Microbial quality of some medicinal herbal products in Kashan, Iran


Mazroi Arani N et al. Journal of HerbMed Pharmacology, Volume 3, Number 2, December 2014 http://www.herbmedpharmacol.com 114 supplements are produced in the United Kingdom in a safe and risk-free form (3,4). In addition to public people, clinical microbiologists tend highly to use these drugs in the treatment of infections in that comparatively the side effects of these drugs are significantly lower than chemical drugs. In a study it was shown that consumption of medicinal plants increased 38% in 1990 and 1997, and revenue from the sale of these products was about 5.1 billion dollars in 1997 (5). Bacteria are of the major causes of microbial infections and food borne poisoning. Over the past decade, the incidence of food borne microbial diseases in the developing and developed countries has been overwhelming, while there is no exact statistics of incidence and the occurrence of infections and food borne poisoning, especially in developing countries (6). The evaluation of existence of a variety of the microorganisms in herbal extracts is a mean to determine their hygienic status of this product. Also, pathogenic microbes such as enterobacter, enterococcus, shigella and streptococcus have been shown to grow on herbal materials (2). Therefore, of the most important evaluation of these products is the existence of coliforms indicating its fecal contamination (1). Escherichia coli is a normal flora in the intestines of humans and many warm-blooded animals and they are reported to be 109 CFU/g. Thus, it was suggested as the specific indicator of fecal contamination in 1982. In microbial tests, searching for coliforms spp. are utilized as an important indicator of health products such as herbal extracts (7). Enterococcus is resistant bacteria to bile salts and sodium azide and these bacteria can confirm fecal contamination of food material through the presence or absence of Coliform. Hence, these are used as a secondary indicator of fecal contamination in foodstuffs (8). Regarding to the spending too much time and money in identifying different types of foodborne pathogens, identification of indicator microorganisms was carried out. As an example, sulphite-reducing Clostridia are anaerobic bacteria, producing spores and belong to genus Clostridia and Bacillaceae family. Spores of sulfite-reducing Clostridia are widely distributed in the environment. They are found in human and animal faeces as well as in sewage and soil. Unlike coliforms and E. coli, they remained alive in water for a long time and are more resistant against chemical and physical agents compared to vegetative forms and can indicate the presence of defects in the refining process of the products and are presented as an indicator of intermittent contamination (7,9). In a study in Birjand, the microbial contamination of herbal waters was reported to be 80% (1). So far, a few studies on microbial and hygienic quality of herbal waters and rosewaters have been done in Iran. According to the consumption approach to this product in raw as a medicinal herbal product, this study was carried out to examine the microbial quality of herbal waters and rose waters distributed in Kashan, Iran. Materials and Methods Sample collection This is a descriptive study carried out on 256 samples of herbal waters and 191 samples of rose waters (totally 447 samples) distributed in Kashan during the 2012 to 2013. Food samples were taken to Kashan University of Medical Sciences for laboratory tests. Then microbial tests were performed according to Iranian national standards with the code numbers as follow: 3270, 3545, 5272, 7724-2, 7725-1, 5353, 4207 and 8869 (10-17). Total aerobic bacterial count 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 (10). Mold and yeast count To count mold and yeast, 100 ml of sample 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 (11). Detection of coliforms spp. and E.coli 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 MacConkey agar medium (Merck ink, Darmstadt, Germany) by means of a sterile forceps. It was incubated at 37 °C for 24 to 48 h 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) (16). Detection of Enterococcus spp. To detect Enterococcus spp., 100 ml of the sample was passed through sterile filter (0.22 μm). The filter was placed on the KF Enterococcus (Merck ink, Darmstadt, Germany) specific culture using sterile forceps. The plates were incubated at 37 °C for 48 h. The Formation of colonies with red, cherry and pink color demonstrated the existence of Enterococcus (15). Detection of Pseudomonas Pseudomonas presence was monitored by passing 100 ml of samples through sterile filter (0.22 μm), cultivating the filter retentive on the surface of cetrimide agar (Merck ink, Darmstadt, Germany) using sterile forceps. Then plates were incubated for 24 to 48 h at 37 °C. The formation of colonies with green color indicates the presence of Microbial quality of herbal products Journal of HerbMed Pharmacology, Volume 3, Number 2, December 2014 http://www.herbmedpharmacol.com 115 Pseudomonas (14). Detection of sulfite-reducing Clostridia Samples were put into water bath to monitor sulfite-reducing Clostridia presence. Heating at 70 °C for 20 min causes heat shock and spores transformation to vegetative form. After cooling, 100 ml of the sample was passed through a membrane filter. By means of sterile forceps, filter was placed on specific Sulfite Polymyxin Sulfadiazine culture (SPS). Then the medium was incubated in an anaerobic jar at 42 °C for 48 h. If sulfitereducing Clostridia, are present in the sample, black colonies will be observed (17). Statistical analysis The data were analyzed using SPSS software version 11.5 through Fisher’s exact test. Results Based on the results gained from the total sample, 43.84 percent (196 cases) of the herbal waters and rosewaters were usable according to national standards of Iran and 56.16% (251 cases) were non-usable. 108 cases (42.18%) of herbal water samples were uncountable regarding to the total count of mesophilic bacteria and total bacteria count for 116 (45.32%) cases was less than 100 cfu/ml. Also, 32 cases (12.5%) of samples had total bacterial count between 102 to 6 ×103 cfu/ml which had an average of 1.153×103±1.738×103 cfu/ml. Nine cases (3.5%) of samples shown contamination combined to mold and yeast and high contamination count to mesophilic bacteria. Only one case (0.3%) of samples had contamination of combined with high counts of mesophilic bacteria and identified Enterococcus. Besides, one sample (0.3%) associated with high count of contamination of mesophilic bacteria, mold, yeast and Pseudomonas was observed. Of rose water samples, 72 (37.7%) of the total count of mesophilic bacteria were uncountable and 85 (44.5%) of their total bacteria counts were less than 100. Total bacterial counts were between 102 to 6×103 cfu/ml for 34 (17.8%) of samples, which had an average of 1.656×103±2.031×103 cfu/ml. The comparative distribution of contamination of herbal water and rose water samples based on microbial tests are presented in Table 1. Based on the obtained results, there was no significant difference between usability and non-usability of distributed herbal waters and rose waters (p>0.05). The results gained from the microbial tests presented as acceptability and unacceptability of herbal water and rosewater samples based on the national standard of Iran are shown in Table 2. Also, there was significant difference between usability and non-usability conventionally herbal water samples and the industrially herbal water samples (p<0.05). 24.68% conventionally herbal waters and 52.52% industrially herbal water samples were usable. The comparative distribution of herbal water samples which were consumable based on traditional and industrial process are presented in Table 3. The maximum permitted number of bacteria, based on the national standards of Iran, can be seen in Table 4. Table 1. Comparative distribution of contamination of herbal water and rose water samples based on Microbiological tests Microbiological tests Total Total bacteria count (cfu/ml)* Coliforms Enterococcus Pseudomonas Sulfite-reducing Clostridia Mold Yeast

Cite this paper

@inproceedings{Chaleshtori2014MicrobialQO, title={Microbial quality of some medicinal herbal products in Kashan, Iran}, author={Reza Sharafati Chaleshtori and Navid Mazroi Arani and Mahmoud Rafieian-Kopaei}, year={2014} }