Evaluating the microbial contamination of some Iranian dried medicinal plants and distillates

Document Type: Original Article

Authors

1 Biology Dept., Shahrekord Branch, Islamic Azad University, Shahrekord, I.R. Iran

2 Food and Drug Dept, Shahrekord University of Medical Sciences, Shahrekord, I.R. Iran

3 Medicinal Plants Dept., Shahrekord Branch, Islamic Azad University, Shahrekord, I.R. Iran

4 Food Hygiene and Public Health Dept., Shahrekord Branch, Islamic Azad University, Shahrekord, I.R. Iran

5 Microbiology Dept., Shahrekord Branch, Islamic Azad University, Shahrekord, I.R. Iran

Abstract

Background and aims: In Iran herbal water and dried herb are as traditional medicinal and are consumed widely. Therefore, microbial evaluation of these products in term of public health is important. The aim of the present study was to study the contamination of some dried medical plants and distillates in the groceries of Shahrekord city.
Methods: In this descriptive study, 35 samples of herbal waters and 35 samples of
7 species (lavender, lemon balm, valerian, savory, borage, mint and thyme) dried medicinal plant (total samples=70) distributed in Shahrekord during spring to summer of 2012 were purchased and transferred to laboratory. Then, microbial tests such as total aerobic bacterial count mold and yeast count, total coliforms, and detection were evaluated based on national standard of Iran.
Results: Contamination to mesophilic bacteria and yeast was observed in the 100% of distilled samples, based on the national standard of Iran. Additionally, none of the medicinal dried plant was contaminated according to the national standard of Iran.
Conclusion: According to contamination of all distilled, It is proposed that producers of herbal distillates consider the hygiene conditions, using correct and suitable pasteurization, considering the structural condition of workshop and also appropriate packaging in order to reduce the secondary contamination and increase the quality of the finished product.

Keywords

Main Subjects


INTRODUCTION

 Medicinal Plants are of great value and importance in providing the health of communities in both treatment and prevention of disease. The use of traditional medicine, especially the plant originated medicines, has increased in the world widely since 1990 because of being cost effective, availability and their role in providing the basic necessities of healthcare in many developing countries in Africa and Asia.1 Consumers of the traditional medicines in South Africa and Asia are estimated 200,000, in consultation with 60% of the consumers in South Africa and Asia, it was concluded that the traditional medicines are as their basic source of health and hygiene.2 In Iran,  herbal water and dried herb are as traditional medicinal and are consumed widely. Therefore, microbial evaluation of these products in term of public health is important.3

To date, much of the herbal research in the corresponding field is focused on the isolation and the evaluation of active and toxic compounds in the herbal medicine to validate and strengthen the security and efficiency. However, the researches on the assessment of toxic substances and other contaminants which have detrimental effects on plants have been carried out.4 The contamination of micro-organisms may lead to the disease.5 In previous research on herbal medicines, pathogenic microorganisms, and several fungal species such as Aspergillus, Penicillium and Fusarium resistant to methicillin and vancomycin have been found.6-8 So, the safety of infected plants in the external markets should be paid attention in which the conditions for microbial proliferation are provided.

Most previous studies have been carried out on the infection of medicinal plants by microbes and fungal species, that focus on the bacterial infections and microbial loads. Collecting and handling the medicinal plants are not always in sanitary conditions that can lead to a lots of microbes and thus cause some damages.9,10 Most herbs are dried in exposed to air that may develop the infections caused by the bacterial and fungal in the air and soil. The microbial infections of plants limit the use in food products, pharmaceutical and cosmetics industries. The hygienic quality of medicinal plants, as well as the use of antiseptic methods, is an important step towards the consumer health and treatment effectiveness. Microorganisms important in the public health(as pathogens) including Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), Clostridium perfringens (C. perfringens), Bacillu scereus and Salmonella, can exist in spiceplants. The final microbial status of plants is determined by the natural numbers of microorganisms in the plants, and by operations such as harvesting, drying, transporting and packing process.11-13 The study of Khodadadi in Birjand City found that 80% of traditional distillates were positive for coliforms and non coliforms.14 Also, Mazroii and colleagues reported herbal extracts distributed in Kashan were contaminated with mesophilic aerobic bacteria and yeast (51/52%, 11/6% respectively).3

Considering the importance of this issue and expand the use of herbal medicine and medicinal plants distilled in our country, the study of the possibility of microbial and fungal becomes necessary.

 

METHODS

A total of 35 medicinal dried plants and 35 distillates samples from 7 plants (lavender, lemon balm, valerian, savory, borage, mint and thyme) were collected randomly from 5 different groceries in Shahrekord city, in the spring and summer 2012. Then, all of samples were transported to the microbiology laboratory of the Islamic Azad University of Shahrekord to investigate the microbial status to load and were tested as soon as possible.

The results were analyzed by SPSS and Chi-square and Fisher exact models with a confidence level of 95%. The test was considered significant correlation between the two data.

Microbial load of medicinal dried plant samples, the number of total aerobic bacterial count, coliforms, E. coli, S.aureus, molds and yeasts were tested. The basic dilution of 10-1 was prepared firstly from the samples.14 For this purpose, 25 grams of the solid samples were added into 225 ml peptone water broth, and the bacterial counting was performed in each case as follows: To count the total number of bacteria, according to ISO 4833 reference method, 5 tubes containing 9 ml of water peptone broth were selected and by transferring1 ml of the basic dilution into the first tube, and preparing the serial dilutions, the dilutions of 10-1 to 10-6 were prepared from samples.16-18 1 ml of the dilutions (10-1 to 10-6) were added into two empty plates, followed by adding melt plate count agar medium (45°C), and incubation at 30°C for 72 hours. In the following, the two plates related to the dilutions containing approximately 30 to 300 colonies were chosen and their numbers were multiplied by the inversely related dilutions. The number of bacteria in 1 ml of the sample was reported in terms of CFU.19-21 The counting of coliform bacteria was performed according to ISO 21528-2 and ISO-4832 reference methods similar to previous steps, and melt MacConkey agar medium (Merck ink, Darmstadt, Germany) was used.22,23 It was incubated at 37°C for 48 h. to count the mold and yeasts, the medium of Extract Glucose Chloramphenicol Agar (YGCA; Merck Ink,Darmstadt, Germany) and the steps were performed as above.then incubated at 25 °C for 3 for 5 days.

For microbial load assay for distillates samples, the total bacteria were counted by pour plate method in the plate count agar (Merck ink, Darmstadt, Germany). Then, they were incubated for 72 h in the temperature of 30±1°C.24

To count mold and yeast, 100 ml of sample was passed through a sterile filters (0.22 μm) and through using a forceps, filter was placed onto the surface of the Yeast Extract Glucose Chloramphenicol Agar (YGCA; Merck Ink, Darmstadt, Germany), and then, the media were incubated at 25 °C for 3 to 5 days.25

In order to detect coliforms spp. and E.coli, 100 ml of the sample was passed through sterile filter (0.22 μm).

The filter was placed on the surface of Mac Conkey agar medium (Merck ink, Darmstadt, Germany) by means of a sterile forceps. It was incubated at 37°C for 24 to 48h at 37°C. In the case of colonies growth, they were counted and reported in the samples of 100 ml. The grown colonies were used for differential diagnosis of E. coli by TSI, SIM, MR-VP and urea Tests (Merck ink,Darmstadt, Germany).26

Finally, after counting the bacteria colonies, if present, to analyze the data, and compare mean, fisher test method and SPSS Ver.20 Statistical software were applied.

 

 

Table 1: List of the medicinal plants that their microbial load was investigated

Plant name

Family

Scientific name

TheUsed section

Mint

Lamiaceae

Mentha spicata

Leaf

Shirazi Thyme

Lamiaceae

Zataria multiflora

Leaf

Savory

Lamiaceae

Satureja hortensis

Leaf

Valerian

Valerianaceae

Valeriana wallichii

Rhizome

Lemon balm

Lamiaceae

Melissa officinalis

Leaf

Borage

Boraginaceae

Echium amoenum

Leaf and flower

Lavender

Labiatea

Lavandula stoechas L

Leaf


 


RESULTS

 The result of microbial evaluation of dried plant and its distillates distributed in Shahrekord were shown in Table (2 and 3).

 

 

Table2: Evaluation of themicrobial loadof various types of dried aromatic vegetables

Sample Type

No. of samples

The total number ofbacteria

Coliforms

Esherishia.coli

Mold

Lavender

5

4.78×102

7.6×101

-

-

Lemon balm

5

1.41×103

6.8×101

-

-

Valerian

5

5.95×102

7.8×101

-

-

Savory

5

9.86×102

2.8×101

-

-

Borage

5

2.27×103

9.72×101

-

-

Mint

5

1.81×103

7.24×101

-

-

Thyme

5

3.8×103

0.92×101

-

-

 

Table 3: Evaluation of themicrobial load of various types of distillates

Sample Type

No. of samples

The total number of bacteria

Coliforms

Eshirishia.coli

Mold

Yeast

Lavender

5

1.28×105

Negative

Negative

Negative

1.418×103

Lemon balm

5

6×104

Negative

Negative

Negative

1.78×102

Valerian

5

4.88×106

Negative

Negative

Negative

3.025×103

Savory

5

4.328×106

Negative

Negative

Negative

1.236×103

Borage

5

7.1246×106

Negative

Negative

Negative

2.02×102

Mint

5

4.103×106

Negative

Negative

Negative

1.53×103

Thyme

5

4.124×106

Negative

Negative

Negative

4.84×102

 

 

Based on the results gained from the total sample of dried plant, despite observation contamination count to mesophilic bacteria, according to national standards of Iran, all samples were usable (Table 4).27

 

Table 4: The microbial standard of microbiolog of dehydrated vegetables specifications

Properties

Admissible limit (gr)

Total count

105

Coliforms

103

E. coli

Negative

Mold

103

21 cases (60%) of distillates showed contamination to yeast and high contamination count to mesophilic bacteria (P<0.05). Total bacterial counts and yeast were between 102 to 3.2×107 cfu/ml and negative to 7.5×103 respectivly. 40% of sample were usable based on the national standard of Iran (Table 5).28

 

Table5: The microbial standard of herbaceous distillates specifications

Properties

Admissible limit in 100 ml

Total count

200 cfu/ml

Coliforms

Negative

E. coli

Negative

Mold

Negative

Yeast

Negative


DISCUSSION

 Based on the obtained results, dry medicinal herbs were consumable according to Iran National Standard. While 60% of distillate samples were contaminated with aerial bacteria neutrophil and yeast, theywere not consumable. However, contamination with Coliform and E.coli was not seen in any distillate sample, showing the individual hygiene of personnel and non-fecal contamination.

Chen et al found in a research on medicinal plants and spices that ginger is free of bacteria Sospedra et al observed in a study on the microbial contamination of medicinal plants and spices no bacterial contamination in thyme, basil, cloves, ginger and parsley has seen.29,30

Microorganisms existing in dry herb are among the native microorganisms of soil and plants. Dust, inset, wastes of birds and rodents and consumed water in the process are some other contamination resources of this product.

Microbiological quality of dry herbs is mostly depended on the culture region, transportation condition and storage of these products. It is not possible to grow the pathogen microorganisms in this product due to its water activity. If drying process and storage condition of dry herb are suitable, microorganisms can not grow in it.27

Contamination in the distillate can be resulted from lack of pasteurization, inappropriate pasteurization, using the contaminated glass or plastic bottles and lack of suitable sealing.31

There are limited studies about microbial quality of herbal distillates. In a study on the microbial quality of industrial and traditional produced rosewater in Kashan, Iran, 63.97% samples were reported as non-consumable according to Iran National Standard, so that
all traditional-produced samples were contaminated and non-consumable.31 In a research done by Khodadadi et al on the supplied distillates in Birjand, it was observeed 80% contamination in the traditional distillates and 60% in the industrial distillates.14 Studies done by Mazrui et al on 132 samples of herbal distillates supplied in Kashan, Iran showed 51.52% and 11.6% contamination to aerial bacterial neutrophil and yeast, respectively, but contamination with California and E.coli was not seen.3 Researchers studying the traditional-produced distillates in Owino market reported that all samples have neutrophil aerial bacterial contamination.32

These studies are all consistent with the results of the recent study showing that distillate samples have contaminated with aerial bacteria mesophil and yeast.

In general, in addition to the previous reasons for contamination of distillates, the following cases can be mentioned. Disrespecting the hygiene hints during collecting and providing the distillates and also dilution of these distillates result in contamination in the distillates.14 These contaminations can have undesirable effects on the health of consumer.32

 

CONCLUSION

Then, based on the obtained results, it is proposed that producers of herbal distillates consider the hygiene conditions, using correct and suitable pasteurization, considering the structural condition of workshop and also packaging appropriate in order to reduce the secondary contamination and increase the quality of the finished product.

 

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest.

 

ACKNOWLEDGEMENTS

The authors would like to thank from the Professor Afshin Akhondzadeh Basti in the Department of Food Hygiene of the University of Tehran for his technical support. This work was support by the Islamic Azad University of Shahrekord by grant number: MSAB 220145.

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