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Fungal profiles in various milk thistle botanicals from US retail
International Journal of Food Microbiology 164 (2013) 87–91
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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
Fungal profiles in various milk thistle botanicals from US retail V.H. Tournas a,⁎, J. Rivera Calo b, C. Sapp c a b c
Center for Food Safety and Applied Nutrition/Food and Drug Administration, College Park, MD, USA University of Arkansas, Fayetteville, AR, USA Johns Hopkins School of Medicine, Baltimore, MD, USA
a r t i c l e
i n f o
Article history: Received 26 December 2012 Received in revised form 21 March 2013 Accepted 31 March 2013 Available online 6 April 2013 Keywords: Milk thistle botanicals Fungal contaminants Enumeration Identification
a b s t r a c t Milk thistle (MT) dietary supplements are widely consumed due to their possible beneficial effect on liver health. As botanicals, they can be contaminated with a variety of fungi and their secondary metabolites, mycotoxins. This study was conducted in an effort to determine the mycological quality of various MT botanical supplements from the US market. Conventional plating methods were used for the isolation and enumeration of fungi, while conventional microscopy as well as molecular methods were employed for the speciation of the isolated strains. Results showed that a high percentage of the MT samples tested were contaminated with fungi. Total counts ranged between b 2.00 and 5.60 log10 colony forming units per gram (cfu/g). MT whole seeds carried the highest fungal levels followed by MT cut herb. No live fungi were recovered from MT seed tea bags, liquid extracts, capsules or soft gels. Potentially toxigenic molds from the Aspergillus sections Flavi and Nigri as well as Eurotium, Penicillium, Fusarium and Alternaria species were isolated from MT supplements. The predominant molds were Eurotia (E. repens, E. amstelodami and E. rubrum), A. flavus, A. tubingensis, A. niger and A. candidus. To our knowledge, this is the first study reporting on fungal contamination profiles of MT botanicals. Published by Elsevier B.V.
1. Introduction Various milk thistle (MT) derivatives have been used as healthpromoting remedies for centuries; these products were consumed as far back as ancient Greece and China. The edible part of thistle was named Silybum by the ancient Greek physician, Dioscorides. In recent years, the use of MT botanical supplements by consumers in the Western Societies has increased markedly due to a trend for use of natural therapies. In the US the marketing and consumption of various dietary supplements including milk thistle derivatives have greatly increased after the enactment of the Dietary Supplement Health and Education Act (DSHEA) (USFDA, 1994). The main health-promoting property of MT supplements is the support of liver health due to their high content of silymarin (a complex of flavonolignans and polyphenols containing silibin, isosilibin, silichristin and silidianin) (Morazzoni and Bombardelli, 1995; Saller et al., 2001). Other potentially-beneficial attributes of MT botanicals include anti-hypercholesterolemic and anti-oxidant properties, and chemoprotective effects against lung and prostate cancers (Wellington and Jarvis, 2001; Skottova and Krecman, 1998; Singh et al., 2006, 2008). Contamination of various botanical supplements with potentially toxigenic fungi has been occasionally reported in the literature. Trucksess and Scott (2008) mentioned the presence of penicillia, Aspergillus flavus and A. parasiticus in botanicals, while Rizzo et al. (2004) documented the ⁎ Corresponding author. E-mail address: [email protected] (V.H. Tournas). 0168-1605/$ – see front matter. Published by Elsevier B.V. http://dx.doi.org/10.1016/j.ijfoodmicro.2013.03.026
occurrence of Fusarium, A. flavus and A. parasiticus in Argentinean medicinal plants. Sato et al. (1992) recovered several molds including Alternaria alternata and Fusarium spp. from medicinal plants, while Raman et al. (2004) reported that several botanical supplements from the US market contained molds and bacteria. Mold secondary metabolites, mycotoxins (e.g. aflatoxins, fumonisins, ochratoxin A, etc.) have also been reported in past studies. Aflatoxins were found in traditional Chinese herbal medicines, medicinal plants and in kava kava (Yang et al., 2005; Selim et al., 1996; Weaver and Trucksess, 2010). Fumonisins have been detected in black tea and in medicinal plants (Martins et al., 2001; Omurtag and Yazicioglu, 2004), while the presence of ochratoxin A in medicinal plant materials from Tilia grandifolia was reported by Halt (1998). A study on the occurrence of aflatoxins (AFs) in MT supplements revealed the presence of these toxins at low levels (0.04–2.00 ng/g) in MT seeds and oil-based liquid seed extracts (Tournas et al., 2012). Further studies by the same investigators showed that certain A. flavus strains could produce high levels of AFs in artificially-infected MT seed powder and lesser amounts in MT herb powders. The mycological quality of MT supplements, however, has not been investigated. Preliminary tests had shown that about 25% of the MT samples tested contained potential aflatoxin-producing Aspergillus section Flavi isolates (sometimes at levels exceeding 4.50 log10 cfu/g). Other toxin producing molds with similar growth requirements (environmental and nutritional) could also be present in these commodities. Therefore, we conducted this study to investigate the presence and levels of potentially-toxigenic molds in MT botanical supplements.
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2. Materials and methods 2.1. MT sample preparation Organic MT supplements (whole and powdered seed, 113.5 or 454 g/sample; cut and powdered herb, 113.5 or 454 g/sample; ground seed in tea bags, 30 g/sample; oil-based liquid seed extracts, 30 ml/sample; alcohol-based seed extracts, 120 ml/sample; capsules, 100 units/sample; and soft gels, 60 units/sample) were purchased from commercial sources. Samples were mixed well in their sealed containers before the portions for analysis were taken out as follows: liquid samples were mixed in a vortex mixer for 1 min; seed and herb powders, whole seeds and minced herb were manually shaken 10 times in a 180° angle. 2.2. Chemicals, reagents and other supplies PCR reagents and DNA ladder were purchased from Fisher Scientific (Houston, TX, USA); DNA extraction kits were obtained from Norgen Biotek Corp. (Thorold, ON, Canada); and primers were purchased from Integrated DNA Technologies (IDT) (Coraville, IA, USA). Agarose gels were obtained from Bio-Rad (Hercules, CA, USA). PD broth, PBS and all the mycological media utilized for the isolation and conventional plating identification of fungal specimens were prepared in-house according to procedures and formulas described in the Bacteriological Analytical Manual (BAM online, 2001) and in Fungi and Food Spoilage (Pitt and Hocking, 2009). 2.3. Fungal isolation and enumeration Isolation and enumeration of viable fungi were accomplished following the method described in BAM online (2001) as follows: twenty five-gram portions from each MT seed and herb sample were aseptically transferred into sterile blender jars and 225 ml of 0.1% peptone water was added to each jar. Ten-gram portions from each MT extract, capsule, soft gel and tea bag sample (+90 ml 0.1% peptone) were analyzed. The samples were blended for 45 s to achieve complete homogenization. Serial dilutions were prepared by adding 1 ml of homogenate to 9 ml 0.1% peptone water. Subsequently, 0.1 ml portions were inoculated onto duplicate pre-poured DG18 agar plates. The inoculum was spread with sterile bent glass rods and the plates were incubated upright at 25 °C for 5 days. Then plates containing 10–150 colonies were counted and the yeast and mold (YM) counts were calculated as described in BAM online (2001). Counts were expressed as colony forming units per gram (cfu/g).
30 °C for 24 h. After the incubation period, samples were centrifuged for 10 min at 10,000 rpm to pellet and the supernatants were discarded. Subsequently, 10 ml of PBS buffer were added to each tube and the pellets were homogenized using a vortex. The tubes were centrifuged again under the same as above conditions and the supernatants were discarded. Then, 1 ml of PBS buffer was added to each sample and the tubes were vortexed. Subsequently, 50 μl were removed from each tube and transferred to 2.0-ml microcentrifuge tubes to proceed with DNA extraction. DNA extraction was facilitated using the Norgen Biotek Fungi/Yeast Genomic DNA Isolation Kit according to manufacturer's instructions. 2.5.2. PCR The primers encoding the β-tubulin gene, Bt2a (5′-GGTAACCAAATCG GTGCTGCTTTC-3′) and Bt2b (5′-ACCCTCAGTGTAGTGACCCTTGGC-3′) (Glass and Donaldson, 1995) were used and a slight modification of the method described by Asefa et al. (2009) was utilized as follows: PCR was performed in reaction tubes with a final volume of 50 μl consisting of 25 μl Promega GoTaq Hot Start colorless master mix, 21.5 μl Promega nuclease-free water, 1.5 μl DNA template, and 1 μl of each primer. The mixture was spun and the PCR reaction was run in an Eppendorf 2231 thermocycler (Eppendorf North America, Hauppauge, NY, USA) programmed with the following conditions: denaturation at 95 °C for 10 min, 38 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 1 min, and final extension at 72 °C for 7 min. 2.5.3. Gel electrophoresis The PCR products were subjected to electrophoresis in 1% agarose gels (Bio-Rad Ready Agarose ethidium bromide precast gels) to determine the band sizes and to confirm amplification of the DNA. Gels were run using Tris–borate–EDTA (TBE) buffer, at 60 V for 70 min and subsequently, were visualized and photographed under UV light in an Alpha Imager (Alpha Innotech Corp., Santa Clara, CA, USA). 2.5.4. Sequencing and species identification Purification and sequencing of the PCR products were performed by MCLAB (South San Francisco, CA, USA) and the resulted sequences were trimmed and edited using FinchTV 1.4.0 software. Subsequently, sequence comparisons and speciation were done by utilizing the basic local alignment search tool (BLAST) in Genbank (www.ncbi. nlm.nih.gov/BLAST). 3. Results and discussion 3.1. Yeast and mold (YM) levels
2.4. Speciation of isolated fungal strains Recovered isolates were purified by re-culturing on PDA (DIFCO, Detroit, MI, USA) agar plates and identified to genus or species level using the conventional methods and keys described in “Identification of Common Aspergillus Species” (Klich, 2002), “A Laboratory Guide to Common Penicillium Species” (Pitt, 1985), “Fusarium Species: An Illustrated Manual for Identification” (Nelson et al., 1983), “Fungi and Food Spoilage” (Pitt and Hocking, 2009) and “Introduction to Foodand Airborne Fungi” (Samson et al., 2004). Isolates, that could not be speciated by conventional plating methods, were identified using molecular techniques as described below. 2.5. Molecular method 2.5.1. DNA extraction DNA from fungal strains recovered from MT supplements was isolated as follows: the fungal isolates were grown on solid media at 25 °C for 5 days; then a small amount from each isolate was transferred to a 15-ml conical tube containing potato dextrose broth and incubated at
A total of 223 fungal strains (214 molds and 9 yeasts) were isolated from the MT supplements tested in this study. Sixty percent of the MT samples tested here were contaminated with fungi. Eighty eight percent of whole seed, 100% of seed powder, 57% of cut herb and 100% of herb powder samples had YM levels higher than 2.00 log10 cfu/g, the limit recommended by the US Pharmacopeia (USP, 2011). The highest levels were observed in whole seed samples (up to 5.60 log10 cfu/g) followed by cut herb (up to 4.59 log10 cfu/g). The powdered samples had somewhat lower YM counts reaching as high as 4.34 and 4.36 log10 cfu/g for seed and herb powders, respectively. All MT herb powder samples, however, carried higher than 4.00 log10 cfu/g. No molds or yeasts were recovered from MT tea bags, liquid seed extracts, capsules or soft gels (Table 1). 3.2. Potentially-toxigenic molds Potentially-toxigenic molds from the Aspergillus sections Flavi and Nigri as well as Eurotium, Penicillium, Fusarium and Alternaria spp. were isolated from MT supplements. The predominant molds were
V.H. Tournas et al. / International Journal of Food Microbiology 164 (2013) 87–91 Table 1 Fungal contamination of various MTa botanicals. MT supplement type
Frequencyd Number of samples YMb counts range (log10 cfuc/g) tested
Whole seed Seed powder Cut herb Herb powder Tea bags Alcohol-based seed extract Oil-based seed extract Capsules Soft gels
34 14 14 7 4 2 8 13 6
a b c d e
2.18–5.60 2.70–4.34 2.00–4.59 4.04–4.36 NDe ND ND ND ND
88 100 71 100 0 0 0 0 0
MT = milk thistle. YM = yeast and mold. cfu = colony forming units. Percent contaminated samples. ND = none detected.
eurotia (E. rubrum, E. repens and E. amstelodami), aspergilli (A. flavus, A. tubingensis, A. versicolor, A. foetidus, A. candidus and A. tritici) and Alternaria spp. Fusarium, Penicillium and Cladosporium spp. were isolated less often (Table 2). A. flavus was found in 29% of the whole seed and 43% of the seed powder samples but was absent from all MT herb samples, while A. parasiticus was isolated only from one herb powder sample. The presence of these organisms is of concern because they have the potential of producing aflatoxins in a wide variety of substrates. Low levels (0.04–2.04 ng/g) of aflatoxins in MT
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seed samples and oil-based extract were reported by Tournas et al. (2012). The same investigators also reported that an A. parasiticus strain isolated from MT herb produced high amounts of aflatoxins (above 49,000 ng/g total AFs) in artificially-contaminated rice and MT seed powder, while a toxigenic A. flavus isolate recovered from MT seed only produced AFs in rice. A. candidus was recovered from 15% of the whole seed, 14% of the seed powder and 21% of the herb powder samples and its counts ranged between 2.18 and 4.96 log10 cfu/g. Twelve percent of the whole seed samples had A. candidus levels ≥4.00 log10 cfu/g. Such elevated counts indicate established growth of the organism on the commodity. This organism is associated with the production of highly cytotoxic secondary metabolites (e.g. terpenins) which have immunomodulating properties (Krysinska-Traczyk and Dutkiewicz, 2000). There are also reports suggesting that some A. candidus strains may be able to produce citrinin — a mycotoxin associated with kidney disease (EMAN, 2012). Another member of the Aspergillus section Candidi, A. tritici, was also encountered in one MT seed powder and two whole seed samples. The A. tritici levels in whole seeds were quite high (over 4.00 log10 cfu/g). Although it is not known if this organism produces mycotoxins, it grew at 37 °C and could possibly cause infections in humans, especially when it is present at high levels. A. penicillioides was found in 15% of the whole seed and 7% of seed powder samples. One whole seed sample had 5.04 log10 cfu/g while two others (6%) contained over 3.00 log10 cfu/g. A. penicillioides levels in MT herb were lower than in seed samples. This organism has not been reported to produce mycotoxins. A. versicolor was found in both, seed and herb, samples. Its highest levels (5.08 log10 cfu/g) were encountered in
Table 2 Fungal species and levels recovered from various MTa botanical supplements. Organism
Alternaria spp. Aspergillus spp. Aspergillus awamori Aspergillus candidus Aspergillus flavus Aspergillus foetidus Aspergillus niger Aspergillus ochraceus Aspergillus parasiticus Aspergillus penicillioides Aspergillus sydowii Aspergillus tamarii Aspergillus tritici Aspergillus tubingensis Aspergillus versicolor Cladosporium spp. Eurotium spp. Eurotium amstelodami Eurotium chevalieri Eurotium repens Eurotium rubrum Fusarium spp. Fusarium proliferatum Fusarium subglutinans Fusarium verticillioides Penicillium spp. Penicillium brevicompactum Penicillium chrysogenum Penicillium dierckxii Penicillium polonicum Rhizopus spp. Yeasts No growth
Contamination levels (log10 cfub/g-range) in MT supplements Whole seeds (n = 34)c
Seed powder (n = 14)
Cut herb (n = 14)
Herb powder (n = 7)
2.30–4.30 (15)d 2.00–2.30 (6) 2.0 (3) 2.74–4.96 (15) 2.00–5.51 (29) 2.00–5.23 (6) 2.00–4.54 (12) ND ND 2.40–5.04 (15) 3.08 (3) 3.30–3.72 (6) 4.11–4.41 (6) 2.65 (3) 2.30–5.08 (15) ND 3.00–4.00 (9) 2.00–5.08 (12) ND 2.30–5.40 (29) 2.30–4.63 (26) 2.30–4.32 (18) 2.95–4.48 (6) ND 2.00 (3) 2.48–3.00 (6) ND 2.00–4.95 (21) ND 2.00 (3) 2.00–3.78(15) 2.65–4.38 (6) (12)
2.30–2.30 (14) 2.00–2.54 (14) ND 2.54–3.11 (14) 2.40–3.48 (43) ND ND ND ND 3.85 (7) ND 2.30 (7) 3.34 (7) 2.30–3.48 (14) 2.00–2.30 (14) 2.90 (7) 2.85 (7) 2.30–3.95 (36) 2.48–2.70 (14) 2.30–3.18 (21) 2.00–3.30 (29) 2.48–3.20 (29) 2.00 (7) 2.48–3.08 (14) ND 2.00–2.30 (14) 2.48 (7) 2.00–2.60 (14) ND ND 2.00 (7) 2.00–3.15 (36) (0)
NDe 2.30 (7) ND 2.18–3.11 (21) ND ND 2.00 (7) 2.60 (7) ND ND ND ND ND 2.00–4.38 (14) 2.30 (7) ND 2.00 (7) 2.00–3.85 (14) 3.48 (7) 2.95–4.18 (29) 2.00–3.30 (50) 2.60 (7) ND ND ND 3.32 (7) 2.00 (7) ND ND ND 3.00 (7) 3.30–4.57 (14) (29)
ND 2.00–3.70 (57) ND ND ND 3.20–3.26 (29) 3.34 (14) ND 3.30 (14) ND ND ND ND 3.30–3.95 (29) 3.20 (14) ND 3.85–3.85 (29) 3.30 (14) ND 3.74–3.95 (57) 3.30–4.00 (57) ND ND ND ND ND ND ND 3.30–3.60 (43) ND ND 3.30 (14) (0)
Aspergilli and eurotia were identified using PCR; all other species were identified by conventional plating methods. a MT = milk thistle. b cfu = colony forming units. c n = number of samples tested. d Numbers in parentheses indicate % samples contaminated with respective organisms. e ND = none detected.
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whole seeds. This organism also has the potential of producing the mutagenic toxins, sterigmatocystin, and versicolorin (Engelhart et al., 2002; Frisvad et al., 2006; Jakšić et al., 2012) and its presence in food and feed commodities should be monitored and controlled. Black aspergilli (A. niger, A. foetidus and A. tubingensis) were isolated from both MT seed and herb samples. The highest levels of A. niger (4.54 log10 cfu/g) and A. foetidus (5.23 log10 cfu/g) were found in whole seeds, whereas the highest concentration of A. tubingensis (4.38 log10 cfu/g) was encountered in cut herb. Some black aspergilli have the ability to produce the mycotoxin, ochratoxin A, which was linked to kidney disease in humans (Perrone et al., 2006). Therefore, their presence at high levels in consumer products is a cause of concern. Some eurotia (i.e. E. amstelodami, E. rubrum and E. repens) were found in high concentrations in MT seeds. The most abundant was E. rubrum found in 52% of the MT herb and 27% of the seed samples at levels ranging between 2.00 and 4.63 log10 cfu/g. E. repens was also quite common, found in 27% of the seed and in 38% of the herb samples. One whole seed sample had E. repens counts of 5.40 log10 cfu/g. E. amstelodami was encountered in 19% of the seed and 14% of the herb samples. The highest E. amstelodami counts (5.08 log10 cfu/g) were observed in a whole seed sample. This organism was found to exert toxic effects on chicken embryos (Frisvad and Samson, 1991). Most of the Penicillia recovered during the course of this study were encountered at low levels and they were probably random contaminants. Two whole seed samples, however, had very high levels (above 4.25 log10 cfu/g) of P. chrysogenum. This organism has the ability of producing antibiotics and some isolates could also elaborate the mycotoxin, citrinin (Devi et al., 2009). Fusarium spp. were isolated from a few MT seed and herb samples at relatively low levels, but one whole seed sample had 4.48 log10 cfu/g of F. proliferatum. This level is quite high and considering the fact that F. proliferatum has the potential of producing mycotoxins such as fumonisins (Desjardins et al., 2007), its presence in MT supplements is a cause of concern. Alternaria species were recovered only from 15% of the MT whole seed and 14% of the seed powder samples, but two whole seed samples contained high numbers of this organisms (≥ 4.00 log10 cfu/g). Such levels are troublesome because some Alternaria species could produce a variety of mycotoxins (e.g. alternariol, alternariol methyl ether, altenuenes, tenuazonic acid, etc.) which cause a wide spectrum of adverse effects on humans and animals (Tournas and Stack, 2001). The counts of potentially-toxigenic molds such as A. flavus, A. versicolor, A. candidus and Fusarium spp. recovered from some samples were above 4.0 log10 cfu/g; this fact suggests that the organisms were growing on the product at some stage of production or marketing. Although there are no reports on the mycological quality of MT supplements, Aspergillus, Penicillium and Fusarium species were previously isolated from dried chamomile (herb and flowers), a member of the same family as milk thistle and perhaps of similar biochemical composition as the MT supplements analyzed in this study (Tournas and Katsoudas, 2008). A. flavus, A. parasiticus, A. niger, F. oxysporum and various Penicillia were also isolated from chamomile and other medicinal plants by Aziz et al. (1998). The mold incidence reported in this study was high, sometimes occurring in 100% of the samples tested. Most of the molds recovered during the course of our study thrive under reduced water activity (aw) conditions. Since the predominant species were xerophilic, it appears that growth took place when the products were incompletely dried or came in contact with and acquired some moisture after complete drying, during storage. Therefore, in order to avoid fungal growth and possible mycotoxin production, care should be taken to quickly dry the MT botanicals after harvest and store them under such conditions that will prevent increase of aw. Since some of the contaminants could have been added to the products during and after harvest (during cleaning, washing, drying, transport, packing, etc.), adherence to good manufacturing practices (GMPs) at
all postharvest stages of production would contribute to minimizing microbial contamination and substantially improving product quality. 3.3. Yeasts Overall, yeasts were recovered from 10% of the tested samples. Yeast counts reached as high as 4.57 log10 cfu/g. Relatively high levels of viable yeasts (≥3.0 log10 cfu/g) were recovered from 14% of herb powder, cut herb and seed powder samples, and from 3% of the whole seed samples. Some of the yeasts isolated in the present study probably originated from the personnel handling the commodity during harvesting, processing and packaging and could indicate lack of strict adherence to GMPs; such yeast species could be opportunistic human pathogens and should be eliminated from the final product (Hazen, 1995). Other species are part of the natural plant microbiota, residing in various plant parts (during plant growth) where they can attach and acquire nutrients from the plant without causing disease. 4. Conclusions A high percentage of the tested MT samples contained potentiallytoxigenic molds (i.e. A. flavus, A. parasiticus, A. niger, A. versicolor and various penicillia and fusaria) at levels above 3.0 log10 cfu/g. Most of the isolated species grew well at low aw; this perhaps indicates that mold growth took place after harvest when the products were partially dried but they still retained enough moisture to support growth of xerophilic species. Therefore, these botanicals should be cleaned and dried quickly after harvest and packaged and stored under such conditions that would not allow increase of aw above 0.65 in order to prevent mold growth and mycotoxin production. Acknowledgments We are grateful to Eugenia Katsoudas and Jeffrey Kohn (Office of Regulatory Affairs/FDA) for their constructive suggestions during the preparation of this manuscript. References Asefa, D.T., Gjerde, R.O., Sidhu, M.S., Solveig Langsrud, S., Kure, C.F., Nesbakken, T., Skaar, I., 2009. Moulds contaminants on Norwegian dry-cured meat products. International Journal of Food Microbiology 128, 435–439. Aziz, N.H., Youssef, Y.A., El-Fouly, M.Z., Moussa, L.A., 1998. Contamination of some common medicinal plant samples and spices by fungi and their mycotoxins. Botanical Bulletin of Academia Sinica 39, 279–285. BAM Online, 2001. Bacteriological Analytical Manual. http://www.cfsan.fda.gov/~ebam/ bam-toc.html. Desjardins, A.E., Busman, M., Proctor, R.H., Stessman, R., 2007. Wheat kernel black point and fumonisin contamination by Fusarium proliferatum. Food Additives and Contaminants 24, 1131–1137. Devi, P., DeSouza, L., Kamat, T., Rodrigues, C., Naik, C.G., 2009. Batch culture fermentation of Penicillium chrysogenum and a report on the isolation, purification, identification and antibiotic activity of citrinin. Indian Journal of Marine Sciences 38, 38–44. EMAN, 2012. European Mycotoxins Awareness Network: Citrinin. http://mycotoxins.org/. Engelhart, S., Loock, A., Skutlarek, D., Sagunski, H., Lommel, A., Färber, H., Exner, M., 2002. Occurrence of toxigenic Aspergillus versicolor isolates and sterigmatocystin in carpet dust from damp indoor environments. Applied and Environmental Microbiology 68, 3886–3890. Frisvad, J.C., Samson, R.A., 1991. Mycotoxins produced by species of Penicillium and Aspergillus occurring in cereals. In: Chelkowski, J. (Ed.), Cereal Grains: Mycotoxins. Fungi and Quality of Drying and Storage. Elsevier, Amsterdam, pp. 441–476. Frisvad, J.C., Nielsen, K.F., Samson, R.A., 2006. Recommendations Concerning the Chronic Problem of Misidentification of Mycotoxigenic Fungi Associated With Foods and Feeds. http://bilder.buecher.de/zusatz/20/20752/20752598_lese_1.pdf. Glass, N.L., Donaldson, G.C., 1995. Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Applied and Environmental Microbiology 61, 1323–1330. Halt, M., 1998. Moulds and mycotoxins in herb tea and medicinal plants. European Journal of Epidemiology 14, 269–274. Hazen, K.C., 1995. New and emerging yeast pathogens. Clinical Microbiology Reviews 8, 462–478.
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Sato, T., Matsuhashi, M., Iida, O., 1992. Fungi isolated from diseased medicinal plants. Eisei Shikenjo Hōkoku 110, 60–66. Selim, M.I., Popendorf, W., Ibrahim, M.S., El Sharkawy, S., El Kashory, E.S., 1996. Aflatoxin B1 in common Egyptian foods. Journal of AOAC International 79, 1124–1129. Singh, R.P., Deep, G., Chittezhath, M., Kaur, M., Dwyer-Nield, L.D., Malkinson, A.M., Agarwal, R., 2006. Effect of silibinin on the growth and progression of primary lung tumors in mice. Journal of the National Cancer Institute 98, 846–855. Singh, R.P., Raina, K., Sharma, G., Agarwal, R., 2008. Silibinin inhibits established prostate tumor growth, progression, invasion, and metastasis and suppresses tumor angiogenesis and epithelial–mesenchymal transition in transgenic adenocarcinoma of the mouse prostate model mice. Clinical Cancer Research 4, 7773–7780. Skottova, N., Krecman, V., 1998. Silymarin as a potential hypocholesterolaemic drug. Physiological Research 47, 1–7. Tournas, V.H., Katsoudas, E.J., 2008. Microbiological quality of various medicinal herbal teas and coffee substitutes. Microbiology Insights 1, 47–55. Tournas, V.H., Stack, M.E., 2001. Production of alternariol and alternariol methyl ether by Alternaria alternata grown on fruits at various temperatures. Journal of Food Protection 64, 528–532. Tournas, V.H., Sapp, C., Trucksess, M.W., 2012. Occurrence of aflatoxins in milk thistle herbal supplements. Food Additives and Contaminants 29, 994–999. Trucksess, M.W., Scott, P.M., 2008. Mycotoxins in botanicals and dried fruits: a review. Food Additives and Contaminants 25, 181–192. US Food and Drug Administration, 1994. Dietary Supplement Health and Education Act of 1994.USFDA, College Park, Maryland, USA. USP, 2011. United States Pharmacopeia. The United States Pharmacopeial Convention. Rockville, MD, USA, pp. 1199–1202. Weaver, C.M., Trucksess, M.W., 2010. Determination of aflatoxin in botanical roots by a modification of AOAC Official Method 991.31: single-laboratory validation. Journal of AOAC International 93, 184–189. Wellington, K., Jarvis, B., 2001. Silymarin: a review of its clinical properties in the management of liver disorders. BioDrugs 15, 465–489. Yang, M.-H., Chen, J.-M., Zhang, X.-H., 2005. Immunoaffinity column clean-up and liquid chromatography with post-column derivatization for analysis of aflatoxins in traditional Chinese medicine. Chromatographia 62, 499–504.
Contents lists available at SciVerse ScienceDirect
International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
Fungal profiles in various milk thistle botanicals from US retail V.H. Tournas a,⁎, J. Rivera Calo b, C. Sapp c a b c
Center for Food Safety and Applied Nutrition/Food and Drug Administration, College Park, MD, USA University of Arkansas, Fayetteville, AR, USA Johns Hopkins School of Medicine, Baltimore, MD, USA
a r t i c l e
i n f o
Article history: Received 26 December 2012 Received in revised form 21 March 2013 Accepted 31 March 2013 Available online 6 April 2013 Keywords: Milk thistle botanicals Fungal contaminants Enumeration Identification
a b s t r a c t Milk thistle (MT) dietary supplements are widely consumed due to their possible beneficial effect on liver health. As botanicals, they can be contaminated with a variety of fungi and their secondary metabolites, mycotoxins. This study was conducted in an effort to determine the mycological quality of various MT botanical supplements from the US market. Conventional plating methods were used for the isolation and enumeration of fungi, while conventional microscopy as well as molecular methods were employed for the speciation of the isolated strains. Results showed that a high percentage of the MT samples tested were contaminated with fungi. Total counts ranged between b 2.00 and 5.60 log10 colony forming units per gram (cfu/g). MT whole seeds carried the highest fungal levels followed by MT cut herb. No live fungi were recovered from MT seed tea bags, liquid extracts, capsules or soft gels. Potentially toxigenic molds from the Aspergillus sections Flavi and Nigri as well as Eurotium, Penicillium, Fusarium and Alternaria species were isolated from MT supplements. The predominant molds were Eurotia (E. repens, E. amstelodami and E. rubrum), A. flavus, A. tubingensis, A. niger and A. candidus. To our knowledge, this is the first study reporting on fungal contamination profiles of MT botanicals. Published by Elsevier B.V.
1. Introduction Various milk thistle (MT) derivatives have been used as healthpromoting remedies for centuries; these products were consumed as far back as ancient Greece and China. The edible part of thistle was named Silybum by the ancient Greek physician, Dioscorides. In recent years, the use of MT botanical supplements by consumers in the Western Societies has increased markedly due to a trend for use of natural therapies. In the US the marketing and consumption of various dietary supplements including milk thistle derivatives have greatly increased after the enactment of the Dietary Supplement Health and Education Act (DSHEA) (USFDA, 1994). The main health-promoting property of MT supplements is the support of liver health due to their high content of silymarin (a complex of flavonolignans and polyphenols containing silibin, isosilibin, silichristin and silidianin) (Morazzoni and Bombardelli, 1995; Saller et al., 2001). Other potentially-beneficial attributes of MT botanicals include anti-hypercholesterolemic and anti-oxidant properties, and chemoprotective effects against lung and prostate cancers (Wellington and Jarvis, 2001; Skottova and Krecman, 1998; Singh et al., 2006, 2008). Contamination of various botanical supplements with potentially toxigenic fungi has been occasionally reported in the literature. Trucksess and Scott (2008) mentioned the presence of penicillia, Aspergillus flavus and A. parasiticus in botanicals, while Rizzo et al. (2004) documented the ⁎ Corresponding author. E-mail address: [email protected] (V.H. Tournas). 0168-1605/$ – see front matter. Published by Elsevier B.V. http://dx.doi.org/10.1016/j.ijfoodmicro.2013.03.026
occurrence of Fusarium, A. flavus and A. parasiticus in Argentinean medicinal plants. Sato et al. (1992) recovered several molds including Alternaria alternata and Fusarium spp. from medicinal plants, while Raman et al. (2004) reported that several botanical supplements from the US market contained molds and bacteria. Mold secondary metabolites, mycotoxins (e.g. aflatoxins, fumonisins, ochratoxin A, etc.) have also been reported in past studies. Aflatoxins were found in traditional Chinese herbal medicines, medicinal plants and in kava kava (Yang et al., 2005; Selim et al., 1996; Weaver and Trucksess, 2010). Fumonisins have been detected in black tea and in medicinal plants (Martins et al., 2001; Omurtag and Yazicioglu, 2004), while the presence of ochratoxin A in medicinal plant materials from Tilia grandifolia was reported by Halt (1998). A study on the occurrence of aflatoxins (AFs) in MT supplements revealed the presence of these toxins at low levels (0.04–2.00 ng/g) in MT seeds and oil-based liquid seed extracts (Tournas et al., 2012). Further studies by the same investigators showed that certain A. flavus strains could produce high levels of AFs in artificially-infected MT seed powder and lesser amounts in MT herb powders. The mycological quality of MT supplements, however, has not been investigated. Preliminary tests had shown that about 25% of the MT samples tested contained potential aflatoxin-producing Aspergillus section Flavi isolates (sometimes at levels exceeding 4.50 log10 cfu/g). Other toxin producing molds with similar growth requirements (environmental and nutritional) could also be present in these commodities. Therefore, we conducted this study to investigate the presence and levels of potentially-toxigenic molds in MT botanical supplements.
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2. Materials and methods 2.1. MT sample preparation Organic MT supplements (whole and powdered seed, 113.5 or 454 g/sample; cut and powdered herb, 113.5 or 454 g/sample; ground seed in tea bags, 30 g/sample; oil-based liquid seed extracts, 30 ml/sample; alcohol-based seed extracts, 120 ml/sample; capsules, 100 units/sample; and soft gels, 60 units/sample) were purchased from commercial sources. Samples were mixed well in their sealed containers before the portions for analysis were taken out as follows: liquid samples were mixed in a vortex mixer for 1 min; seed and herb powders, whole seeds and minced herb were manually shaken 10 times in a 180° angle. 2.2. Chemicals, reagents and other supplies PCR reagents and DNA ladder were purchased from Fisher Scientific (Houston, TX, USA); DNA extraction kits were obtained from Norgen Biotek Corp. (Thorold, ON, Canada); and primers were purchased from Integrated DNA Technologies (IDT) (Coraville, IA, USA). Agarose gels were obtained from Bio-Rad (Hercules, CA, USA). PD broth, PBS and all the mycological media utilized for the isolation and conventional plating identification of fungal specimens were prepared in-house according to procedures and formulas described in the Bacteriological Analytical Manual (BAM online, 2001) and in Fungi and Food Spoilage (Pitt and Hocking, 2009). 2.3. Fungal isolation and enumeration Isolation and enumeration of viable fungi were accomplished following the method described in BAM online (2001) as follows: twenty five-gram portions from each MT seed and herb sample were aseptically transferred into sterile blender jars and 225 ml of 0.1% peptone water was added to each jar. Ten-gram portions from each MT extract, capsule, soft gel and tea bag sample (+90 ml 0.1% peptone) were analyzed. The samples were blended for 45 s to achieve complete homogenization. Serial dilutions were prepared by adding 1 ml of homogenate to 9 ml 0.1% peptone water. Subsequently, 0.1 ml portions were inoculated onto duplicate pre-poured DG18 agar plates. The inoculum was spread with sterile bent glass rods and the plates were incubated upright at 25 °C for 5 days. Then plates containing 10–150 colonies were counted and the yeast and mold (YM) counts were calculated as described in BAM online (2001). Counts were expressed as colony forming units per gram (cfu/g).
30 °C for 24 h. After the incubation period, samples were centrifuged for 10 min at 10,000 rpm to pellet and the supernatants were discarded. Subsequently, 10 ml of PBS buffer were added to each tube and the pellets were homogenized using a vortex. The tubes were centrifuged again under the same as above conditions and the supernatants were discarded. Then, 1 ml of PBS buffer was added to each sample and the tubes were vortexed. Subsequently, 50 μl were removed from each tube and transferred to 2.0-ml microcentrifuge tubes to proceed with DNA extraction. DNA extraction was facilitated using the Norgen Biotek Fungi/Yeast Genomic DNA Isolation Kit according to manufacturer's instructions. 2.5.2. PCR The primers encoding the β-tubulin gene, Bt2a (5′-GGTAACCAAATCG GTGCTGCTTTC-3′) and Bt2b (5′-ACCCTCAGTGTAGTGACCCTTGGC-3′) (Glass and Donaldson, 1995) were used and a slight modification of the method described by Asefa et al. (2009) was utilized as follows: PCR was performed in reaction tubes with a final volume of 50 μl consisting of 25 μl Promega GoTaq Hot Start colorless master mix, 21.5 μl Promega nuclease-free water, 1.5 μl DNA template, and 1 μl of each primer. The mixture was spun and the PCR reaction was run in an Eppendorf 2231 thermocycler (Eppendorf North America, Hauppauge, NY, USA) programmed with the following conditions: denaturation at 95 °C for 10 min, 38 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 1 min, and final extension at 72 °C for 7 min. 2.5.3. Gel electrophoresis The PCR products were subjected to electrophoresis in 1% agarose gels (Bio-Rad Ready Agarose ethidium bromide precast gels) to determine the band sizes and to confirm amplification of the DNA. Gels were run using Tris–borate–EDTA (TBE) buffer, at 60 V for 70 min and subsequently, were visualized and photographed under UV light in an Alpha Imager (Alpha Innotech Corp., Santa Clara, CA, USA). 2.5.4. Sequencing and species identification Purification and sequencing of the PCR products were performed by MCLAB (South San Francisco, CA, USA) and the resulted sequences were trimmed and edited using FinchTV 1.4.0 software. Subsequently, sequence comparisons and speciation were done by utilizing the basic local alignment search tool (BLAST) in Genbank (www.ncbi. nlm.nih.gov/BLAST). 3. Results and discussion 3.1. Yeast and mold (YM) levels
2.4. Speciation of isolated fungal strains Recovered isolates were purified by re-culturing on PDA (DIFCO, Detroit, MI, USA) agar plates and identified to genus or species level using the conventional methods and keys described in “Identification of Common Aspergillus Species” (Klich, 2002), “A Laboratory Guide to Common Penicillium Species” (Pitt, 1985), “Fusarium Species: An Illustrated Manual for Identification” (Nelson et al., 1983), “Fungi and Food Spoilage” (Pitt and Hocking, 2009) and “Introduction to Foodand Airborne Fungi” (Samson et al., 2004). Isolates, that could not be speciated by conventional plating methods, were identified using molecular techniques as described below. 2.5. Molecular method 2.5.1. DNA extraction DNA from fungal strains recovered from MT supplements was isolated as follows: the fungal isolates were grown on solid media at 25 °C for 5 days; then a small amount from each isolate was transferred to a 15-ml conical tube containing potato dextrose broth and incubated at
A total of 223 fungal strains (214 molds and 9 yeasts) were isolated from the MT supplements tested in this study. Sixty percent of the MT samples tested here were contaminated with fungi. Eighty eight percent of whole seed, 100% of seed powder, 57% of cut herb and 100% of herb powder samples had YM levels higher than 2.00 log10 cfu/g, the limit recommended by the US Pharmacopeia (USP, 2011). The highest levels were observed in whole seed samples (up to 5.60 log10 cfu/g) followed by cut herb (up to 4.59 log10 cfu/g). The powdered samples had somewhat lower YM counts reaching as high as 4.34 and 4.36 log10 cfu/g for seed and herb powders, respectively. All MT herb powder samples, however, carried higher than 4.00 log10 cfu/g. No molds or yeasts were recovered from MT tea bags, liquid seed extracts, capsules or soft gels (Table 1). 3.2. Potentially-toxigenic molds Potentially-toxigenic molds from the Aspergillus sections Flavi and Nigri as well as Eurotium, Penicillium, Fusarium and Alternaria spp. were isolated from MT supplements. The predominant molds were
V.H. Tournas et al. / International Journal of Food Microbiology 164 (2013) 87–91 Table 1 Fungal contamination of various MTa botanicals. MT supplement type
Frequencyd Number of samples YMb counts range (log10 cfuc/g) tested
Whole seed Seed powder Cut herb Herb powder Tea bags Alcohol-based seed extract Oil-based seed extract Capsules Soft gels
34 14 14 7 4 2 8 13 6
a b c d e
2.18–5.60 2.70–4.34 2.00–4.59 4.04–4.36 NDe ND ND ND ND
88 100 71 100 0 0 0 0 0
MT = milk thistle. YM = yeast and mold. cfu = colony forming units. Percent contaminated samples. ND = none detected.
eurotia (E. rubrum, E. repens and E. amstelodami), aspergilli (A. flavus, A. tubingensis, A. versicolor, A. foetidus, A. candidus and A. tritici) and Alternaria spp. Fusarium, Penicillium and Cladosporium spp. were isolated less often (Table 2). A. flavus was found in 29% of the whole seed and 43% of the seed powder samples but was absent from all MT herb samples, while A. parasiticus was isolated only from one herb powder sample. The presence of these organisms is of concern because they have the potential of producing aflatoxins in a wide variety of substrates. Low levels (0.04–2.04 ng/g) of aflatoxins in MT
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seed samples and oil-based extract were reported by Tournas et al. (2012). The same investigators also reported that an A. parasiticus strain isolated from MT herb produced high amounts of aflatoxins (above 49,000 ng/g total AFs) in artificially-contaminated rice and MT seed powder, while a toxigenic A. flavus isolate recovered from MT seed only produced AFs in rice. A. candidus was recovered from 15% of the whole seed, 14% of the seed powder and 21% of the herb powder samples and its counts ranged between 2.18 and 4.96 log10 cfu/g. Twelve percent of the whole seed samples had A. candidus levels ≥4.00 log10 cfu/g. Such elevated counts indicate established growth of the organism on the commodity. This organism is associated with the production of highly cytotoxic secondary metabolites (e.g. terpenins) which have immunomodulating properties (Krysinska-Traczyk and Dutkiewicz, 2000). There are also reports suggesting that some A. candidus strains may be able to produce citrinin — a mycotoxin associated with kidney disease (EMAN, 2012). Another member of the Aspergillus section Candidi, A. tritici, was also encountered in one MT seed powder and two whole seed samples. The A. tritici levels in whole seeds were quite high (over 4.00 log10 cfu/g). Although it is not known if this organism produces mycotoxins, it grew at 37 °C and could possibly cause infections in humans, especially when it is present at high levels. A. penicillioides was found in 15% of the whole seed and 7% of seed powder samples. One whole seed sample had 5.04 log10 cfu/g while two others (6%) contained over 3.00 log10 cfu/g. A. penicillioides levels in MT herb were lower than in seed samples. This organism has not been reported to produce mycotoxins. A. versicolor was found in both, seed and herb, samples. Its highest levels (5.08 log10 cfu/g) were encountered in
Table 2 Fungal species and levels recovered from various MTa botanical supplements. Organism
Alternaria spp. Aspergillus spp. Aspergillus awamori Aspergillus candidus Aspergillus flavus Aspergillus foetidus Aspergillus niger Aspergillus ochraceus Aspergillus parasiticus Aspergillus penicillioides Aspergillus sydowii Aspergillus tamarii Aspergillus tritici Aspergillus tubingensis Aspergillus versicolor Cladosporium spp. Eurotium spp. Eurotium amstelodami Eurotium chevalieri Eurotium repens Eurotium rubrum Fusarium spp. Fusarium proliferatum Fusarium subglutinans Fusarium verticillioides Penicillium spp. Penicillium brevicompactum Penicillium chrysogenum Penicillium dierckxii Penicillium polonicum Rhizopus spp. Yeasts No growth
Contamination levels (log10 cfub/g-range) in MT supplements Whole seeds (n = 34)c
Seed powder (n = 14)
Cut herb (n = 14)
Herb powder (n = 7)
2.30–4.30 (15)d 2.00–2.30 (6) 2.0 (3) 2.74–4.96 (15) 2.00–5.51 (29) 2.00–5.23 (6) 2.00–4.54 (12) ND ND 2.40–5.04 (15) 3.08 (3) 3.30–3.72 (6) 4.11–4.41 (6) 2.65 (3) 2.30–5.08 (15) ND 3.00–4.00 (9) 2.00–5.08 (12) ND 2.30–5.40 (29) 2.30–4.63 (26) 2.30–4.32 (18) 2.95–4.48 (6) ND 2.00 (3) 2.48–3.00 (6) ND 2.00–4.95 (21) ND 2.00 (3) 2.00–3.78(15) 2.65–4.38 (6) (12)
2.30–2.30 (14) 2.00–2.54 (14) ND 2.54–3.11 (14) 2.40–3.48 (43) ND ND ND ND 3.85 (7) ND 2.30 (7) 3.34 (7) 2.30–3.48 (14) 2.00–2.30 (14) 2.90 (7) 2.85 (7) 2.30–3.95 (36) 2.48–2.70 (14) 2.30–3.18 (21) 2.00–3.30 (29) 2.48–3.20 (29) 2.00 (7) 2.48–3.08 (14) ND 2.00–2.30 (14) 2.48 (7) 2.00–2.60 (14) ND ND 2.00 (7) 2.00–3.15 (36) (0)
NDe 2.30 (7) ND 2.18–3.11 (21) ND ND 2.00 (7) 2.60 (7) ND ND ND ND ND 2.00–4.38 (14) 2.30 (7) ND 2.00 (7) 2.00–3.85 (14) 3.48 (7) 2.95–4.18 (29) 2.00–3.30 (50) 2.60 (7) ND ND ND 3.32 (7) 2.00 (7) ND ND ND 3.00 (7) 3.30–4.57 (14) (29)
ND 2.00–3.70 (57) ND ND ND 3.20–3.26 (29) 3.34 (14) ND 3.30 (14) ND ND ND ND 3.30–3.95 (29) 3.20 (14) ND 3.85–3.85 (29) 3.30 (14) ND 3.74–3.95 (57) 3.30–4.00 (57) ND ND ND ND ND ND ND 3.30–3.60 (43) ND ND 3.30 (14) (0)
Aspergilli and eurotia were identified using PCR; all other species were identified by conventional plating methods. a MT = milk thistle. b cfu = colony forming units. c n = number of samples tested. d Numbers in parentheses indicate % samples contaminated with respective organisms. e ND = none detected.
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whole seeds. This organism also has the potential of producing the mutagenic toxins, sterigmatocystin, and versicolorin (Engelhart et al., 2002; Frisvad et al., 2006; Jakšić et al., 2012) and its presence in food and feed commodities should be monitored and controlled. Black aspergilli (A. niger, A. foetidus and A. tubingensis) were isolated from both MT seed and herb samples. The highest levels of A. niger (4.54 log10 cfu/g) and A. foetidus (5.23 log10 cfu/g) were found in whole seeds, whereas the highest concentration of A. tubingensis (4.38 log10 cfu/g) was encountered in cut herb. Some black aspergilli have the ability to produce the mycotoxin, ochratoxin A, which was linked to kidney disease in humans (Perrone et al., 2006). Therefore, their presence at high levels in consumer products is a cause of concern. Some eurotia (i.e. E. amstelodami, E. rubrum and E. repens) were found in high concentrations in MT seeds. The most abundant was E. rubrum found in 52% of the MT herb and 27% of the seed samples at levels ranging between 2.00 and 4.63 log10 cfu/g. E. repens was also quite common, found in 27% of the seed and in 38% of the herb samples. One whole seed sample had E. repens counts of 5.40 log10 cfu/g. E. amstelodami was encountered in 19% of the seed and 14% of the herb samples. The highest E. amstelodami counts (5.08 log10 cfu/g) were observed in a whole seed sample. This organism was found to exert toxic effects on chicken embryos (Frisvad and Samson, 1991). Most of the Penicillia recovered during the course of this study were encountered at low levels and they were probably random contaminants. Two whole seed samples, however, had very high levels (above 4.25 log10 cfu/g) of P. chrysogenum. This organism has the ability of producing antibiotics and some isolates could also elaborate the mycotoxin, citrinin (Devi et al., 2009). Fusarium spp. were isolated from a few MT seed and herb samples at relatively low levels, but one whole seed sample had 4.48 log10 cfu/g of F. proliferatum. This level is quite high and considering the fact that F. proliferatum has the potential of producing mycotoxins such as fumonisins (Desjardins et al., 2007), its presence in MT supplements is a cause of concern. Alternaria species were recovered only from 15% of the MT whole seed and 14% of the seed powder samples, but two whole seed samples contained high numbers of this organisms (≥ 4.00 log10 cfu/g). Such levels are troublesome because some Alternaria species could produce a variety of mycotoxins (e.g. alternariol, alternariol methyl ether, altenuenes, tenuazonic acid, etc.) which cause a wide spectrum of adverse effects on humans and animals (Tournas and Stack, 2001). The counts of potentially-toxigenic molds such as A. flavus, A. versicolor, A. candidus and Fusarium spp. recovered from some samples were above 4.0 log10 cfu/g; this fact suggests that the organisms were growing on the product at some stage of production or marketing. Although there are no reports on the mycological quality of MT supplements, Aspergillus, Penicillium and Fusarium species were previously isolated from dried chamomile (herb and flowers), a member of the same family as milk thistle and perhaps of similar biochemical composition as the MT supplements analyzed in this study (Tournas and Katsoudas, 2008). A. flavus, A. parasiticus, A. niger, F. oxysporum and various Penicillia were also isolated from chamomile and other medicinal plants by Aziz et al. (1998). The mold incidence reported in this study was high, sometimes occurring in 100% of the samples tested. Most of the molds recovered during the course of our study thrive under reduced water activity (aw) conditions. Since the predominant species were xerophilic, it appears that growth took place when the products were incompletely dried or came in contact with and acquired some moisture after complete drying, during storage. Therefore, in order to avoid fungal growth and possible mycotoxin production, care should be taken to quickly dry the MT botanicals after harvest and store them under such conditions that will prevent increase of aw. Since some of the contaminants could have been added to the products during and after harvest (during cleaning, washing, drying, transport, packing, etc.), adherence to good manufacturing practices (GMPs) at
all postharvest stages of production would contribute to minimizing microbial contamination and substantially improving product quality. 3.3. Yeasts Overall, yeasts were recovered from 10% of the tested samples. Yeast counts reached as high as 4.57 log10 cfu/g. Relatively high levels of viable yeasts (≥3.0 log10 cfu/g) were recovered from 14% of herb powder, cut herb and seed powder samples, and from 3% of the whole seed samples. Some of the yeasts isolated in the present study probably originated from the personnel handling the commodity during harvesting, processing and packaging and could indicate lack of strict adherence to GMPs; such yeast species could be opportunistic human pathogens and should be eliminated from the final product (Hazen, 1995). Other species are part of the natural plant microbiota, residing in various plant parts (during plant growth) where they can attach and acquire nutrients from the plant without causing disease. 4. Conclusions A high percentage of the tested MT samples contained potentiallytoxigenic molds (i.e. A. flavus, A. parasiticus, A. niger, A. versicolor and various penicillia and fusaria) at levels above 3.0 log10 cfu/g. Most of the isolated species grew well at low aw; this perhaps indicates that mold growth took place after harvest when the products were partially dried but they still retained enough moisture to support growth of xerophilic species. Therefore, these botanicals should be cleaned and dried quickly after harvest and packaged and stored under such conditions that would not allow increase of aw above 0.65 in order to prevent mold growth and mycotoxin production. Acknowledgments We are grateful to Eugenia Katsoudas and Jeffrey Kohn (Office of Regulatory Affairs/FDA) for their constructive suggestions during the preparation of this manuscript. References Asefa, D.T., Gjerde, R.O., Sidhu, M.S., Solveig Langsrud, S., Kure, C.F., Nesbakken, T., Skaar, I., 2009. Moulds contaminants on Norwegian dry-cured meat products. International Journal of Food Microbiology 128, 435–439. Aziz, N.H., Youssef, Y.A., El-Fouly, M.Z., Moussa, L.A., 1998. Contamination of some common medicinal plant samples and spices by fungi and their mycotoxins. Botanical Bulletin of Academia Sinica 39, 279–285. BAM Online, 2001. Bacteriological Analytical Manual. http://www.cfsan.fda.gov/~ebam/ bam-toc.html. Desjardins, A.E., Busman, M., Proctor, R.H., Stessman, R., 2007. Wheat kernel black point and fumonisin contamination by Fusarium proliferatum. Food Additives and Contaminants 24, 1131–1137. Devi, P., DeSouza, L., Kamat, T., Rodrigues, C., Naik, C.G., 2009. Batch culture fermentation of Penicillium chrysogenum and a report on the isolation, purification, identification and antibiotic activity of citrinin. Indian Journal of Marine Sciences 38, 38–44. EMAN, 2012. European Mycotoxins Awareness Network: Citrinin. http://mycotoxins.org/. Engelhart, S., Loock, A., Skutlarek, D., Sagunski, H., Lommel, A., Färber, H., Exner, M., 2002. Occurrence of toxigenic Aspergillus versicolor isolates and sterigmatocystin in carpet dust from damp indoor environments. Applied and Environmental Microbiology 68, 3886–3890. Frisvad, J.C., Samson, R.A., 1991. Mycotoxins produced by species of Penicillium and Aspergillus occurring in cereals. In: Chelkowski, J. (Ed.), Cereal Grains: Mycotoxins. Fungi and Quality of Drying and Storage. Elsevier, Amsterdam, pp. 441–476. Frisvad, J.C., Nielsen, K.F., Samson, R.A., 2006. Recommendations Concerning the Chronic Problem of Misidentification of Mycotoxigenic Fungi Associated With Foods and Feeds. http://bilder.buecher.de/zusatz/20/20752/20752598_lese_1.pdf. Glass, N.L., Donaldson, G.C., 1995. Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Applied and Environmental Microbiology 61, 1323–1330. Halt, M., 1998. Moulds and mycotoxins in herb tea and medicinal plants. European Journal of Epidemiology 14, 269–274. Hazen, K.C., 1995. New and emerging yeast pathogens. Clinical Microbiology Reviews 8, 462–478.
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