World Journal of Science and Technology 2011, 1(10): 48-53 ISSN: 2231 2587 www.worldjournalofscience.

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ANTIBACTERIAL POTENTIAL OF SOME NATURAL FOOD PRESERVATIVES AGAINST STAPHYLOCOCCUS AUREUS ISOLATED FROM VARIOUS FOOD SAMPLES OF HIMACHAL PRADESH (INDIA)
Amit Kumar1, Ajay Kumar2, Vandana Kaushal3, Sandip Patil4, Chandani Payal4, and Anil Kumar4 Professor, Faculty of Biotechnology, Shoolini University of Biotechnology and Management Sciences, Solan, Himachal Pradesh, 173212, India 2Head, Department of Microbiology, Shoolini Institute of Life Sciences and Business Management, Solan, Himachal Pradesh, 173212, India 3Shoolini Institute of Life Sciences and Business Management, Solan, Himachal Pradesh, 173212, India 4Faculty of Biotechnology, Shoolini University of Biotechnology and Management Sciences, Solan, Himachal Pradesh, 173212, India Corresponding author e-mail: amit.thakur2035@gmail.com Abstract
Various Indian spices are reported to possess antimicrobial activity as well as medicinal properties. In the present study, the antibacterial activity of various natural preservatives i.e. spices was checked against Staphylococcus aureus isolated from different food samples. A total of fifteen different food samples were collected from the local market of Solan (H.P). All the samples were processed and checked for the contamination of S.aureus, the common organism associated with food spoilage as well as food borne diseases by using standardized protocols of isolation. Out of fifteen different food samples, six samples showed the presence of S.aureus. Six different spices such as Cucurma longa (turmeric), Zingiber officinale (ginger), Piper nigrum (black pepper), Trigonella foenum-graecum (methi), Syzygium aromaticum (clove) and Ferula assafoetida (hinge) were collected randomly. Antibacterial assay of the aqueous, methanolic and ethanolic extracts of the above six spices was done using disk diffusion method. Different volumes i.e. 10L, 20L and 30L of each extract of the spices were tested against all the six bacterial isolates by loading them onto separate sterile filter paper discs. Almost all the spices used, showed significant antibacterial activity against S.aureus isolated from food samples. Out of all extracts, ethanolic extracts of all the spices showed maximum inhibitory effect against S.aureus followed by methanolic and aqueous extracts. The study showed that Syzygium aromaticum and Ferula assafoetida are the most potent inhibitors of S.aureus while Piper nigrum and Trigonella foenum-graecum have least activity. Keywords: Staphylococcus aureus, Antibacterial activity, Food preservatives, Extracts
1Assistant

Introduction
Food spoilage and food borne diseases are considered as area of concern and importance to humans (George et al., 2009). In modern industrialized world, food safety is a big issue of fundamental concern to both food consumers as well as food industries. Man has developed many

different methods in order to preserve the food since ancient times (Oonmetta-aree et al., 2005). Many interesting and useful means of food preservation invented by man have been found to be cited in ancient historical books. Herbs and spices are well known for their preservative and medicinal value as well as for boosting the flavor, color and aroma of the food (Chaudhry and Tariq,

World Journal of Science and Technology | www.worldjournalofscience.com | 2011 | 1(10): 48-53

2006). In modern world, food preservation is achieved by the extensive use of chemicals such as food additives and food preservatives. In the global food industry more priority is given to natural preservatives as there is increase occurrence of resistance in pathogenic strains against chemical food preservatives (George, 2009). The Staphylococcus genus includes at least forty species, of these, nine have two subspecies and one has three subspecies. (Harris, 2002). Staphylococcus aureus have two types of effects: infections and intoxications. In order to cause infection, cocci enter the damaged skin, mucosal or tissue sites, colonize by adhering to cells or extracellular matrix, evade host defense mechanisms, multiply and cause tissue damage. In intoxications, bacterial toxins cause the illness by producing endotoxins during its multiplication in the food, when kept at room temperature (Panikar, 2007).

Materials and Methods

Sample collection A total of 15 food samples were collected from the Solan market and examined for the contamination of Staphylococcus aureus. About 25 g of each sample was collected in a wide mouthed, capped autoclavable plastic sample container of about 100ml capacity using sterile spoon. After collection of the sample the container was capped tightly. The samples were immediately brought to the laboratory for processing or stored at 4C if to be processed later. Isolates A total of 15 food samples were collected from the Solan market and examined for contamination of Staphylococcus aureus. Six different spices, Cucurma longa (turmeric), Zingiber officinale (ginger), Piper nigrum (black pepper), Trigonella foenum-graecum (methi), Syzygium aromaticum (clove) and Ferula assafoetida (hinge) commonly used kitchen ingredients and natural preservatives were purchased from different manufacturers located at Solan (H.P) and screened for antibacterial activities against S. aureus. Processing All the food samples were processed by three methods as described by Parkash et al., 2007

and Sasidharan et al., 2011. Serial dilution was prepared by taking nine test-tubes containing 9ml of distilled water each were autoclaved at 121C for 15 minutes. One gm of sample was transferred in first test-tube containing 9ml of autoclaved distilled water aseptically inside the laminar air flow. The test-tube was vortexed for 2-5 minutes. 1ml of sample was transferred from the first tube with the help of micropipette into the second test-tube to prepare 10-1 dilution of the sample. Further dilutions were prepared and appropriate dilution was spread over Nutrient agar, Blood agar, MacConkey agar and Mannitol salt agar respectively with the help of sterile L-shaped spreader. Following incubation at 37C for 24 hours, the growth and colony characteristics of bacterial colonies were observed on the Petri-plates. Only the bacterial colonies which were suspected to be Staphylococcus aureus were further processed. The suspected colonies were marked on the plate. The single marked colony was picked up with the help of sterile inoculating loop and further streaked onto nutrient agar, mannitol salt agar, blood agar and MacConkey agar separately. The plates were incubated for 24 hours at 37C. The colony growth and its characteristics were observed. Bacteriological identification Morphological examination of isolated bacterial colonies was done using microscopic and macroscopic methods as described by Sasidharan, 2011. Grams staining was done for primary morphological characterization of isolated bacterial colonies as per methodology given by Parkash, 2007; Gundogan, 2010 and Singh P. 2008 For more precise identification of observed colonies various biochemical tests viz., Catalase, Oxidase, MR-VP, Indole, Citrate, Nitrate Reduction, Urease, Sugar fermentation were performed as per methodology described by Parkash, 2007; Gundogan, 2010; Sasidharan, 2011 and Singh, 2008. Preparation of various extracts Various food preservatives viz., Cucurma longa (turmeric), Zingiber officinale (ginger), Piper nigrum (black pepper), Trigonella foenum-graecum (methi), Syzygium aromaticum (clove), Ferula assafoetida (hing) were collected randomly from Solan market. The aqueous extract of the following six spices was prepared by mixing 20 g of dry powder of plant leaves with 100ml of sterile distilled

World Journal of Science and Technology | www.worldjournalofscience.com | 2011 | 1(10): 48-53

water in a round bottom flask with occasional shaking. The extract was then filtered through a muslin cloth for coarse residue and finally filtered through Whatmann No.1 filter paper and stored in an airtight container at 4oC until use (Aarati, et al., 2011; Liu et al., 1996). For the preparation of methanolic extract the air-dried and powdered spice material (20g of each) was extracted with 100ml methanol (95%) and kept on a rotary shaker for 24hr, filtered through Whatmann No.1 filter paper and centrifuged at 10000 rpm for 20 minutes. The supernatant was collected and stored in a bottle at 4oC until use (Parekh 2008; Liu 1996). For the preparation of ethanolic extract, air-dried and powdered spice materials (20g of each) was extracted with 100ml ethanol (70%) and kept on a rotary shaker for 24hr. Filtered through Whatmann No.1 filter paper and centrifuged at 10000 rpm for 20 minutes. The supernatant was collected and stored in bottle at 4oC until use (Parekh 2008; Liu 1996). Preparation of Inoculum Four-five colonies from pure growth of each organism was transferred to 5 ml of Mueller- Hinton broth. The broth was incubated at 37C for 18-24 hours. The turbidity of the culture was compared with 0.5 McFarland Nephelometer standard to get 150 106

CFU/ml. The standardized inoculum suspension was inoculated within 15-20 minutes (Saeed, 2007; Kunwar, 2010). Antibacterial assay Antibacterial activities of the different extracts of various spices were determined by disc diffusion method as described by Malu, 2008; Upadhyay, 2010; Chaudhry, 2006 and Singh, 2011. Mueller-Hinton agar (MHA) was used as antibacterial susceptibility test medium. A sterile cotton swab was dipped into the standardized bacterial test suspension and used to evenly inoculate the entire surface of MHA plates. Aqueous, methanolic as well as ethanolic extracts of Cucurma longa, Zingiber officinale, Piper nigrum, Trigonella foenum-graecum, Syzygium aromaticum and Ferula assafoetida with varying volumes i.e. 10, 20, 30 L were loaded onto sterile filter paper discs (5 mm diameter). Control discs were similarly prepared using distilled water, 95% methanol and 70% ethanol. These were placed on the surface of inoculated plates with sterile forceps. All plates were incubated at 37C for 24 hours. The diameters of the zones of inhibition appearing around the discs were measured to the nearest millimeter (mm) and recorded.

Table 1.Biochemical tests results for colonies of S.aureus Test

Dal (isolate I)

Isolate number and sample name

Burfi (isolate I) Rasmalai (isolate II) Kulfi (isolate II) Panipuri (isolate I) Cake (isolate I)

Catalase Oxidase Coagulase Indole MR VP Citrate Lactose Glucose Sucrose Mannitol Urease Nitrate reduction

+ + + + + A A A A + +

+ + + + + A A A A + +

+ + + + + A A A A + +

+ + + + + A A A A + +

+ + + + + A A A A + +

+ + + + + A A A A + +

World Journal of Science and Technology | www.worldjournalofscience.com | 2011 | 1(10): 48-53

Results
Various Indian spices are reported to possess antimicrobial activity as well as medicinal properties. In the present study, the antibacterial activity of various natural preservatives i.e. spices was checked against Staphylococcus aureus isolated from different food samples. A total of fifteen different food samples were collected from the local market of Solan (H.P). Out of fifteen different food samples, six samples showed the presence of S.aureus. Six different spices i.e. Cucurma longa, Zingiber officinale, Piper nigrum, Trigonella foenum-graecum, Syzygium aromaticum and Ferula assafoetida were

collected from the Solan market randomly. Antibacterial assay of the aqueous, methanolic and ethanolic extracts of all the above six spices was done using disk diffusion method. Almost all the spices used showed significant antibacterial activity against S.aureus isolated from food samples. Out of aqueous, methanolic and ethanolic extracts, ethanolic extracts of all the spices showed maximum inhibitory effect against S.aureus followed by methanolic and aqueous extracts. The study showed that Syzygium aromaticum (clove) and Ferula assafoetida (hinge) are the most potent inhibitors of S.aureus while Piper nigrum (black pepper), Trigonella foenum-graecum (methi) have least activity against S. aureus.

Table-2. Zone of inhibition in mm for different extracts of six natural food preservatives
S.aureus isolated from
Clove

Size of zones of inhibition (mm) for different extracts Aqueous extracts (30l)
Turmeric Ginger Black papper Methi Hinge Clove

Methanolic extracts(30l)
Turmeric Ginger Black papper Methi Hinge Clove

Ethanolic extracts(30l)
Turmeric Ginger Black papper Methi Hinge

RASMA LAI KULFI CAKE PANIPU RI BURFI DAL

12 10 10 10.5 11 8.5

3 1 4 5 2 2

9 10 0 9 10 12

2 3 3 0 5 5

2 2 0 1 3 2

12.5 13 12 12 12 11 11 11 12 12

5 4 6 7

12 14 12 11 11 12

5 7 6 4 6 7

4 5 3.5 3 3 3.5

13 12 11 12 13 11

15 13 12 12 13 12

6 5 7 7 5 5

14 14 14 13 14 14

8 8 7 5 8 6

5 5 4 3.5 3.5 4

15 11 12 12 13 12

10. 3 5 11 4

Fig 1. Colonies of S. aureus on Mannitol Salt Agar (2) Antibacterial activity of Ferula assafoetida ethanolic extract against S. aureus.

World Journal of Science and Technology | www.worldjournalofscience.com | 2011 | 1(10): 48-53

Discussion Food-borne illness is an important public health problem as it not only affects human health but also has a significant effect on world economic and trade issues (Sasidharan S. 2011). Contamination of food by various contaminating sources especially improper handling and unhygienic practices among food handlers is one of the major factors responsible for the outbreak of food-borne diseases (Gundogen N. 2006; Sasidharan S. 2011). Staphylococcus aureus is one of the common contaminant of food. Antibacterial activity of six different spices (i.e. Cucurma longa, Zingiber officinale, Piper nigrum, Trigonella foenumgraecum, Syzygium aromaticum and Ferula assafoetida) was checked against S. aureus isolated from various food samples in the current study. Various plant products, particularly spices and extracts of various herbal plants were widely utilized as natural antimicrobials and antioxidants (George, 2009). The demand of these herbal products is increasing because of their antimicrobial activity against many human as well as animal pathogens (Gur, 2006). The major components of ginger are gingerone and gingerol which have strong inhibitory activity against pathogenic bacteria. The major antimicrobial compound in garlic is allicin (Conner, 1993). (Hirasa, 1998). All the products vary in their pattern of sensitivity against S. aureus (Boerlin, 2002). Present findings indicated considerable variations in the antibacterial activity of various extracts of the six spices against S. aureus. The results of antibacterial testing revealed that the ethanolic extracts of all spices had maximum inhibitory effect due to their better solubility in the organic solvents as compared to water, followed by methanolic and aqueous extracts (Malu, 2009 and Gur, 2006).

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Acknowledgements
We are thankful to Prof. M.P. Vohra (Dean life sciences SILB Solan) for their help and encouragement from time to time.

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