EFFECTIVENESS OF VARIOUS SPICES IN INHIBITING

THE SPOILAGE RATE OF FOOD

*IRUOLAJE, F. O., & OGBEBA, J. AND **DOGO, B. A.

*Department of Science Laboratory Technology, Federal Polytechnic, Bauchi.
**National Agency for Food and Drug Administration and Control (NAFDAC). Federal
Secretariat Complex, Oke-Mosan, Abeokuta, Ogun state.

ABSTRACT
The effectiveness of locally used spices Zingber officinale (ginger), Myristrica fragrams
(nutmeg), Allium sativum (Garlic), Thymus vulgaris (thyme), and Curry powder in
inhibiting the spoilage rate of food were determined. The spoilage organisms isolated
from control plates were Bacillus cereus and Staphylococcus aureus from jellof rice,
Proteus mirabilis and Bacillus cereus from fish sauce, Enterobacter aerogenes
,Klebisella pneumonia and Escherichia coli from bean cake(moi moi). In the plates
containing spices in all the food samples only Bacillus cereus was able to grow. The
result of colony count expressed in cfu/ml showed that plates containing curry powder
and thyme had no growth. Ginger had 1.0 x 102, garlic 2.0 x 102, and nutmeg 5.0 x 102,
while control had 1.28 x 104 from jellof rice. In bean cake, curry powder had 4.0 x 10 2,
thyme 1.2 x 103, ginger 9.0 x 102, garlic 5.0 x 102, and nutmeg 7.0 x 102 while control
plate had 2.25 x 104. Curry powder and garlic had nil, thyme 9.0 x 10 2,ginger 3.0 x 102,
nutmeg 9.0 x 102 while control had 1.15 x 104, from fish sauce. The plates containing
curry powder in all the food samples showed highest inhibitory effect on the food
spoilage organisms. Nutmeg, Garlic, Ginger, and Thyme also showed strong
antimicrobial effect against food pathogens in all the food samples. There was a gross
reduction in microbial load in the experimental plates (with spices) compared with
control plates (without spices) as indicated by the number of colonies in eachplate. The
result of this study showed that spices have inhibitory effect on food borne pathogens, so
they could be used in food preservation as main antimicrobial compounds in order to
assure the production of microbiologically stable food.

Introduction
Spices are mixtures of several or dozens of phytochemicals from indigenous or exotic
origin with the major bioactive compounds constituting up to 85%, while other
components are found at trace levels (Burt, 2004 and Moreno et al.,2006), and according
to Lamber et al., (2001) these bioactive compounds may evolve multiple modes of
antimicrobial action including degradation of the cell wall, disruption of the cytoplasmic
membrane, leakage of cellular components, alteration of fatty acid and phospholipid
constituents, changes in the synthesis of DNA and RNA and destruction of protein

1
translocation (Shan et al., 2007). Spices include leaves (bay, mint, rosemary, coriander,
laurel, oregano), flowers (clove), bulbs (garlic, onion), fruits (cumin, red chili, black
pepper), stems (coriander, cinnamon), rhizomes (ginger) seed (nutmeg) and other plant
parts (Smid & Gorris, 2005). Spices contain products of secondary metabolism such as
phenolics, phenolic acids, quinones, flavonoids, tannins (Alvarez et al., 2008). Many of
these phytochemicals are rich sources of antioxidants (Moreno et al., 2006). Many
workers have reported a high correlation between antimicrobial efficacy and the level of
phenolic components present in certain spice preparations. Indeed, compounds such as
eugenol, carvacrol and carnosic acid present in clove, oregano and rosemary respectively,
have been identified as being responsible for antimicrobial activity.
Spices have been currently used as a food additive for the purpose of flavoring and as a
preservative by killing or preventing the growth of harmful bacteria in food (Agaoglu et
al., 2007). Besides adding to the taste, spices have multifarious functions that include
combating food borne microorganisms, reducing food poisoning, (Bajpai et al., 2008),
antioxidant function, and antimicrobial activity (Alvarez et al., 2008). Spices are also
known to possess a wide range of medicinal values, such as fight against cancer causing
cells, reduction of cholesterol level in the blood and prevention of several skin diseases
(Burt, 2004).
Despite the high degree of awareness of food preservation methods there is increasing
occurrence of disease outbreaks caused by pathogenic and spoilage microorganisms in
foods (Witkowska et al., 2011). Consumer awareness and concern that synthetic chemical
additives may have some toxic or even carcinogenic effects, has increased the demand for
high-quality, minimally processed foods with extended shelf-life, preferably free from or
with a reduced level of added chemical antimicrobial agents (Desmond, 2006). There is
growing interest in using natural antimicrobial compounds, including extracts of herbs
and spices, as salt replacers or alternatives to synthetic compounds for food preservation
(Smid & Gorris, 2005).
Food spoilage is a metabolic process that causes foods to be undesirable or unacceptable
for human consumption due to changes in sensory characteristics such as changes in
texture, smell, taste, or appearance (George, 2009).
Food borne diseases and food spoilage by microorganisms is responsible for food
poisoning and is unhealthy for food producers, retailers, consumers and regulatory
authorities(Lanciotti et al., 2004).
There are also increasing occurrence of food-borne disease outbreaks caused by
pathogenic microorganisms. Recently in Bauchi state there was an outbreak of cholera - a
food borne disease caused by Vibrio cholera which claimed many lives. Information on
the effectiveness of locally used spices in inhibiting the spoilage organisms in food is
scarce in Bauchi. Spices are abundantly available in Bauchi state all year round and are
cheap. It is important to investigate the effectiveness of spices in inhibiting the spoilage
organisms in foods commonly consumed in Bauchi metropolis.

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Materials and Methods
Collection of Samples
Zingiber officinale (Ginger), Myristica fragrams (Nutmeg), Allium sativum (Garlic),
Thymus vulgaris (Thyme) and Curry powder (Ducrose) used in this study were purchased
from wunti market Bauchi state, likewise the rice, beans, fresh fish and other condiments.
The samples were packed separately in cellophane bags to microbiology laboratory,
Federal polytechnic Bauchi immediately and were kept in the cupboard.

Preparation of Culture Media

The media used were nutrient agar (oxoid) for isolation and enumeration of bacteria;
MacConkey agar (Difco) as a differential media; potato dextrose agar (oxoid) for the
isolation and enumeration of fungi and blood agar (oxoid) as a differential media. They
were prepared according to the manufacturer’s instructions.

Processing of samples
Sample 1: Jellof rice
The rice were per boiled, washed and mixed with fried tomatoes and other condiments
without salt ,onions and pepper.120g of the per boiled rice were weighed into five sterile
beakers and 0.2g of each grounded spice was mixed one to each five beakers of rice and
were cooked separately and then kept at room temperature for 36 hours.

Sample 2: Bean cake

The beans were soaked in water for one hour, washed and grounded without salt, onions
and pepper. Other condiments were added and 120g of the grounded beans were weighed
into five sterile beakers and 0.2g of each grounded spice were added one to each the five
beakers and were cooked separately and then kept at room temperature for 36hours.

Sample 3: Fish sauce

Fresh fish were washed thoroughly, pieced and 120g of the washed fish was weighed into
five sterile beakers, 0.2g of each grounded spice was added to each of the five beakers
and was cooked separately and kept at room temperature for 36hours.
One beaker of each of the food samples was prepared without any spice added to it and
was kept at room temperature for 36hours and this served as control.

Isolation of spoilage organisms

Isolation of food spoilage organisms from food samples was done using the standard
method of (Teramota et al., 2005). A tenfold serial dilution(10-1-10-5) of food samples in
each of the five beakers were prepared using distilled water. The test tubes containing
9ml of distilled water were autoclaved at 121 0C for 15minuts prior to serial dilution and
1ml from dilution 10-2 and 10-4 of each samples were inoculated on nutrient agar and
potato agar using spread plate method and incubated at 370C for 24hours for bacteria
growth and at 28°C for 5 days for fungal growth. The growth and colony characteristics

3
of the colonies were observed on the Petri plates and the colonies were counted using
colony counter. Each of the different colonies that grew on the plates was sub cultured by
streaking onto a fresh nutrient agar, blood agar and mac Conkey agar separately. The
plates were incubated for 24hours at 370C.The colony growth and its characteristics were
macroscopically and microscopically observed.

Identification of bacterial isolates

Morphological examination of isolated bacterial colonies was done using microscopic
and macroscopic methods as described by Sasidharan, (2011). Gram staining was done
for primary morphological characterization of isolated bacterial colonies, for more
precise identification of observed colonies various biochemical test viz; catalase, indole,
coagulase, methyl red test were performed (Gaffa, 2005). Bacteria colonies that appeared
on the plates were counted using a digital illuminated colony counter. The colony counts
from the plates were obtained and expressed as colony forming unit per millitre (cfu/ml).
Final identification of bacteria isolates was by comparison of results obtained with
literature standard using Bergey’s manual of determinative bacteriology (Buchanan and
Gibbons, 1975).

Gram Staining Reaction

A loopful of the bacteria culture was taken up with the inoculating wire loop and spread
thinly on a clean grease free glass slide. The film was fixed by passing the dried slide
with film upwards over a bunsen flame for a few seconds, the slide was then covered
with crystal violet solution and allowed to act for 30seconds. The stain was poured off by
holding the slide at an angle downwards and pouring water to wash away the crystal
violet. The slide was then covered with fresh Lugol’s iodine for one minute and washed
off with water. The smear was decolorized rapidly with acetone and washed off with
clean water .The smear was then counter stained with 30% safranin for 1minute and was
washed with water ,the slide was then air dried and was examined under the microscope
with the oil immersion objective lens.

Biochemical tests:
For more precise identification of isolated organisms, the following biochemical test
were carried out according the method described by (Gaffa, 2005).

Motility Test
A ring of plasticine was made on a glass slide and an inoculum of the organism was made
at the center of the plasticine. The inoculums was emulsified with distilled water the slide
was then observed using the microscope

Methyl Red Test

The isolates were inoculated in tubes of prepared and sterilized glucose phosphate
peptone broth and incubated at 370C for 48hours. 5 drops of methyl solution was added to

4
each tubes and the color change was observed. The positive result gave bright red color
while the negative gave yellow color with the indicator

Voges Proskauer Test

The isolates were inoculated in test tubes of prepared and sterilized methyl red Voges
proskauer medium and incubated at 37 0C for 5days. 5ml of 40% potassium hydroxide
was added to each tube. A bright pink color appearing within 5minute showed a positive
reaction.

Oxidase Test
A piece of filter paper was placed in a clean Petri dish and 2 drops of freshly prepared
oxidase reagent was added using a wire loop. Colony of the isolates was smear on the
filter paper. The development of a blue-purple color within a few seconds shows a
positive result.

Litmus Milk Test

A sterile loop was used to inoculate 0.5ml of sterile litmus milk medium with the isolate.
Heavy inoculums of the isolates were used and the loop was scrapped three times across
an area of heavy growth. It was then incubated at 37 0C for 4hours. It was examined half
hour intervals for a reduction reaction as shown by a change in color from mauve to
white or pale yellow which indicate positive while no color change or a pink color
indicate negative.

Indole Test
Tubes of tryptone broth were inoculated with the test organisms and incubated at 35 0C for
48hours. After incubation, 2ml of kovac’s reagent was added to each tubes and shaken
gently. The tubes were then kept on test tube rack and allowed to stand for 20minute.A
red color at the reagent layer indicates indole production.

Citrate Utilization Test

Citrate tablet was used, a dense bacteria suspension of the isolates was prepared in
0.25ml of sterile physiological saline in a small tubes. A citrate tablet was added to it and
the tubes were covered with a stopper. It was then incubated over night at 35-37 0C.
Appearance of red color shows positive reaction while yellow to orange color shows
negative.

Sugar Fermentation Test

15mls of peptone water was dispensed into ten test tubes with an inverted Durham tubes
in each. The test tubes were capped and sterilized at 121 0C for 15minute.Then 1.5g of
each sugar were weighed and added into each test tubes containing peptone water and test
tubes were shaken and a few drops of methyl red was added to test tubes. The cap was
then replaced and sterilized at 100 0C for 30minute in water bath and was allowed to cool

5
to about 450C and was aseptically inoculated with the isolates and was incubated at 37 0C
for 48hours.A positive result shows change in color to yellow while gas production was
evidence by media displacement in the Durham tubes.

Coagulase Test
Nutrient broth was prepared and dispensed into test tubes and sterilized at 121 0C for
15minute. 0.2ml of blood plasma was added to each of the tubes and inoculated with the
test organism.The tubes were incubated in a water bath for 6hours and examined at
intervals clothing shows a positive result

Result
The effectiveness of the spice in inhibiting the spoilage organisms in food as shown by
the reduction in microbial load as indicated by the number of bacterial colony counts
from each food were presented in table 1. Bacterial load were higher in the controls
while curry powder show the higher inhibitory action in all the food samples.
The morphological, physiological and biochemical characteristics of the identified
organisms were presented in table 2. As can be seen, only Bacillus cereus was isolated
from all the food samples with spices while in the control Staphylococcus aureus and
Bacillus cereus were isolated from jollof rice, bacillus cereus and Proteus mirabilis were
isolated from fish sauce and Enterobacter aerogenes, Klebisella pneumoniae and
Escherichia coli were isolated from bean cake (moi moi)

Table 1: Result of Bacterial load after the addition of spices.

Food samples S p i c e s Mean colony count CFU/ml

Jellof Rice
4
C o n t r o l 1 . 2 8 x 1 0
C u r r y N i l
T h y m e N i l
G i n g e r 1.0 x
2
G a r l i c 1 0
N u t m e g 2.0 x
2
1 0
2
5 . 0 x 1 0

Bean cake
4
C o n t r o l 2 . 2 5 x 1 0
2
C u r r y 4 . 0 X 1 0
3
T h y m e 1 . 2 X 1 0
2
G i n g e r 9 . 0 X 1 0
2
G a r l i c 5 . 0 x 1 0

6
 2
N u t m e g 7 . 0 x 1 0
Fish Sauce
C o n t r o l 1 . 1 5 x 1 0 4
C u r r y N i l
T h y m e 9.0 x
2
G i n g e r 1 0
G a r l i c 3.0 x
2
N u t m e g 1 0
N i l
2
9 . 0 x 1 0

7
 Table 2: Morphological and Biochemical characteristics of suspected organisms
Food samples M o r p h o l o g i c a l Gram strain reaction Motility C C I M C L G
Characteristics of the isolate s a o n . it a l
t a d R r c u
a g ol a t c
l u e te a o
a l s s
s a e e
e s
e
Jellof Rice Typical colonies, pinkish red, ar anged in chain on MacConkey Agar, on blood agar, it produced haemolytic colonies, non-capsulated. White, large and flat colonies in nutrient agar.

+ + + - - - + - + +
Bean cake Typical colonies, pinkish red, ar anged in chain on MacConkey Agar, on blood agar, it produced haemolytic colonies, non-capsulated. White, large and flat colonies in nutrient agar. + + + - - - + - + +

Fish Sauce Typical colonies, pinkish red, ar anged in chain on MacConkey Agar, on blood agar, it produced haemolytic colonies, non-capsulated. White, large and flat colonies in nutrient agar. + + + - - - + - + +

Jellof Rice Ye l l o w r a i s e d r o u n d c o l o n i e s + - + + - + + A A -

Fish sauce C r e a m y c o l o r e d , r o d i n c h a i n - + + - - + + - - -

Bean cask R o d i n c h a i n - - + - - - + + + -
Rod raised circular colorless colonies - + + - + + - + + -
R o d i n c h a i n - + + - - - + + + -
LMD – Litmus milk decolorization text
VP – Voges proskauer test
MR – Methyl red
A –Acid production flowing fermentation of sugar

8
Discussion
From the result obtained, the colony count in the plates containing curry powder in all the
food samples showed highest inhibitory effect on the food spoilage organisms. This high
efficacy of antimicrobial activity of curry spice may be due to synergistic effect as curry
powder contained turmeric, coriander, fenugreeks, salt, onion, fennel, garlic, cumin,
caraway, paprika, chilipepper and black pepper. This agreed with the work carried out by
Thomas and Isak (2006) who reported that this combination enhance the inhibitory
activity of spice. Burt (2004) also reported the synergic effect of spices against B.cereus
and Al-jedal et al., (2006) analyzed the action of combined spices on food pathogens
count in fish sauce and their results showed that spice mixture were able to exert static
effect on all assayed bacteria. nutmeg, garlic, ginger, and thyme also showed strong
antimicrobial effect against food pathogens in all the food samples. There was a gross
reduction in load in the experimental plates as compared with control plates as indicated
by the number of colonies in each plate (table 1). This may be due to the antimicrobial
effectiveness of their chemical compounds such as gingerone and gingerol in ginger,
allicin in garlic, and thymol in thyme.
It was noticed that in plate where growth occurred, low colony counts were observed on
the low dilution plates while higher counts were obtained on the higher dilution plates.
This may be due to reduction in the concentration/strength of spice as effectiveness of
spice is determined by its concentration.
The spoilage organisms isolated from control plates were Bacillus cereus and
Staphylococcus aureus from jellof rice, Proteus mirabilis and Bacillus cereus from fish
sauce, Enterobacter aerogene, Klebisella pneumonia and Escherichia coli from bean
cake and in the plates containing spice in all food samples only Bacillus cereus that was
able to grow, the rest of the organisms were inhibited by the spices .This may be due to
their antimicrobial activity/effect on the microorganisms. This is in accordance with the
work carried out by Gundogen et al., (2006) in six different spices which inhibited the
growth of staphylococcus aureus. Al-jedal et al., (2006) also analyzed the action of
combined spices on food pathogen count in fish sauce which shows that spice mixture
were able to exert inhibitory effect on all assayed bacterial.

Conclusion
The result obtained in this study showed that spices are very effective in inhibiting food
pathogens by exerting antimicrobial effect as proved by gross reduction in microbial load.

Recommendation
Proper hygienic should be maintained during harvesting, processing and storage of spices
as spices could act as vehicles for micro for microbial contamination of foods. Spices
should be washed before use. Further studies should be carried out on the mode of action
of these spices.
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