International Journal of Microbiological Research 3 (1): 53-58, 2012
ISSN 2079-2093
© IDOSI Publications, 2012
DOI: 10.5829/idosi.ijmr.2012.3.1.619
Bacteria Associated with Common Spices and Their Possible Implications
Mahbubar Rahman Khan, Mihir Lal Saha and Farhana Islam Khan
Department of Botany, University of Dhaka, Dhaka, 1000, Bangladesh
Abstract: Twenty one different spices collected from local markets of Dhaka Metropolitan City were used to
study the associated microorganisms and their possible implications. Out of 202 organisms initially isolated,
42 were selected and characterized in detail. All the selected isolates were rods, Gram positive and Spore-former
except one, which was a non spore former rod. Thirty-nine isolates were provisionally identified as members
of the genus Bacillus. Two isolates belonged to the genus Sporolactobacillus while the remaining one
belonged to the genus Kurthia. Fifteen isolates showed different inhibitory effect on 10 tested pathogenic
organisms. Antibacterial activity of the selected spices against 8 pathogenic bacteria was also studied. Extract
of black cumin showed considerable antibacterial activity against 5 pathogenic bacteria.
Key words: Antimicrobial Activity
Associated Bacteria
INTRODUCTION
Extracts
Spices
Additionally, the presence of moulds creates a potential
risk for human health due to mycotoxins production.
Numerous naturally occurring antimicrobials are
present in animal and plant tissues [10], where they
probably evolved as part of the defense mechanisms of
the host against microbial invasion. There are many plants
that demonstrate antimicrobial activity [11] and these
plants have found application in the food industry as
antibacterial and antifungal agents [12]. Being of plant
origin, spices appeal to consumers who question the
safety of synthetic preservatives [13]. Although, the
primary purpose of spices is to impart flavor and
piquancy to food, the medicinal, antimicrobial and
antioxidant properties of spices are also being exploited
[14]. The antimicrobial activity of spices is documented
and interest continues to the present [11, 15, 16].
The
present
work was aimed to study the
microorganisms associated with our common spices to
understand their morphological, physiological characters
as well as any possible harmful effect they could impart
on the consumers.
Spices constitute an important group of agricultural
commodities, which are virtually indispensable in the
culinary art. The spices are produced from roots, barks,
leaves, bulbs, stems, flowers and seeds of certain plants
and mostly grown and harvested in warm, humid areas of
the world [1]. Spices are used for flavor, color, aroma,
taste and preservation of food and beverages. Spices are
often dried and used in a processed but complete state.
Another option is to prepare extracts such as essential
oils by distilling the raw spice material (wet or dry), or to
use solvents to extract oleoresins and other standardized
products [2, 3].
Like any other object of this planet spices and
condiments are also not free from microbial
association. The spices may acquire microorganisms
during their growth and development or during later
stages when these are passed through collection,
processing, storage and marketing. Finally at consumers'
level these are obviously found with multitude of
organisms.
Several workers reported the microbiological quality
of spices [4, 5]. Satchell et al. [6] performed a
microbiological survey on four selected spices viz. black
and white pepper, coriander and fennel seeds. Antai [7]
reported the Bacillus flora of Nigerian spices. Several
spices, including turmeric, paprika, aniseed and pepper
have been linked with salmonellosis outbreaks [8, 9].
MATERIALS AND METHODS
Twenty one different spices were collected from
various local markets of Dhaka metropolitan city (Table 1).
Samples were collected in sterilized glass bottles,
immediately plugged, brought into the laboratory and
preserved for detailed study.
Corresponding Author: Farhana Islam Khan, Department of Botany, University of Dhaka , Dhaka-1000, Bangladesh.
53
Intl. J. Microbiol. Res., 3 (1): 53-58, 2012
Table 1: Particulars of the collected samples.
Sample No.
English Name
Scientific Name
Bengali Name
Parts used
Place of Collection
01.
Black Cumin
Nigella sativa L.
Kalo-jira
Seed
Chawk Bazar,Dhaka
02.
Cumin
Cuminum cyminum L.
Jira
Seed
Chawk Bazar,Dhaka
Chawk Bazar,Dhaka
03.
Black pepper
Piper nigrum L.
Golmorich
Drupe
04.
Coriander
Coriandrum sativum L.
Dhania
Seed
Chawk Bazar,Dhaka
05.
Fenugreek
Trigonella foenum-graecum L.
Methi
Seed
Chawk Bazar,Dhaka
06.
LesserCardamom
Elettaria cardamomum Maton
Elachi
Seedpod
Chawk Bazar,Dhaka
07.
Black Mustard
Brassica nigra Koch
Rai Sarisha
Seed
Chawk Bazar,Dhaka
08.
White Mustard
Brassica hirta Moench
ShadaSarisha
Seed
Chawk Bazar,Dhaka
09.
White pepper
Piper nigrum L.
Golmorich
Seed
Chawk Bazar,Dhaka
10.
Bay leaf
T. Nees and Eberm.
B.D.R. Market,Uttara,Dhaka.
Cinnamomum tamala (Buch.-Ham.)
Tejpata
Leaf
11.
Turmeric
Curcuma domestica Valet
Halud
Rhizome
B.D.R. Market,Uttara,Dhaka.
12.
Ginger
Zingiber officinale Rose.
Ada
Rhizome
B.D.R. Market,Uttara,Dhaka.
13.
Nutmeg
Myristica fragrans Houtt.
Jaiphal
Nut
B.D.R. Market,Uttara,Dhaka.
14.
Mace
Myristica fragrans Houtt.
Joitri
Aril
B.D.R. Market,Uttara,Dhaka.
15.
Aniseed
Pimpinella anisum L.
Muhuri
Seed pod
B.D.R. Market,Uttara,Dhaka.
16.
GreaterCardamom
Amomum subulatum Roxb
Bara elachi
Seedpod
B.D.R. Market,Uttara,Dhaka.
17.
Red pepper,Chillies
Capsicum frutescens L.
Lal-marich
Fruit
B.D.R. Market,Uttara,Dhaka.
18.
Ammi,Lovage
TrachyspermumAmmi L.
Jowan
Seed
B.D.R. Market,Uttara,Dhaka.
B.D.R. Market,Uttara,Dhaka.
19.
Cinnamon
Cinnamomum zeylanicum Bl.
Dalchini
Bark
20.
CommonBasil
Ocimum basilicum L.
Babui tulsi
Leaf
Botanical garden, Department of Botany, D. U.
21.
Holy Basil
Ocimum sanctum L.
Tulsi
Leaf
Botanical garden, Department of Botany, D. U.
Isolation of Bacteria: Two different techniques viz. i)
impression method and ii) direct inoculation method
were followed for the isolation of bacteria. For the
impression method the spices (washed and raw) were
pressed on the surface of three culture media and
after one minute samples were removed from the
media. The inoculated plates were then incubated at 37°C
for 24 h in an incubator.
For the direct inoculation method the spices were
inserted in the solidified media and the inoculated plates
were then incubated at 37°C for 24 h in an incubator.
Three different media viz. Nutrient Agar (NA), Yeast
Extract Malt Extract Agar (YM) and Glucose Asparagine
Agar (GA) were used for the cultivation of
microorganisms.
[22] and
Understanding
Microbes
[23],
the
physiological and biochemical tests of the isolated
bacteria were carried out.
Antibacterial Activity of Selected Isolates: By using giant
colony technique [24] the antagonistic activity of the
selected isolates was determined. The selected isolates
were streaked as a narrow band across the center of the
MH agar plates. The plates were then incubated until a
heavy growth occurred. The test organisms were then
streaked from the edges of the plates up to the growth but
not touching it. The plates were further incubated to allow
growth of each test organism and the inhibition distance
was measured and recorded. In this study, 10 bacteria viz.
Alcaligenes sp., Enterobacter sp., Enterobacter
intermedium,
Bacillus
fastidiosus,
Enterobacter
amnigenus,
Staphylococcus
aureus,
Bordetella
bronchiseptica, Escherichia coli, Micrococcus luteus
and Staphylococcus epidermidis were used to determine
the antimicrobial activity of the selected microorganisms.
Identification of the Isolates: After development of
bacterial colony on the agar surface, colonies were
randomly selected and isolated on respective agar
slants. The selected bacterial colonies were studied for
various characters viz. color, form, elevation, margin,
surface, optical characters etc. [17, 18]. Following Bergey's
Manual of Systematic Bacteriology [19], Bergey’s Manual
of Determinative Bacteriology [20], Manual of
Microbiological Methods [21], Microbiological Methods
Antibacterial Activity of Selected Spices: The extracts of
four different spices viz. black cumin, black mustard, black
pepper and fenugreek were used to evaluate their
antibacterial activity. For this purpose methanolic extract
54
Intl. J. Microbiol. Res., 3 (1): 53-58, 2012
Under the genus Bacillus there were 16 distinct species,
viz. B. alvei (2.56%), B. alcalophilus (15.38%), B.
brevis (5.12%), B. circulans (7.69%), B. coagulans
(12.82%), B. epiphytes (5.12%), B. firmus (2.56%), B.
fastidiosus (5.12%), B. globisporus (2.56%), B.
licheniformis (7.69%), B. laevolacticus (2.56%), B.
macerans (5.12%), B. polymyxa
(12.82%),
B.
subtilis (5.12%), B. stearothermophilus (5.12%) and
B. thuringiensis (2.56%).
Freire and Offord [27] reported that most bacteria
present in spices are aerobic spore-former. In the present
work also most of the isolates were found to be aerobic,
spore former and rod shaped. Thirty-nine isolates were
identified as members of the genus Bacillus (92.86%).
Baxter and Holzapfel [28] also found that approximately
90% of colonies in viable counts were Bacillus spp.
Bacteriological examination of 230 spice samples by
Antai [7] showed that Bacillus spp. including B. subtilis,
B. polymyxa and B. coagulans occurred in significant
numbers. During the present research, isolated Bacillus
were provisionally identified as B. subtilis, B. polymyxa
and B. coagulans. Similar result was reported earlier by
Zuha et al [29].
Inhibition spectrums of 42 isolates were studied
employing giant colony technique against 10 pathogenic
bacterial strains. Out of these, 18 isolates showed
considerable inhibition (Table 3). Isolate No. CA-1 and
CI-1 showed strong antagonistic activity against 4 tested
pathogenic bacteria.
of spices were used. Extracts were prepared by the
modified method of Shan et al. [25]. An agar-well
diffusion method was employed for determination of
antibacterial activities [26]. The methanolic extracts of
spices and herbs were dissolved in phosphate buffered
saline (PBS, pH 6.5) to the final concentration of 100
mg/ml. All pathogenic bacteria were suspended in sterile
water and the suspension was spread onto the surface of
Mueller Hinton medium. Wells were cut from the agar with
a sterile borer and extract solutions were delivered into
them. Negative controls were prepared using PBS solution
and 80% methanol. The inoculated plates were incubated
at 35 °C for 24 h. Antibacterial activity was evaluated by
measuring the diameter of inhibition zone (DIZ) of the
tested bacteria. DIZ was expressed in millimeters. All tests
were performed in duplicate.
RESULTS AND DISCUSSION
Most of the aerobic heterotrophic bacterial isolates
were rod shaped, Gram positive and spore former.
By Comparing all tested morphological and biochemical
characters of the isolated organisms, the isolates were
provisionally identified with the help of Bergey’s Manual
of Systematic Bacteriology [19] and Bergey’s Manual of
Determinative Bacteriology [20].
Among the 42
bacterial isolates, 39 belonged to the genus
Bacillus (92.86%), 2 to the genus Sporolactobacillus
(4.76%) and one to the genus Kurthia (2.38%) (Table 2).
Table 2: Provisional identification of the selected isolates.
Isolate No.
Identified Members
Number ofSpecies
Frequency (%)
CA-1, CA-2, BL-1, TU-1, TU-2, CI-1
B. alcalophilus
6
15.38
CH-1, CU-1, FG-7, FG-8, FG-10
B. coagulans
5
12.82
CU-2, FG-4, FG-9, WP-1, WP-2
B. polymyxa
5
12.82
BM-1, BM-2, BM-3
B. licheniformis
3
7.69
FG-3, ZI-1, BC-1
B. circulans
3
7.69
BP-1, NT-1
B. macerans
2
5.12
FG-2, FG-5
B. stearothermophilus
2
5.12
CS-1, FG-1
B. fastidiosus
2
5.12
WM-2, AJ-1
B. brevis
2
5.12
AM-1, CP-1
B. subtilis
2
5.12
AJ-2, CL-3
B. epiphytes
2
5.12
BA-2, BA-3
Sporolactobacillus inulinus
2
4.76
CH-2
B. firmus
1
2.56
ZI-2
B. globisporus
1
2.56
NU-1
B. thuringiensis
1
2.56
CL-1
B. alvei
1
2.56
TL-1
B. laevolacticus
1
2.56
WM-1
Kurthia gibsonii
1
2.38
Total
42
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Intl. J. Microbiol. Res., 3 (1): 53-58, 2012
Table 3: Inhibition spectrum of the selected isolates against tested pathogenic bacteria.
Isolate No.
CH-1
CH-2
CU-1
CU-2
BP-1
BP-2
CS-1
FG-1
FG-2
FG-3
FG-4
FG-5
FG-7
FG-8
FG-9
FG-10
CA-1
CA-2
BM-1
BM-2
BM-3
WM-1
WM-2
WP-1
WP-2
BL-1
TU-1
TU-2
ZI-1
ZI-2
NT-1
AM-1
BC-1
CP-1
AJ-1
AJ-2
CI-1
NU-1
BA-2
BA-3
CL-1
CL-3
TL-1
Inhibition Zone measured in mm.
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------AlcaliEnteroE. interme Bacillus
Bordetella
Escherichia Micrococcus
genes sp. bacter sp. dium
fastidiosus E. amnigenus S. aureus bronchiseptica coli
luteus
S. epidermidis
2
3
3
6
3
1
13
5
10
7
5
9
4
16
13
10
9
9
9
7
13
12
15
8
3
4
6
5
4
2
4
5
4
2
3
2
3
3
10
10
2
6
15
3
21
1
2
1
2
3
3
7
4
5
3
8
8
13
15
9
3
13
17
3
17
6
3
12
6
1
9
10
5
3
5
2
5
3
12
8
1.5
3
8
3
“-” indicates no inhibition
Antibacterial activity of the methanolic extract of
selected spices against 8 pathogenic bacteria was also
studied. Extract of black cumin showed antibacterial
activity against 2 pathogenic bacteria (Figure 1).
Extract of black mustard and fenugreek showed no
antibacterial activity. Thomas et al. (30) have reported
that methanol extracts of some medicinal plants have
remarkable antimicrobial activity against various Gram
positive and Gram negative bacteria. In the present
study antibacterial activity of the methanolic extract
of black cumin showed positive result against 5
pathogenic
bacteria
viz.
Bacillus
fastidiosus,
Enterobacter amnigenus, Bordetella bronchiseptica,
Escherichia coli and Staphylococcus epidermidis.
Methanolic extract of black pepper inhibited the growth
of Staphylococcus epidermidis.
56
Intl. J. Microbiol. Res., 3 (1): 53-58, 2012
Fig. 1: (A-F): Effects of phosphate buffer (PO42-), methanol (80%) and methanolic extract of black cumin on the growth
of Staphylococcus aureus and Micrococcus luteus. A & B plates having only buffer solution in the agar well
(AW), C & D. only methanol was poured in the agar well. E & F plates showing Zone of inhibition (ZI) around
the agar well in which methanolic extract of black cumin was poured
The methanolic extracts of the tested spices showed
considerable antimicrobial activity. Thus the consumption
of these spices did not pose any threat to the consumer
provided that they are properly stored.
2.
3.
ACKNOWLEDGEMENT
4.
The kind cooperation of Dr. Nasima Akhtar (P.S.O.)
and Dr. Miskat Ara Akhter Jahan (P.S.O.) of the BCSIR, is
gratefully acknowledged. The test organisms were
obtained from the Culture collection of the Institution of
Nutrition and Food science (INFS), Dhaka University.
5.
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