Research Article | Open Access
Cleusa Fernandes Ramos1, Andreia Assuncao Soares2 , Isabela Carvalho dos Santos3, Felipe Andre Pereira Ramos4, Odair Alberton5, Luciana Kazue Otutumi6, Lidiane Nunes Barbosa6, Ricardo de Melo Germano7, Daniela Dib Goncalves8and Luiz Sergio Merlini9
1Master in Animal Science with an Emphasis on Bioactive Products, Paranaense University (UNIPAR), Umuarama, Parana, Brazil.
2Professor at Post-Graduation Programs in Animal Science with an Emphasis in Bioactive Products and Professional Master’s Degree in Medicinal and Phytotherapic Plants at Primary Care, Paranaense University (UNIPAR), Umuarama, Parana, Brazil.
3Doctoral student in the Post-Graduation Program in Animal Science with an Emphasis on Bioactive Products, (Scholarship holder PROSUP / CAPES, UNIPAR-PR); Paranaense University (UNIPAR), Umuarama, Parana, Brazil.
4Master’s student of the Post-Graduation Program in Medicinal Plants and Herbal Medicines at Primary Care, Paranaense University (UNIPAR), Umuarama, Parana, Brazil.
5Professor at Post-Graduation Program – Professional Master’s Degree in Medicinal Plants and Phytotherapies at Primary Care, Paranaense University (UNIPAR), Umuarama, Parana, Brazil.
6Professor of the Post-Graduation Program in Animal Science with an Emphasis on Bioactive Products, Paranaense University (UNIPAR), Umuarama, Parana, Brazil.
7Professor of Post-Graduation Programs in Animal Science with an Emphasis in Bioactive Products and Professional Master’s Degree in Medicinal and Phytotherapeutic Plants at primary care, Paranaense University (UNIPAR), Umuarama, Parana, Brazil.
8Professor of Post-Graduation Programs in Animal Science with an Emphasis in Bioactive Products and Professional Master’s Degree in Medicinal and Phytotherapeutic Plants at Atencao Basica, Paranaense University (UNIPAR), Umuarama, Parana, Brazil.
9Professor of the Post-Graduation Program in Animal Science with an Emphasis on Bioactive Products, Paranaense University (UNIPAR), Umuarama, Parana, Brazil.
J Pure Appl Microbiol. 2020;14(3):1817-1823 | Article Number: 6293
https://doi.org/10.22207/JPAM.14.3.20 | © The Author(s). 2020
Received: 01/05/2020 | Accepted: 24/08/2020 | Published: 16/09/2020
Abstract

The verification of microorganisms that indicate contamination of medicinal plants cultivated in vegetable gardens is fundamental to contribute and improve the conditions and management of the garden, providing safer and healthier food for the consumer population. The study aims to evaluate the bacteriological quality of Cymbopogon citratus, Aloysia citriodora, Plectranthus barbalus, Aloe vera (L.) Burm. F., Sedum dendroideum, and Peumus boldus cultivated in community gardens. Six samples were randomly collected from each plant and sent to the Laboratory of Veterinary Preventive Medicine and Public Health at Universidade Paranaense-UNIPAR, for bacteriological analysis (aerobic mesophilic microorganisms, total coliforms and coliforms at 45ºC. The cold extract (room temperature) of the medicinal plants evaluated, the Plectranthus barbalus had the highest mean for aerobic mesophiles (1.26 CFU/g x 106) and the Cymbopogon citratus second highest mean for aerobic mesophiles (9.80 CFU/g x 105). In the coliform count at 45°C, the highest mean was found in the Aloysia citriodora (2.90 CFU/g x 103) and it presented the second lowest mean for coliforms at 45°C (0.63 CFU/g x 103) and was zero for total coliforms. Regarding the hot extract (95°C-100°C) of the medicinal plants evaluated, a significantly higher aerobic mesophiles count was found for Aloysia citriodora when compared with Aloe vera (L.) Burm. F., Sedum dendroideum and Peumus boldus, not differing statistically from Cymbopogon citratus and Plectranthus barbalus. It was found that medicinal plants are contaminated but, since they are indicated as teas (infusion), they are safe for their consumers because the risk of bacteriological contamination can be considerably eliminated or reduced due to the boiling process.

Keywords

Aerobic Mesophiles, Food Safety, Microbiology, Public Health, Total Coliforms

Introduction

The cultivation of food species in home gardens favors access to fresh food in quantity and quality, which contributes to food and nutritional security1. These characteristics of vegetables contribute positively to health, as they act as functional/nutraceutical foods, helping to improve health and well-being and/or reduce the risk of diseases, in addition to providing pleasure/taste of planting, cultivar, occupation and therapy2.

With the growing search for natural products and their derivatives, a more careful assessment of the quality of the products offered to the consumer must be carried out, based on specific legislation, controlling all stages of production. The control and evaluation of the microbiological and parasitological load of medicinal plants are determinant for quality assurance and, if they are not properly complied with, may compromise their use as adjuvant in improving the individual’s health status3-5.

Henz and Alcantara6 defines a community garden as one that is grown jointly by groups of families or people from a community, by means of production cooperatives, who are responsible for managing production. In general, they are installed in idle urban areas (public and private), used for the cultivation of vegetables, medicinal plants, production of seedlings, vegetables, fruits and other foods, which supply families living near these lands.

Another positive aspect of agricultural production in urban areas is the formation of eating habits, especially as the relationship between food and health is evident. The production and sale of vegetables benefits not only the families involved in the garden, but also the residents around the unit, who generally consume more food free of agrochemicals7.

The World Health Organization estimates that 80% of people depend on traditional medicine8 and despite major advances in the pharmaceutical industry, the use of this alternative therapy is growing, which increases the responsibility of regulatory agencies and manufacturers, as they must ensure the quality and therapeutic efficacy of these products9. The organization responsible for the safety and efficacy of herbal products in Brazil is the National Health Surveillance Agency, however, these products are not considered with the same vigor as synthetic drugs10 and yet it is desirable and important that such products are within the minimum quality standards11.

Nevertheless, investigations of the microbiological quality of vegetable-based drugs and by-products obtained from them, carried out in other countries, showed rates of microbial contamination in disagreement with internationally accepted standards for drugs12, and in Brazil, microbiological evaluations of herbal medicines marketed in pharmacies showed similar results13.

Thus, the verification of microorganisms that indicate contamination of medicinal plants cultivated in vegetable gardens is fundamental to contribute and improve the conditions and management of the garden, providing safer and healthier food for the consumer population. The present work aims to evaluate the bacterial contamination by aerobic mesophiles, total coliforms and 45°C obtained through heat treatment in six medicinal plants grown in a community garden.

Materials and Methods

The survey was conducted in the community garden of the State School of Campo Benjamim Antonio Motter, located in the district of Central Santa Cruz, Cafelandia, state of Parana.

The Municipality of Cafelandia is located in the western region of Parana, at latitude 24° 36’ 12” and longitude 53° 21’ 09”, and its altitude is 550 meters and coordinates are 24° 37’ South Latitude, 53° 20’ Longitude W – GK.

Cafelandia is a predominantly agricultural municipality, largely due to the good natural fertility of its soils. Four types of soils are detected in the Municipality (Eutrophic Purple Latoil, Structured Purple Soil, Indiscriminate Hydromorphic Soils and Litholic Soils), with a predominance of Eutrophic Purple Latoil (90.3% of the total area) and Structured Purple Soil (8.0%)14.

The medicinal plants were collected from May to July 2019, using gloves and previously sterilized material. This work was carried out with six species of medicinal plants, which were chosen for their availability at the time of this work and followed the procedure described in Fig. 1.

Fig. 1. Scheme of the processing stages of medicinal plants

From the six species, two samples (to cold extract and hot extract) randomly chosen from different points were collected and the samples were packed in sterile Kraft paper bags with the appropriate identifications until processing in the UNIPAR Laboratory of Veterinary Preventive Medicine and Public Health.

In the laboratory, the samples of each species, after being weighed (10 grams of each sample), were transferred aseptically to different plastic bags, with hermetic closure. Then, the technique known as surface washing or rinsing was used to obtain the microorganisms present in medicinal plants15.

To determine aerobic mesophilic microorganisms, the methodology described by Silva et al.16 and Barbosa et al.17 was used with some modifications.

The culture medium used was Plate Count Agar (PCA) together with the surface seeding technique. Samples were analyzed by pipetting 0.1 mL aliquots or 100 microliters of the 10-1, 10-2 and 10-3 dilutions to sterile petri dishes. The plates were homogenized and incubated inverted in an oven at 35°C for 24-48 hours. After the incubation period, the colony forming units (CFU) were counted, aided by a colony counter where the number of plates between 25 and 250 colonies were considered for counting, multiplying the number of CFU by the respective dilution factor.

The results were expressed in CFU/g (colony forming units/gram), considering the initial weight of the samples.

For the determination of total coliforms and coliforms at 45°C, the methodology described by Bernadin et al.18 was used with some modifications.

An initial volume of 0.1 mL of each dilution (10-1, 10-2 and 10-3) was used. It was inoculated in Brilliant Green (BG) culture medium for total coliform counts and in Escherichia coli (EC) culture medium for coliform analyses at 45°C.

The inoculum was carefully spread over the entire surface until it was completely absorbed with the aid of a “Drigalski” strap. The BG plates were incubated in an incubator at 35°C for 24h and the EC plates were incubated at 45°C for 48h.

Plates containing 15 to 150 colonies were considered for counting and the value corrected according to dilution. Results were expressed in CFU/g (colony forming units/gram), considering the initial weight of the samples.

Bacteriological analysis of medicinal plants (CFU/gram) was performed using the Kruskall-wallis test, using the Bioestat 5.0 statistical program19. When relevant, the Student-Newman-Keuls test was chosen to compare means, and for all tests a 5% significance level was chosen.

RESULTS

There were variations in the count of the microorganisms evaluated in the present study for the different medicinal plants (Table 1).

For the cold extract, there was a higher (P <0.05) aerobic mesophilic count for P. barbalus when compared to A. vera L. Burm. F., P. boldus and S. dendroideum, not differing (P> 0.05) from C. citratus and A. citriodora. On the other hand, in relation to the total coliform count, this plant presented the third lowest count (2.6 x 103 CFU/g) differing (P <0.05) only from C. citratus.

In the hot extract, there was a reduction in the aerobic mesophil count for all medicinal plants evaluated, however, A. citriodora had a higher (P <0.05) count (1.5 x 103 CFU/g) when compared to A. vera (0.07 x 103 CFU/g), S. dendroideum (0.3 x 103 CFU/g) and P. boldus (0.83 x 103 CFU/g).

Regarding total coliforms and at 45°C, counting was found only for the cold extract (Table 1), with C. citratus presenting the highest (P <0.05) total coliform count (1.6 x 105 CFU/g) when compared with A. vera (1.8 x 104 CFU/g), S. dendroideum (2.3 x 103 CFU/g), P. boldus (1.1 x 103 CFU/g) and P. barbalus (2.6 x 103 CFU/g). For coliforms at 45°C there was no significant difference (P> 0.05) in the count for the different medicinal plants evaluated, and the highest mean was found for A. citriodora (2.90 x 103CFU/g).

DISCUSSION

Analyzing the results obtained, it was possible to observe that, with the exception of P. boldus and S. dendroideum, the other medicinal plants showed contamination above that established by Anvisa20, as published in Normative Instruction No. 4 of June 18, 2014, for the total aerobic bacteria count for the cold extract (Table 1).

 

According to this normative instruction, the total count of aerobic bacteria in plant drugs submitted to cold extraction process should be 105 CFU/g, and to hot extraction process should be 107 CFU/g20. The count of aerobic bacteria is used to obtain general information on product quality because products with high bacterial rates may indicate deficiency or low sanitary condition, but they may also be pathogenic16.

A similar work conducted by Marcondes and Esmerino15, showed that 82% of medicinal plants grown in domestic gardens in a district of Ponta Grossa (PR), analyzed before heat treatment, had an aerobic mesophilic count within the recommended by Normative Instruction No. 4 of June 18, 201420.

Similarly, Zaroni et al.21 evaluating 72 samples of medicinal plants without submitting them to the hot extraction process found that 41.67% of the samples met ANVISA’s recommendation for aerobic mesophiles20.

In the present study, it was found that the count of aerobic mesophiles for C. citratus submitted to the cold extraction process was 9.8 x 105 CFU/g, similar to that found by Zaroni et al.21 who verified counts varying between 8.5 x 105 and 1.7 x 107 CFU/g for the same extraction process.

Although there is no specific Brazilian legislation for plant drugs subjected to the cold or hot extraction process, the analysis of total coliforms and at 45°C is important, as they are microorganisms that indicate contamination, and the group of coliforms at 45°C it is most often used as an indicator of faecal contamination, however, it also inhabits environments such as the soil22. Thus, its presence in medicinal plants submitted to the cold extraction process in the present study, may be associated with soil or water contamination.

On the other hand, it should be noted that differences in the degree of contamination may be related to the distance from the soil surface on which the plant grows, presenting greater contamination the greater the proximity to the soil23. Furthermore, according to the same authors the difference in the degree of contamination between medicinal plants can be explained by the fact that certain plants contain natural barriers and antimicrobial substances that have an effect on microbial growth.

Marcondes and Esmerino15 evaluating different medicinal plants grown in home gardens found that the contamination by total coliforms varied between 2.85 x 101 and 1.25 x 103 NMP/g of coliforms at 45°C from 4.3 x 101 to 1.71 x 102 NMP/g. Similarly, Bernadin et al.18 found contamination of total coliforms and coliforms at 45°C in medicinal plants subjected to cold extraction, varying between 4 x 103 to 3.4 x 107 CFU/ g and 1 x 103 to 6.6 x 106 CFU/g, being slightly higher than found in the present study (Table 1).

Table (1):
Mean of aerobic mesophilus count (CFU/gram), total coliforms (CFU/gram) and coliforms at 45°C (CFU/gram) of the cold and hot extract of six different plants (Aloe vera L. Burm. F., Sedum dendroideum, Peumus boldus, Cymbopogon citratus, Aloysia citriodora and Plectranthus barbalus) collected in a community garden.

Medicinal plant Aerobic mesophilesa* Total coliformsa Coliforms at 45oCb
Cold extract
Aloe vera L. Burm. F. 2.1 x 105bc 1.8 x 104cd 0.67 x103
Sedum dendroideum 5.0 x 104bc 2.3 x 103d 0.70 x103
Peumus boldus 2.6 x 103cd 1.1 x 103d 1.03 x103
Cymbopogon citratus 9.8 x 105ab 1.6 x 105ab 0,00
Aloysia citriodora 7.4 x 105ab 4.1 x 104bc 2.90 x103
Plectranthus barbalus 1.26 x 106a 2.6 x 103cd 0.63 x103
Hot extracta
Aloe vera L. Burm. F. 0.07 x 103c
Sedum dendroideum 0.30 x 103bc
Peumus boldus 0.83 x 103bc
Cymbopogon citratus 1.83 x 103ªb
Aloysia citriodora 3.17 x 103ª
Plectranthus barbalus 1.50 x 103ªb

aAverages followed by different letters in the column differ by Student-Newman-Keuls test (P<0.05); b Not significant by Kruskal-wallis test (P>0.05). *Standard to count of aerobic bacteria in plant drugs submitted to cold extraction process (105 CFU/g) and hot extraction process (107 CFU/g)

One of the important points to be taken into consideration was that after the heat treatment, coliforms were absent in all samples surveyed.

According to Alterthum and Carvalhal24, heat is one of the most widely used methods for inactivating microorganisms, because high temperatures cause them to be inactivated and irreversibly lose their ability to multiply. When a microbial population is heated, the reduction in the number of viable organisms occurs at high rates, justifying what was found in the present work.

With the considerable increase in medicinal plants or derived products, there is always a concern about their quality, which may lead to public health problems, due to the possibility of access to products without adequate conditions of consumption, resulting from the great potential for contamination linked to these products25.

Microbiological quality control is one of the essential steps within this process, since microbiological contamination becomes a problem when it results in undesirable effects. Therefore, before using them in natura or commercialization, they must go through a rigorous control from collection and storage to final production, so that the quality and safety of the plants can be guaranteed26. With these results, it is important to use medicinal plants in natura, as infusion or tea, to reduce or eliminate the microorganisms present.

CONCLUSION

Under the conditions in which the present research was carried out, it is concluded that in relation to the count of aerobic mesophiles, only two plants (P. boldus and S. dendroideum) met the Brazilian legislation in force for the process of cold extraction of plant drugs.

In relation to the total coliforms and at 45°C, counting was verified only in the plants submitted to the cold extraction process. The hot extraction process (infusion or tea) reduced the count of aerobic mesophiles and did not allow the detection of coliforms by the microbiological method, demonstrating that this is a safe way to use plant drugs.

Therefore, it is recommended that the quality control of vegetable drugs be more rigorous in addition to the adoption of good practices in the management of community gardens in order to comply with the relevant legislation and offer a safe product to the population.

Declarations

ACKNOWLEDGMENTS
We would to thanks the Executive Board of Research and Post-Graduate Management – DEGPP UNIPAR, and the Coordination for the Improvement of Higher Level Personnel – Brazil (CAPES/CNPq) and the State College of Campo Benjamim Antonio Motter, district of Central Santa Cruz, Cafelandia-PR for the collaboration in the development of work.

CONFLICT OF INTEREST
The authors declare that there is no conflict of interest.

AUTHORS’ CONTRIBUTION
All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

FUNDING
This work was supported by Executive Board of Research and Post-Graduate Management – DEGPP UNIPAR, and the Coordination for the Improvement of Higher Level Personnel – Brazil (CAPES/CNPq).

ETHICS STATEMENT
This article does not contain any studies with human participants or animals performed by any of the authors.

AVAILABILITY OF DATA
All datasets generated or analyzed during this study are included in the manuscript.

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