± 0.58 | 10.00 ± 0.00 | ||
P. aeruginosa | - | 7.00 ± 0.00 | 8.33 ± 0.58 | 9.33 ± 0.58 | 9.33 ± 0.58 |
Key: Hex = Hexane, DCM = Dichloromethane, MeOH = Methanol (n = 3), (-) = No zone of inhibition.
polarity of solvent used for extraction. A polar compound will extracted by polar solvent and non-polar compound will extracted by non-polar solvent. Thus, the biological activity shown may be different between extracts since different compounds will be extracted [
Interestingly, hexane extract of C. verum and S. aromaticum showed antibacterial properties against all five tested organisms while hexane extract from P. minus gave positive result against three bacteria; S. aureus, B. cereus and P. aeruginosa. Even though essential oil is usually extracted by steam distillation technique, in this study, essential oil, waxes and fats might be dissolved in non polar solvent such as hexane [
Previous studies indicate that eugenol and cinnamaldehyde, were two major chemical components found in the spice oil of Cinnamom species and S. aromaticum that showed inhibition against gram negative and gram positive bacteria [
The results from disc diffusion methods show three extracts that exhibited very active antibacterial activity; dichloromethane extract of C. verum, hexane extract of S. aromaticum and hexane extract of P. minus Huds. leaves (
Test organisms | Extracts/Minimum inhibition concentration (mg/ml) | ||
---|---|---|---|
DCM extract of C. verum | Hex extract of S. aromaticum | Hex extract of P. minus Huds. | |
E. coli | - | 25 | - |
S. aureus | 25 | 25 | 25 |
S. typhi | 50 | 75 | - |
B. cereus | 25 | 50 | 25 |
P. aeruginosa | 25 | 25 | 25 |
mg/ml against tested bacteria S. aureus, B. cereus and P. aeruginosa, whereas 50 mg/ml for S. typhi. Hexane extract of S. aromaticum showed MIC of 25 mg/ml against E. coli, S. aureus and P. aeruginosa, whereas 50 mg/ml against B. cereus and 75 mg/ml against S. typhi. The MIC of 25 mg/ml was obtained against S. aureus, B. cereus and P. aeruginosa when tested with hexane extract of P. minus Huds. From the results, antibacterial activity of the selected herbs and spices against both gram positive and gram negative bacteria show the presence of broad-spectrum antibacterial properties. Therefore, it is hypothesized that this local herbs and spices can be used as biopreservative agent in food products.
Herbs, spices and edible plants have long been given most attention for their medicinal properties and low toxicity to human. They also show potential as antibacterial agents that are relatively safer than synthetic alternatives like sodium benzoate. From the findings, the most significant antibacterial activities were found in all four extracts of C. verum, hexane extracts of S. aromaticum and P. minus Huds. which can form basis information for further studies. Hence, these herbs and spices that are of interest for antibacterial agents are suggested for further study on toxicity testing, further isolation of active compounds, identification of its chemical structure and evaluation against a wider range of biological activities like in vivo testing with the aim for use in food preservation.
The authors acknowledge Ministry of Higher Education, Malaysia and Universiti Pendidikan Sultan Idris (UPSI) for provision of financial support for this research work, FRGS (2014-0030-101-02) and GPU (2013-0068- 102-01).