Ahvaz, Iran
Ahvaz, Iran
Ahvaz, Iran
Introduction. Medicinal herbs are susceptible to microbial contamination which can have profound effects on the consumer’s health. Our study aimed to evaluate microbial contamination of common medicinal herbs in Ahvaz. Study objects and methods. We collected 80 samples of traditional and industrial medicinal plants from the supply market, namely valeriana, fennel, licorice, and shirazi thyme. The reference method was used to determine microbial indices such as the total count of microorganisms, yeast and mold, Bacillus cereus, coliforms, and Escherichia coli. Results and discussion. We found that the total microbial count, yeast and mold, B. cereus, and coliform contamination accounted for 45, 77, 55, and 55% of the total samples, respectively, exceeding the allowed limits. There was a significant difference between the industrial and traditional samples in fungal and coliform contamination, with the traditional samples being more highly contaminated. However, no significant difference was observed between them in total count and B. cereus contamination. E. coli contamination was detected in 31.2% of the samples, mostly in traditional. Total microbial count and yeast and mold contamination were highest among valeriana plants. Fennel showed the highest B. cereus and coliform contamination. The lowest contamination was observed in licorice. Conclusion. The results showed that a considerable percentage of the medicinal herbs under study were contaminated at levels exceeding the standard limits. Plants could be contaminated during harvesting, processing or storage. Finally, different species of plants have different antimicrobial activities that affect their microbial contamination.
Microbiology, microbial contamination, quality control, medicinal herbs, total microbial count
INTRODUCTION
Medicinal plants become contaminated by a variety
of sources such as heavy metals, insect larvae and seed,
different bacteria, and fungi [1]. Heat and humidity
of the environment, long-time drying, irrigation with
contaminated water, and lack of farmer training may
result in considerable microbial contaminations and
reduce the quality of plants. Moreover, microbial
contamination of plants may take place during unhealthy
collection, cleaning, storage, transportation, and
packaging. Contact of herbal products with external
factors such as plastic, glass, and other materials may
lead to cross contamination.
Medicinal plants can be contaminated by a
wide range of microorganisms, such as fungi,
yeasts, protozoa, and viruses, most of which are
transferred from soil [2, 3]. Total microbial count
is an important factor in determining the health
status or probable detection of a contamination
source [4]. Yeast and mold are the most common
contaminants of medicinal herbs. Various species
of molds and yeasts that proliferate on food stuff
secrete metabolic toxic materials such as mycotoxins,
which are harmful for humans and animals [5].
The WHO (World Health Organization) has a large
amount of data in this direction [6].
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Coliforms (Escherichia, Enterobacter, and
Klebsiella) from the Enterobacteriaceae family
inhabit human and animal intestines. Most of them are
not pathogenic, although some E. coli strains could
be highly pathogenic and cause food poisoning [7].
According to Iran’s national standard, coliform
contamination in most dried vegetables should not
exceed the maximum level of 1000 CFU/g, while the
presence of E. coli is not allowedI. Bacillus cereus is
widely distributed in the environment and some its
strains are harmful for human health and can cause
food poisoning. This bacterium secretes enterotoxin,
hemolysin, and lecithinase C which are responsible
for disease [9]. Dried vegetables contamination by
Bacillus cereus should not exceed the maximum level of
100 CFU/g.
Valeriana (Nardostachys jatamansi L.) from the
Valerianceae family is known for its anticonvulsant,
sedative, anti-asthmatic, and cardiotonic properties [9].
Fennel (Foeniculum vulgare L.) from the
Umbelliferae family has culinary and medicinal
properties (anti-inflammatory, anti-spasmodic,
diuretic, laxative, analgesic, antioxidant, and woundhealing)
[10]. Licorice (Glycyrrhiza glabra L.)
growing in Mediterranean countries, Central Asia,
and Europe has a wide range of pharmacological
effects such as antioxidant activity, liver protection,
and regulation of the immune system [11].
Shirazi thyme (Zataria multiflora Boiss L.) is used in the
south of Asia as tea or spice and in traditional medicine
as a gastrointestinal disinfectant, diuretic, or an antiinflammatory
remedy [12].
Contaminants such as microorganisms, heavy
metals, and pesticides affect the quality and the efficacy
of herbal products. Since it is impossible to remove all
contaminants, precautionary measures should be taken
to prevent or limit contamination [2, 3]. Therefore, our
study aimed to show the effect of these contaminations
on consumer’s health.
STUDY OBJECTS AND METHODS
Collection and preparation of samples. For this
study, samples were randomly collected from medicinal
herb retailers and drugstores of Ahvaz (Iran) from
December 2017 for 6 months. A total of 80 samples
were used: 40 traditional (10 samples for each traditional
herb) and 40 industrial herbs from different companies
(19 shirazi thyme, 6 fennel, 8 valeriana, and 7 licorice
samples). The amounts of industrial samples were not
equal due to their insufficient availability.
Total microbial count. Total microbial count was
performed as described by Standard No. 5272, IranII.
Different dilutions of medicinal herbs were prepared and
I Standard No. 5939, Microbiology of dehydrated vegetablesspecifications.
Institute of Standards and Industrial Research of Iran;
2008.
II Standard No. 5272, Microbiology of food and animal feeding
stuffs-horizontal method for the enumeration of microorganismscolony
count technique at 30°C. Institute of Standards and Industrial
Research of Iran; 2007.
cultured on Plate count agar (PCA, Merck, Germany).
Triplicate plates for each dilution were cultured and
incubated for 72 h at 30°C. Then, the average of counted
colonies was measured taking into account the dilution
coefficient.
Mold and yeast count. Fungal count was performed
according to Standard No. 10899, IranIII. Different
dilutions of medicinal herbs were inoculated on
Sabouraud dextrose agar (SDA, Merck, Germany) in
triplicates and incubated at 25°C for 5 days. Then, the
average number of molds and yeasts per gram of herb
was estimated.
Bacillus cereus detection. To detect and count
B. cereus (Standard No. 2324, Iran), dilutions of
medicinal herbs were prepared and cultured in triplicate
on Mannitol-egg yolk-polymyxin (MYP) agar (Merck,
Germany) at 30°C for 48 h. The agar contained an egg
yolk emulsion and polymyxin B sulfate (Shijiazhuang
Pharma, China)IV. Large and pink colonies (lack
of manitol fermentation) with a sedimentary halo
(lecithinase producer) were counted as probable
B. cereus. To confirm the suspected colonies, a
hemolysis test was performed on Blood agar (Merck,
Germany).
Coliform detection and enumeration. Coliform
detection and enumeration were performed according
to Standard No. 9263, IranV. Different dilutions of
medicinal herbs were inoculated (pour plate and twolayer
culture) in triplicate on Crystal violet neutral
red bile lactose (VRBL, Merck, Germany) agar and
incubated at 37°C for 24 h. Typical red purple colonies
were confirmed on Brilliant green bile lactose (BGBL)
broth (Merck, Germany) contained in Durham tubes at
two temperatures (37 and 44°C) for 24 h.
Escherichia coli detection. Following coliform
detection, positive BGBL tubes (gas production) were
inoculated into peptone water and incubated at 44°C for
48 h. Gas production in BGBL and production of indole
in peptone water were recorded for presence of E. coliVI.
Statistical analysis. Analysis of data was performed
using SPSS statistical software. The significance of the
results was evaluated by McNemar nonparametric test
with significance level of P < 0.05.
RESULTS AND DISCUSSION
Total microbial contamination. According to the
results of total microbial count (Fig. 1), 45% of the total
III Standard No. 10899, Microbiology of food and animal feeding
stuffs-horizontal method for the enumeration of yeasts and molds.
Institute of Standards and Industrial Research of Iran; 2008.
IV Standard No. 2324, Microbiology of food and animal feeding
stuffs-horizontal method for the enumeration of presumptive Bacillus
cereus-colony count technique at 30°C. Test method. Institute of
Standards and Industrial Research of Iran; 2006.
V Standard No. 9263, Microbiology of food and animal feeding stuffshorizontal
method for the enumeration of coliforms-colony count
technique. Institute of Standards and Industrial Research of Iran; 2007.
VI Standard No. 2946, Microbiology of food and animal feeding
stuffs-detection and enumeration of presumptive Escherichia
coli. Most probable number technique. Institute of Standards and
Industrial Research of Iran; 2005.
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herbs (36 samples out of 80) showed contamination
over the limit (105 CFU/g). Of them, 19 (48%) and
17 (43%) were traditional and industrial, respectively.
The microbial load in the samples with contamination
over the limit varied from 5.03 ± 0.03 to 6.25 ± 0.03 log
CFU/g. This ranged from 5.14 ± 0.01 to 6.25 ± 0.03 in
the traditional samples and from 5.03 ± 0.3 to
6 ± 0.05 log CFU/g in the industrial samples. However,
there was no significant difference between the
total microbial contamination in the traditional and
industrial samples (P > 0.05). Among the studied herbs,
valeriana (Nardostachys jatamansi L.) and licorice
(Glycyrrhiza glabra L.) showed the highest and the
lowest contamination ‒ 56 and 18%, respectively. Also,
the total microbial contamination of shirazi thyme
(Zataria multiflora Boiss L.) and fennel (Foeniculum
vulgare L.) was over the limit in 55% and 44% of the
samples, respectively.
Fungal contamination. The results of fungal
contamination are presented in Fig. 2. As we can
see, 61 (77.5%) out of 80 samples had mold and
yeast contamination over the limit (103 CFU/g).
They comprised 34 (85%) traditional and 27 (67.5%)
industrial samples. The fungal load in the samples with
contamination over the limit varied from 3.02 ± 0.00 to
4.78 ± 0.06 log CFU/g. This reached from 3.02 ± 0.00 to
4.78 ± 0.06 in the traditional samples and from 3.04 ±
0.01 to 4.60 ± 0.01 log CFU/g in the industrial samples.
It should be mentioned that the traditional samples
were significantly contaminated with mold and yeast
(P < 0.05). Meanwhile, valeriana (100%) and licorice
(47%) showed the highest and the lowest contamination,
respectively. Also, 79% and 75% of shirazi thyme
and fennel, respectively, showed over the limit fungal
contamination.
Bacillus cereus contamination. B. cereus
contamination in 44 samples (55%) of the total herbs
exceeded the limit (103 CFU/g) (Fig. 3), including
23 (57.5%) traditional and 21 (52.5%) industrial herbs.
Over the limit B. cereus contamination varied from
2.03 ± 0.03 to 3.84 ± 0.06 log CFU/g. In the traditional
samples, it ranged from 2.03 ± 0.03 to 3 ± 0.06 log
CFU/g and in the industrial samples, from 2.03 ± 0.03
to 3.84 ± 0.06 log CFU/g. Our results showed that there
was no significant difference between the traditional and
industrial samples in B. cereus contamination (P > 0.05).
The contamination in fennel (94%) and valeriana (66%)
was significantly higher (P < 0.05). Shirazi thyme (41%)
and licorice (29%) showed a lower level of B. cereus
contamination.
Coliform contamination. Coliform contamination
was found over the limit (103 CFU/g) in 44 samples
(55%) of the total herbs. Among these samples,
29 (72.5%) and 15 (37.5%) were from traditional and
industrial herbs, respectively (Fig. 4). Over the limit
coliform contamination ranged from 3.01 to 4.16 ±
0.03 log CFU/g, namely from 3.01 to 4.16 ± 0.03 in
the traditional samples and from 3.03 ± 0.02 to 4.15 ±
0.03 log CFU/g in the industrial samples. According to
the results, the traditional samples showed a significantly
higher coliform contamination than the industrial
samples (P < 0.05). Fennel (81%) and valeriana (66%)
Figure 1 Total microbial contamination in medicinal herbs.
Total is number of samples. Positive ‒ contamination over the
limit (105 CFU/g). Negative ‒ contamination below the limit
0
20
40
60
80
Industerial
&Traditional
Traditional Industerial
Samples number
Sample type
Total Posetive Negative
0
20
40
60
80
Industerial
&Traditional
Traditional Industerial
Samples number
Sample type
Total Posetive Negative
0
20
40
60
80
Industerial Traditional
Samples number
Total 0
20
40
60
80
Industerial &
Traditional
Traditional Industerial
Samples number
Sample type
Total Posetive Negative
Positive
Industrial Industrial
Figure 2 Mold and yeast contamination in medicinal herbs.
Total is number of samples. Positive ‒ contamination over the
limit (103 CFU/g). Negative ‒ contamination below the limit.
* P < 0.05
Industerial
Negative
0
20
40
60
80
Industerial
&Traditional
Traditional Industerial
Samples number
Sample type
Total Posetive Negative
0
20
40
60
80
Industerial &
Traditional
Traditional Industerial
Samples number
Sample type
Total Posetive Negative
0
20
40
60
80
Industerial Traditional
Samples number
Total Industerial
Positive
Industrial Industrial
Figure 3 Bacillus cereus contamination in medicinal herbs.
Positive ‒ contamination over the limit (102 CFU/g). Negative
‒ contamination below the limit. * P < 0.05
Industerial
Negative
0
20
40
60
80
Industerial
&Traditional
Traditional Industerial
Samples number
Sample type
Total Posetive Negative
0
20
40
60
80
Industerial &
Traditional
Traditional Industerial
Samples number
Sample type
Total Posetive Negative
0
20
40
60
80
Industerial Traditional
Samples number
Total Industerial
Positive
Industrial Industrial
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revealed a very high level of contamination (P < 0.01),
while the lowest level was recorded in licorice (41%) and
shirazi thyme (41%).
Escherichia coli contamination. There should
be no E. coli contamination in dried vegetables [8].
According to our results, 25 samples (31.2%) of the
total herbs showed E. coli contamination. Of them,
19 (47.5%) and 6 (15%) were from traditional and
industrial herbs, respectively (Fig. 5). E. coli contamination
in the traditional samples was significantly
higher than in the industrial samples (P < 0.05). The
contamination in fennel (43.7%) and valeriana (33.3%)
was significantly higher (P < 0.05) than that in licorice
(23.5%) and shirazi thyme (27.5%).
One of the important aspects is contamination of
medicinal herbs by different types of harmful factors
such as microbes, heavy metals, as well as radioactive
and chemical materials [2]. Our results, in many cases,
indicated high contamination of the herbs under study
with different microbial agents. These contaminations
could occur during different stages of cultivation,
extraction, drying, packing or distribution [2, 3, 13].
In our study, the microbial contamination level in the
traditional samples was higher than that in the industrial
samples. This result could be due to different production
and packaging conditions.
Researchers have reported that the differences
in technological level and preparation, supply and
production of medicinal herbs could affect their
contamination level [13–15]. Worldwide, a high level
of contamination has been reported in a variety of
medicinal herbs. For example, Banerjee et al., in a
study of 154 dried plants collected from shops in India,
showed that the total microbial count was over the
limit in 51% of the samples, and 97% of them had mold
contamination [16].
Moreover, Abba et al., in a study of powdered
medicinal plants in Nigeria, reported that 87% of
them had high microbial contamination [17]. Their
contamination level was significantly higher than that in
our study (45%), which could be due to environmental
factors, soil or inappropriate packing conditions. Some
studies in different locations showed that many of the
investigated medicinal herbs were contaminated with
various fungi [5, 16, 18, 19]. Alwakeel, in a study on 32
samples of various medicinal plants in Saudi Arabia,
showed Bacillus cereus as the most common microbial
contaminant [20]. Martins et al. found the same result
in more than 90% of the studied medicinal plants in
Portugal [21].
In our study, fennel and valeriana showed the highest
and licorice showed the lowest levels of contamination
with B. cereus and coliform. In a study of the
antimicrobial activity of Turkish spices, fennel showed
a lower antibacterial effect on B. cereus [22]. Moreover,
Lang et al. in Austria reported that fennel had a lower
inhibitory effect on coliform than licorice [23]. It seems
that the antimicrobial properties of medicinal herbs
could also explain the differences in their contamination
levels. In the previous studies, high microbial
contaminations were reported in valeriana [19, 24]. We
found that valeriana had the highest contamination level.
This result can be due to the fact that most of its active
medicinal ingredients are in the root of the plant, which
is in direct contact with soil, so more microorganisms
can be transmitted to it [19]. In our study, licorice
showed a considerably lower contamination level than
other plants. It could be due to differences in plant
production, its active constituents, and distribution
processes [3, 25]. In addition, most studies have shown
that licorice had higher antimicrobial and antifungal
activities than other plants, especially against B. cereus
and E. coli [22, 35–38].
CONCLUSION
In our study, we tested 80 samples of traditional
and industrial herbs, such as valeriana, fennel, licorice,
and shirazi thyme, for microbial contamination. The
Figure 4 Coliform contamination in medicinal herbs.
Positive ‒ contamination over the limit (103 CFU/g).
Negative ‒ contamination below the limit. * P < 0.05
Industerial
Negative
0
20
40
60
80
Industerial &
Traditional
Traditional Industerial
Samples number
Sample type
Total Posetive Negative
Positive
Industrial Industrial
Figure 5 Escherichia coli contamination in herbs. Total is
number of samples. Positive ‒ presence of E. coli.
Negative ‒ absence of E. coli. * P < 0.05
0
20
Industerial
&Traditional
Traditional Industerial
Samples Sample type
Total Posetive Negative
0
20
Industerial
&Traditional
Samples Total 0
20
40
60
80
Industerial &
Traditional
Traditional Industerial
Samples number
Sample type
Total PPoosseittiivvee Negative
Industrial Industrial
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results of the experiment showed that microbial indices
in considerable percentage of the samples exceeded
the standard limit. Valeriana had the highest total
microbial count and yeast/mold contamination, fennel ‒
B. cereus and coliforms, while licorice was not
massively contaminated.
We also revealed that fungal, coliform, and E. coli
contamination in traditional herbs was considerably
higher than that in industrial samples. However, there
was no significant difference between them in total
microbial count and B. cereus contamination.
Thus, our results demonstrated the importance of
monitoring medicinal plants contamination to control
the quality of herbal products.
CONTRIBUTION
Maryam Ekhtelat developed the original idea and the
protocol. Abdolghani Ameri, Maryam Ekhtelat and Sara
Shamsaei developed the protocol, analyzed the data, and
wrote the manuscript.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
ACKNOWLEDGMENT
This study was financially supported by Ahvaz
Jundishapur University of Medical Sciences and
extracted from the thesis of Ms Sara Shamsaei (B-9605).
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