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Introduction
Yerba mate (Ilex paraguariensis) is a plant native to South America, specifically from the region between Southern Brazil, Northern Argentina, Paraguay, and Uruguay (Marcowicz et al., 2007), Is one of the products which is deeply rooted in Paraguayan culture. Its aqueous extract is consumed in a massive way in different forms such as: mate (hot water), tereré (with cold water) or cocido (hot infusion of toasted yerba) (Jerke et al., 2009; Pérez Paiva et al., 2013).
In popular medicine, it is recommended to trait arthritis, headache, rheumatism, hemorrhoids, obesity, fatigue, slow digestion, and liver disorders among other health problems (Bastos et al., 2005). Yerba mate has been reported to possess multiple properties, such as vasodilatory effects, reducing cholesterol and triglyceride (Stein et al., 2005), preventing DNA damage (Mohadjerani & Roodgar, 2016), antimutagenic and radioprotective effects (Bracesco et al., 2018), antidiabetic effects (Heck & De Mejia, 2007; Rocha et al., 2018), prevention of the senescence of retinal cells and mobilization of the cellular pathways (Tate et al., 2020), antioxidant and antimicrobial effects (Kungel et al., 2018), preservation of the cardio- vascular health (Cardozo Junior & Morand, 2016), anti-obesity effects (Gambero & Ribeiro, 2015; Kim et al., 2015; Uecker et al., 2019), prevention of liver redox imbalance, high triglycerides and microsteatosis (de Oliveira et al., 2018), among others. It is also an excellent source of polyphenols such as caffeic and chlorogenic acids (Tate et al., 2020).
Yerba mate is a plant rich in vitamins and pro- duces a feeling of well-being, vigor, and intellectual lucidity, based on the presence of the alkaloid matein. It is diuretic and digestive product with laxative properties (Jerke et al., 2009; Riachi & De Maria, 2017).All these held benefits have made yerba mate a popular product, being considered as a nutraceutical and a functional ( Alkhatib et al., 2017; Kujawska, 2018; Riachi & De Maria, 2017) currently consumed in different countries around the world (Alkhatib & Atcheson, 2017).
During its process, the herb undergoes changes in temperature changes and storage periods for aging to improve its flavor. This period can vary from 2 months to 1 year or more, which favors the development of filamentous fungi in the final product (Silva et al., 2019). The Paraguayan regulations establish a limit of 1,8×102 CFU/g of total fungi and yeasts for processed yerba mate (Instituto Nacional de Tecnología Normalización y Metrología, 2007). Several species of filamentous fungi have been reported in yerba mate, among them Aspergillus of the Sections Nigri and Flavi (Jerke et al., 2009; Pérez Paiva et al., 2013; Silva et al., 2019), which are potential producers of mycotoxins.
Considering its beneficial effects, yerba mate has become a popular product around the world (Alkhatib et al., 2017; Alkhatib & Atcheson, 2017), and an increase in its consumption has been seen. Therefore, it is necessary to have a quality control on the commercial products to ensure their safety. The aim of this work was to evaluate the content of fungi and yeasts in the commercial brands of yerba mate being sold in Paraguay.
Materials y Methods
Sample collection
39 brands (1 kg packages) of yerba mate elaborated (Ilex paraguariensis), all products with RSPA (sanitary food product registration) and RE (establishment registration) in force from the National Institute of Food and Nutrition, INAN. Each package, adquired in supermarkets of the country was considered as a unit of analysis.
Fungal analysis for colony forming units
Samples were analyzed by serial dilution plates. A 25 g subsample of each sample was diluted in 225 mL of sterile distilled water. An aliquot of 0.1 ml was spread on Potato Dextrose Agar (PDA) (90 × 15 mm plates) and incubated at 25 °C for 5 days. The number of colony forming units (CFU) and fungal incidence were determined based on morphological characteristics (Pitt & Hocking, 2009).
Fungal analysis for isolation of Aspergillus
Samples were analyzed by serial dilution plates. A 25 g subsample of each sample was diluted in 225 mL of sterile distilled water. An aliquot of 0.1 mL was spread 90 × 15 mm plates with MAAS, (selective medium for isolation of Aspergillus from soil) (5 g of peptone, 1 g of potassium hypophosphite, 30 g of sodium chloride, 10 g of glucose, 0.5 g of magnesium sulfate heptahydrate, 20 g of agar and 1000 mL of distilled water) (Dhingra, OD; Sinclair, 1995; Klich, 2002) and incubated at 25
°C for 5 days. The number of colony forming units (CFU) and fungal incidence was determined based on morphological characteristics (Barnett & Hunter, 1972; Pitt & Hocking, 2009).
Subsequently, each colony was purified, and monosporic cultures were performed using serial dilutions. The fungi were seeded on PDA (90 × 15 mm plates) and incubated at 25 °C for 5 days. Those who presented characteristics of belonging to the genus Aspergillus were identified using a taxonomic key(Raper et al., 1965).
For the analysis, aspects related to the geographical origin of the samples, brand, grain size, handling (conventional and organic) were considered. Maximum, minimum, and median values and were calculated.
Results
A total of 39 yerba mate brands marketed in Paraguay were examined for the presence of fungal contamination. Two thirds (26) of the samples declared conventional processing while the remaining one third (13) were promoted as organic. In terms of granulometry, 74.3% declared intermediate grinding, 12.8% coarse grinding, 10.3% of fine grinding and 2.5% with stick.
Of the total sample analyzed, 15.4% (n=6), were found to be free from the presence of fungi and yeasts. The remaining 84.6% (n=33) of the samples were found to be positive for fungal contamination and the fungal load per sample ranged from 2 × 101 CFU/g to 3.2 × 104 CFU/g.
Almost 40% of the positive samples (n=33) were found to have contamination values over 1.8 × 103 CFU/g, which are higher than the Paraguayan regulations. In Paraguay, in a study, 22 brands of elaborated yerba mate, it was determined that 10 brands exceeded the limits established by the national regulations 8 × 103 CFU/g (Pérez Paiva et al., 2013).
Our study revealed that 54.5% (n=18) of the positive samples exhibited contamination levels below 1.0 × 103 CFU/g. Of these samples, 93.4% were processed conventionally, while 6.6 % were organic. With regards to granulometry, 88.9% declared intermediate grinding, 11.1% of fine grinding, and 5.5% of coarse and stick grinding, respectively. Regarding of 27.3% (n=9) of the samples showed levels, between 1.0×103 and 9.0×103 CFU/g; 11.1% were organic and 88.9% conventional, in other hand granulometry, 22.2% suffered coarse grinding and 88.8% intermediate grinding (Table 1).
Six samples (18.2%),all from different manufacturers, exceeded the contamination limit (1.0x104 CFU/g) by the World Health Organization (WHO, 2007).
Considering the geographical origin, the highest levels of contamination were found in brands produced in the departments of Itapúa and Guairá, where the maximum values of CFU/g were 3 × 104 and 2×104 respectively (Table 2).
With respect to granulometry, fine grinding leads to the highest level of contamination, 3.2 × 104 CFU/g with a median of 6.8 × 102 CFU/g, followed by intermediate grinding with 2.2 × 104 CFU/g and a median of 6 .1 × 102 CFU/g; Coarse grinding, led to a maximum of 7.1 × 103 CFU/g and a median of 1.6 × 102 CFU/g and the product with stick, with a maximum of 8.0 x 101 CFU/g (Table 3).
Table 2. Colony Forming Units per gram (CFU/g) in yerba mate samples considering the geographic origin (place of planting).
| Geographic Origin | n (samples) | Minimum CFU/g | Maximum CFU/g | Median CFU/g |
|---|---|---|---|---|
| Alto Paraná | 3 | 0 | 4.0 x103 | 3.0 x 102 |
| Guairá | 12 | 0 | 2×104 | 5.5 x 102 |
| Itapúa | 21 | 0 | 3 x 104 | 9.0 x 102 |
| Misiones | 2 | 80 | 1.0 x 102 | 9.0 x 102 |
| Central | 1 | 0 | 0 | 0 |
Presence of Aspergillus
The 89.7% of the samples presented variable levels of contamination with fungi of the genus Aspergillus. The presence of Aspergillus Section Nigri, Flavi, Circumdati, Fumigati, Wentii, Usti, Versicolor and Emericella nidulans was determined.
Aspergillus Section Nigri was prevalent, representing 65.3%, followed by Aspergillus Section Flavi with 26.9%. These two groups represented 92.2% of the isolated colonies. The other 7.8% were represented by Aspergillus Section Fumigati (2.9%), Versicolor (2.4%), Circumdati (1.4%), Wentii (0.4%), Emericella nidulans (0.5%), and Usti (0.1%).
Discussion
Yerba mate undergoes extensive processing before it is ready to be packaged including selection of the leaves, blanching and drying, the final product is extremely dehydrated, and for these, impairs the growth of pathogenic organisms but fungal species, are ubiquitous in the environment and may easily contaminate yerba mate leaves during storage or manipulation (Vieira et al., 2010).
A study done in Brazil in 2010 reported substantial growths of fungi in the range of 0.1×101 to 4.9×102CFU/g in 7 of the 8 brands analyzed. Also, in Brazil, in 2019, 33.3% (n=15 ) of the samples studied exhibited contamination levels below 1.0 × 103 CFU/g; 60.1% showed intermediate contamination levels between 1.0 × 103 and 9.0 × 103 CFU/g, and two samples exceeded the contamination limit (1 x 104 CFU/g). In both studies, the levels of contamination detected are higher than those obtained in our study, where 6 brands did not present any type of microbiological contaminant. In Argentina, an analysis of 36 samples of different brands from establishments of the provinces of Misiones, Corrientes, and Buenos Aires determined that on average the fungal count was 6.1 x 103 CFU/g (Jerke et al., 2009), with the highest incidence of molds (89%) than yeasts, higher than that obtained in our study of 5 × 103 CFU/g. Besides evaluating different brands, this study also considered other important aspects such as: handling, geographical origin of the products, and the granulometry of the grinding.
Table 3. Colony Forming Units per gram (CFU/g) in yerba mate samples considering the Granulometry
| n (samples) | Minimum CFU/g | Maximum CFU/g | Median CFU/g | |
|---|---|---|---|---|
| Fine grinding | 4 | 1.1 x 102 | 3.2 x 104 | 6.8 x 102 |
| Intermediate grinding | 29 | 0 | 2.2 x 104 | 6.1 x 102 |
| Coarse grinding | 5 | 0 | 7.1 x 103 | 1.6 x 102 |
| With stick | 1 | 8.0 x 101 | 8.0 x 101 | 8.0 x 101 |
Considering product management, the highest levels of contamination were found in brands produced under conventional process, with the maximum CFU value being 3.0 ×104 CFU/g, and a median of 6 × 102 CFU/g. On other hand, organic management demonstrated much lower contamination, the higher level being 4.0 × 103 CFU/g and the median 1.0×101CFU/g (Table 1).
In soils with organic management, there is a greater heterogeneity of the microbiome when compared to soils with conventional management, this could help control pathogens during cultivation and decrease the initial inoculum present in yerba mate (Lupatini et al., 2017).
In Paraguay, to be able to label a product as organic, it must be demonstrated to have some type of certification that indicates its organic management, although there is no specific certification that is mandatory. The definition of "organic yerba mate" is not standardized or regulated in current system (Instituto Nacional de Tecnología Normalización y Metrología, 2007: Norma Paraguaya NP 35 001 93 C.D.U. : Yerba Mate Elaborada, Requisitos Generales).
In sense of geographic results, Itapúa and Guairá are the Departments with the highest yerba mate production in the country in terms of cultivated area and productivity (Ministerio de Agricultura y Ganadería, 2018). The higher level of contamination can probably be related to the high volumes of product handled in these departments 73,000 and 20,000 tons respectively, compared to Alto Paraná with 6,500 tons and Misiones with 52 tons.
With respect to granulometry, fine grinding leads, the existence of a correlation between the content of molds and yeasts and the size of the Yerba mate particles could not be determined due to the small size of the sample. However, the present data allows us to believe that a fine grinding of the product increases the chances of contamination compared to a coarse grinding.(Silva et al., 2019; Vieira et al., 2010).
In the sense of Aspergillus presence, a study from 2013 including 22 commercial brands of elaborated yerba mate commercialized in Paraguay determined the presence of Aspergillus niger, Aspergillus flavus, Pullularia sp. Penicillium sp. and Cladosporium sp. (Perez Paiva et al., 2013). In Argentina, where 36 samples of processed yerba mate were analyzed, it was determined that the incidence of the Aspergillus genus was 86%. In Brazil, 8 brands of yerba, it was determined that Aspergillus niger and Aspergillus fumigatus were the prevalent fungi (Vieira et al., 2010). At 2019 also in Brazil was reported as prevalent with 76.5% to Aspergillus Section Nigri followed by Aspergillus Section Flavi with 1.6%(Silva et al., 2019). In Argentina,36 samples of yerba mate processed, 1215 strains corresponded to Aspergillus Section Nigri, these being the prevalent fungi (Castrillo et al., 2013). All these results are like those obtained in our study, thus corroborating the importance of this fungal group as a contaminant of yerba mate. The occurrence of fungi belonging to Aspergillus Section Nigri and Section Flavi in yerba mate demands more attention due to the risk of mycotoxin production (Silva et al., 2019). A concerning issue that arise during the development of this work was the unevenness of the labeling of the commercial product. In the various labels, terms such as selected, premium, organic, natural herb medium grinding or fine grinding were found, however none of these definitions are found in current regulations.
This situation is worrying from the point of view of the quality and safety of the products; since there is no legal definition about the different parameters, the final product is therefore highly heterogeneous.
It is important to analyze and consider in the fu ture regulations regarding yerba mate the particle size of the herb and the contents of mycotoxins present in these products.
In yerba mate, high fungal counts may indicate a failure during processing or a recontaminationat some stage of storage, transportation or marketing, due to an increase in moisture content (Silva et al., 2019).
Conclusion
Based on the results obtained, we conclude that yerba mate is a favorable substrate for the growth of fungi and yeasts; therefore, periodic microbiological control is essential. Regarding the presence of fungi of the genus Aspergillus, their importance should be considered due to their potential for mycotoxin production. In our work of 39 samples tested, 84.6% (n=33) were positive for fungal contamination and 39.4% presented values higher than 1.8 × 103. CFU/g, above the limits established by Paraguayan regulations. Aspergillus Section Nigri was the prevalent group 65.3%, followed by Aspergillus Section Flavi with 26. 8%.
