Introduction

Food and waterborne disease are the leading cause of illnesses and death in

developing countries (Roller, 2003). Food diseases contribute to annual economic

losses, in medical costs and lost productivity. Economic loses due to food spoilage

clearly also play important role. Both infection diseases and food spoilage are

caused by the growth of undesired-microbial in foods. So, it is important to control

microbial growth in foods.

One strategic for reduction of food-borne illnesses is adds antimicrobial food

preservatives at the processing stage to inactivate or preventing growth of

microbial (Thongson et. al., 2004). There are natural antimicrobial compound and

synthetic antimicrobial compound. Antimicrobial compound that is usually used as

preservative in foods are the synthetic antimicrobial compounds. People awareness

of healthy living has changed people’s mind to back to nature. This encourages

many researches of natural potential including research of new antimicrobial

compound from nature.

Black cumin (Nigella sativa L.) is a plant whose seeds are used as traditional

medicine in Middle East to treat a variety of health condition. Achyad et. al. (2000)

said that black cumin seeds contain essential oil, fat, saponin melantin, nigellin,

nigellon, dan thimoquinone which known is good for health.

In Indonesia, black cumin is used to treat diarrhea, one symptom of food

poisonings by microbial. So, it is assumed that black cumin has antimicrobial

activity. The aim of this study is to analyze the antimicrobial activity of the crude

extracts of black cumin seeds on several bacteria.

Literature Study

Black Cumin

Black cumin seed (Nigella sativa) is an annual herbaceous plant and a member of the
Ranunculaceae (buttercup) family. The fruit of the plant, the black seeds, accounts for its name.
Black cumin seed (also called black seed) should not be confused with the herb, cumin
(Cumunum cyminum, which is found in many grocery stores.

Considered native to the Mediterranean region, black cumin seed is cultivated in North Africa,
Asia, and southeastern Europe. The largest producers of black cumin seed are Egypt, India,
Pakistan, Iran, Iraq,Indonesia, and Turkey.

The lower leaves are short and supported by slender stems, while the upper leaves generally
grow to approximately 4 inches (10 cm) in length. The stalk of the plant, with its bluish white
flower petals, can grow up to 18 inches (46 cm) in height while its fruit matures. At first, the
seeds (the fruit of the plant) are held in a capsule in the center of the flower. The capsule opens
upon maturity, revealing lightly colored seeds. It is only upon their exposure to air that the seeds
become black.

Black cumin seeds (Nigella sativa) is widely known in treating many kind of disease.

The reason might be found in the complex chemical structure of the seeds. These

little seeds have over one hundred different chemical constituents, including

abundant sources of all the essential fatty acids. Though it is the oil that is most

often used medicinally, the seeds are a bit spicy and are often used whole in

cooking—curries, pastries, and Mediterranean cheeses.

Nigella sativa seeds have very little aroma but are carminative, meaning they tend

to aid digestion and relieve gases in the stomach and intestines. They aid peristalsis

and elimination. The essential oil of black cumin is antimicrobial and helps to rid the

intestines of worms.

The use of The Blessed Seed oil - Black Cumin seed oil is compatible with every
other type of medical treatment. The only time that Black Cumin seed oil should not

be taken is during pregnancy due to its possible dilation of the cervix.

Black Cumin (Nigella sativa) Seed, popularly called Black Seed, is an herb that has

traditionally been used for thousands of years in the Middle East, Far East and Asia

as an herbal health aid. Having so many diverse uses, it has gained the popular

approbation "the seed of blessing". Many other function of black cumin seeds are

under an intensive investigation including the potential of its extracts to be utilized

further as antimicrobial on food products.

Table 1. Proximate analysis of black cumin seeds

Composition Percentage (%)

Water 5.52

Protein 19.69

Fat 31.68

Ash 4.28

Carbohydrate 38.83

Antimicrobial

An antimicrobial is a substance that kills or inhibits the growth of microorganisms such as

bacteria, fungi, or protozoans, as well as destroying viruses.

The history of antimicrobials begins with the observations of Pasteur and Joubert, who
discovered that one type of bacteria could prevent the growth of another. They did not know at
that time that the reason one bacterium failed to grow was that the other bacterium was
producing an antibiotic

Some varieties of plant can be applied as natural antimicrobial due to its bioactive compounds.
Bioactive compounds could kill microbes through some mechanisms. The most frequently
studied mechanism is by destructing cell walls. The destruction of microbes cell wall can result
into the death of microbes. Bioactive compounds have to be extracted first using some extraction
method before purified and further utilized as antimicrobials. The effectiveness of these natural
compounds as antimicrobial depend on the concentration used and the adhesiveness of the
compounds. Prior to the application as antimicrobial, these bioactive compounds have to be
tested to some microbes to observe its ability in killing and inhibiting microbial growth.

Discussion

Three grade polarities of extracts (polar extract, semi polar extract and non

polar extract) were made to screen in which polarity the antimicrobial was

concentrated or in high activities. Polar extract were aqueous extract and

methanolic extract, semi polar extract were ethanolic extract and ethyl acetate

extract, and non polar extract was hexane extract. Reported in Table 3, all of the

black cumin extract, polar extract, semi polar extract and non polar extract showed

antimicrobial activity but specific to the kinds of bacteria. Whatsoever, we can

conclude that black cumin has polar antimicrobial compound, semi polar

antimicrobial compound and non polar antimicrobial compound.

Gram positive bacteria and Gram negative bacteria have different wall cell

structure that affects their resistance to any strange material which attacked them.

In this research we found that Gram positive (Staphylococcus aureus and Bacillus

cereus, except Pseudomonas aeruginosa) were more sensitive to black cumin

extracts than Gram negative (Salmonella Typhimurium and Escherichia coli). The

less sensitivity of Gram negative bacteria may be related to its outer membrane

which makes the bacterial surface strongly hydrophilic and acts as a strong

permeability barrier (Nikaido et. al., 1985 in Rota et. al., 2004). Based on Duncan

test on level p<0.05, Staphylococcus aureus and Pseudomonas aeruginosa were the

most sensitive bacteria to black cumin extracts and Escherichia coli was the most
resistant bacteria. Resistance of Escherichia coli was due to its ability to survive in

poor condition and lack of nutrition.

Aqueous extract was more ineffective than methanolic extract in inhibiting the

growth of bacteria test although both of them have similar polarity. Ineffectiveness

of aqueous extract may be because of its un-concentrated condition. If aqueous

extract has been concentrated, may be it will show better antimicrobial activity.

Components that soluble in methanol commonly are the same as that soluble in

water such as sugars, amino acid, and glycosides. Antimicrobial activity of aqueous

extract and methanolic extract may be due to the presence of glycoside, such

saponin that contained in black cumin seeds (Achyad et. al., 2000). Besides

glycosides, tannin is soluble in water and methanol and it was known has

antimicrobial activity.

Ethanolic extract showed the best antimicrobial activity. Antimicrobial activity

of ethanolic extract may due to the presence of glycosides and tannins. Houghton

and Raman (1998) said that glycoside soluble in ethanol and Ahmad et al. (2006)

reported that tannins were presence in ethanolic extract of black cumin. Ethyl

acetate extracts effective in inhibiting all of bacteria test. Ethyl acetate will extract

alkaloids, aglycones, and glycosides (Houghton and Raman, 1998). Alkaloids and

glycosides are known have antimicrobial activity. In this research, ethanolic extract

and ethyl acetate extract showed broad antimicrobial activity. Inhibition zone of

ethanolic extract against Staphylococcus aureus is showed in Figure 2.

Essential oil of black cumin effective inhibited all of bacteria test. Most of the

antimicrobial activity in essential oils derived from spices derived from phenolics

compounds (Maritini et. al, 1996) whereas other constituents were believed to
contribute little to the antimicrobials effects (Nychas, 1995). It is clear that essential

oil of black cumin contain amounts of phenolics compounds (terpineol, trans-

anethole, citronellol) and hence the antimicrobial activity of could be due to them.

Besides phenolics compounds, antimicrobial activity of essential oil could be due to

the presence of palmitic acid and oleic acid which known have antimicrobial activity

(Davidson et. al, 1993). Hexane extracts was one of the most ineffective extract of

black cumin. It showed no inhibition zone to Salmonella Typhimurium and

Escherichia coli (Table 3). Ineffectiveness of hexane extracts may be because of

hexane is non polar and not much antimicrobial compounds soluble in hexane.

Commonly, antimicrobial compounds come from phenol group which is has high

polarity. Component that common soluble in hexane were wax, fat, volatile oils, and

terpenoids (Houghton et. al., 1998).

The volatile compound of materials that extracted by hexane had been taken.

So, antimicrobial compound which presence in hexane extracts was non volatile

compounds such steroid, triterpenoid and fatty acids. Hinton et. al. (2000) in Ji et.

al. (2002), reported that antibacterial activity of fatty acids was probably due to the

ability of these compounds to disrupt the membranes of bacterial cells and cause

lysis of cells. Ji et. al. (2002) reported that linolenic acid had an antimicrobial effect

for some strain of bacteria. Antimicrobial activity of non polar compounds

performed probably by changing the membrane cell compounds.

Black cumin extracts that showed broad spectrum antimicrobial activity were

ethanolic extracts essential oils and ethyl acetate extract. Ethanolic extract and

ethyl acetate extracts, both of them are semi polar extracts but essential oil is non

polar extracts. So, we concluded that polarity of antimicrobial compound in black

cumin that has broad spectrum was semi polar and non polar. Ethanolic extract
more effective than essential oil and ethyl acetate extracts because ethanolic

extracts contained volatile and non volatile compounds. Essential oil only contained

volatile compound and ethyl acetate extract only contained non volatile

compounds. So, ethanolic extracts will have more various kinds of antimicrobial

compounds that present in black cumin seeds.

According to efficacy in inhibit the growth of bacteria test and the simplicity of

the process, it is better to choose single extraction using ethanol to get

antimicrobial compounds from black cumin seeds. But, economically is better to re-

extract the residue of black cumin that its volatile oils had been taken to get

antimicrobial compounds.

References

Achyad, D. E. dan R. Rasyidah. 2000. Jintan Hitam (Nigella sativa L.).

www.asiamaya.com. [20 Januari 2006].

Ahmad, I. and A. Z. Beg. 2001. Antimicrobial and phytochemical studies on 45

Indian medicinal plants against multi-drug resistant human pathogens.

Journal of Ethnopharmacology 74:113-123.

Apriantono, A., D. Fardiaz, N. L. Puspitasari, S. Yasni dan S. Budiyanto. 1989.

Petunjuk Laboratorium Analisis Pangan. Bogor, IPB Press.

Bloomfield, S. F. 1991. Methods for assesing antimicrobial activity. Di dalam Denyer

S. P. dan W. B. Hugo. (eds.). Mechanism of Action of Chemical Biocides their

Study and Exploitation. Blackwell Scientific Publication, London.

Davidson P. J.; Branen, A. L. Antimicrobials in Foods; Marcel Dekker: New York,

1993.
Garigga, M., M. Hugas, T. Aymerich and J. M. Monfort. 1993. Bacteriocinogenic

Activity of Lactobacilli from Fermentation Sausages. Journal of Applied

Microbiology 7:142-148.

Hinton, A. and K. D. Ingram. 2000. Use of oleic acid to reduce the population of the

bacterial flora of poultry skin. Journal of Food Protection 9:1282-1286.

Houghton P. J. and A. Raman. 1998. Laboratory Handbook for the Fractionation of

Natural Extracts. Chapman and Hall, London.

Ji, Y. L. , S. K. Yong and H. S. Dong. 2002. Antimicrobial synergistic effect of linolenic

acid and monoglyceride against Bacillus cereus and Staphylococcus aureus.

Journal Agricultural and Food Chemistry 50 (7).

Maritini, H.; Weidenborner, M.; Adams, S.; Kunz, B. Eugenol and carvacrol: the main

fungicidal compounds in clove and savory. Ital. J. Food Sci. 1996, 1, 63-76.

Nychas, G. J. E. Natural Antimicrobials from Plants; Chapman & Hall: New York,

1995.

Nikaido, H. and M. Vaara. 1985. Molecular basis of bacterial outer membran permeability.

Microbiology Review 49:1-32.

Roller, S. 2003. Natural Antimicrobials for Minimal Processing of Foods. CRC Press,

New York.

Rota, C., J.J. Carraminana, J. Burillo, A. Herrera. 2004. In vitro antimicrobial activity

of essential oils from aromatic plants against selected foodborne pathogens.

Journal of Food Protection 67:1252-1256.

Thongson, C., P. M. Davidson, W. Mahakarnchanakul and J. Weiss. 2004.

Antimicrobial activity of ultrasound-assisted solvent-extracted spices. Letters

in Applied Microbiology 39:401-406.