Occurrence of Aspergillus chevalieri and A. niger on Herbal Tea and Their Potential to Produce Ochratoxin A (OTA)

: Herbal teas, including Camellia sinensis (black and green teas), are popular beverages with health beneﬁts for consumers worldwide. These products are prepared from natural materials of different plant parts containing antioxidant properties and vitamins. The aim of this study was to investigate fungal contaminants and their ability to produce ochratoxin A (OTA) in herbal tea samples. Seven herbal teas were obtained from local markets in Chiang Rai, northern Thailand. Samples were incubated on potato dextrose agar (PDA), and the growing mycelia were isolated into a pure culture. The cultures were identiﬁed via both morphology and molecular analysis to conﬁrm species identiﬁcation. The identiﬁed species were subjected to OTA analysis using high-performance liquid chromatography (HPLC) with a ﬂuorescence detector. Ochratoxin A was produced by Aspergillus chevalieri and A. niger , isolated from seven herbal tea samples (black tea, green tea, bael fruit, goji berry, jasmine, lavender, and rose). This ﬁnding raises concerns about the safety of herbal tea and should be investigated further for potential health implications.


Introduction
Phytotherapeutic sources have become important for healthy drink and food consumption and healthcare.Herbal teas have become popular beverages [1,2], with Camellia sinensis L. tea being the most consumed drink in the world [3].In this paper, herbal tea refers to the aromatic brewing of diverse parts of plants known as herbs (such as leaves, flowers, seeds, bark, stems, and roots) [4][5][6].
The naturally occurring bioactive substances or phytochemicals in herbal teas are released through infusion [7][8][9].These bioactive compounds include antioxidants and other therapeutic properties, while tea contains rather high amounts of caffeine [1,4,10].Some of the most popular herbal teas include bael fruit, chamomile, chrysanthemum, jasmine, lavender, marigold, pomegranate, safflower, and rose [11][12][13].In Thailand, there are several popular flower teas, such as butterfly-pea, chrysanthemum, jasmine flower, rose, roselle, or safflower.They contain color, flavor, taste, fragrance, aesthetic qualities, and antioxidant activities [14,15].However, most of the herbal teas are produced by local farmers and do not undergo quality inspections, unlike tea products processed by the industry.Some of these herbal plants have been shown to be contaminated by toxigenic fungi in Asian countries such as China, India, Sri Lanka, and Thailand [16].
Herbal tea products include several parts of dried plants, which are suitable substrates for the growth of microorganisms, especially toxicogenic fungi [16][17][18][19].In the natural environment, fungi habitually grow on organic and inorganic substrates [20][21][22].Their presence can negatively impact human health, cause infectious diseases, and contaminate food or food ingredients [23,24].They can also deteriorate agricultural food crops and products under poor post-harvest facilities [25].A large number of foodborne fungi, also known as storage fungi, are able to produce one or more toxic secondary metabolites (mycotoxins) that cause a wide array of negative effects and other complications in animals and humans [26][27][28].
Ochratoxin A, produced by species within Aspergillus and Penicillium, is one of the most significant toxins that affects agricultural products and human health worldwide [48,49].Magan and Aldred [50] reported that A. niger within section Nigri, especially A. carbonarius, can produce OTA contamination in grapes, wine, and vine fruits.Han et al. [51] reported OTA contamination in the Chinese food industry from some strains of A. niger.On the other hand, A. chevalieri produces aflatoxins, citrinin, flavoglaucin, gliotoxin, and sterigmatocystin, but OTA production has not been reported in this species [52][53][54][55].
In this study, we investigated the potential of Aspergillus species isolated from herbal tea samples from local markets in Chiang Rai Province and their ability to produce OTA. A. chevalieri and A. niger isolated from seven herbal tea samples were found to produce OTA.The species were identified and are illustrated using both morphological and molecular data.The implications of detecting these species in such products are discussed.

Samples Collection and Fungal Isolation
Herbal teas were randomly purchased from five local markets in Chiang Rai Province (Doi Mae Salong, Fah Thai, Mae Sai, and Lan Muang).They included bael fruit, Camellia sinensis (black and green teas), jasmine, goji berry, lavender, and rose (Table 1).Isolation of fungi from the samples was performed under sterile conditions following the method described by Senanayake et al. [56].One random piece of each tea sample was placed directly on potato dextrose agar (PDA) and incubated at 25 • C for 5 days.Mycelia growing from the herbal tea samples were individually transferred to fresh PDA plates to obtain pure cultures and for identification.

Macro-and Microscopic Identification of Fungi
Macroscopic and microscopic characteristics were examined by following the identification methods used in previous studies [57][58][59] and structures were measured following Senanayake et al. [56].Macro-and micro-characteristics, such as conidiophores, conidiogenous cells, and conidia, were observed and photographed using the Nikon Eclipse Ni-U compound microscope connected to the Nikon DS-Ri2 digital camera.The photoplates were prepared with Adobe Photoshop CS3 Extended version 10.0 software (Adobe Systems, San Jose, CA, USA).Specimens were deposited at the Fungarium of Mae Fah Luang University (MFLU), and living cultures were deposited at Mae Fah Luang University Culture Collection (MFLUCC), Chiang Rai, Thailand.
Polymerase chain reaction (PCR) was performed in a volume of 25 µL reaction process containing 12.5 µL of 2× Power Taq PCR Master Mix, 1 µL of each primer (20 µM), and 1 µL of 50 ng of DNA template in 9.5 µL of deionized water.PCR amplification conditions for each gene were performed following previous studies (Table 2).The PCR products were purified according to the company protocols and DNA sequencing was performed using Sanger sequencing at Solgent Co., Ltd., Daejeon, South Korea.
Phylogenetic analyses to identify fungal species were performed as described in Dissanayake et al. [69].The fungal sequence data obtained from this study were deposited in GenBank (Tables 3 and 4).Note: The ex-type cultures are marked as " T ", and "N/A" indicates sequence is unavailable.

OTA Extraction and Quantification
Isolates of A. chevalieri and A. niger were grown on yeast extract sucrose agar (YES) [68,70,71] and incubated at 25 • C in darkness for 14 days for OTA production [70].Small pieces of culture agar plugs (6 mm diameter) of each isolate were transferred to 50 mL centrifuge tubes, and 16 mL methanol (HPLC grade) was added, followed by orbital shake at 230 rpm for 60 min, vortexing at every 20 min, and centrifugation at 2683× g (5000 rpm) for 15 min [48,72].Five microliters of the solution were collected and evaporated to dryness under a nitrogen stream at 50 • C [48,73,74].The dried extracts were dissolved in 1 mL of methanol, filtered through a 0.22 µm Polyvinylidene difluoride (PVDF) membrane filter into 2 mL amber vials, and sent to the Scientific and Technological Instruments Center (STIC), Mae Fah Luang University, for HPLC analysis.The analyses were performed on a Waters HPLC System with a 2998 PDA detector, following the manufacturer's instructions.

Mycotoxin Detection
The set of isolates (Figure 5), representing the different locations from where the various teas were bought, were tested for potential toxigenicity (Table 5). A. niger strains were shown to produce OTA with values ranging between 0.328 and 1.660 ng/L, while A. chevalieri strains produced OTA in the range between 0.663 and 39.182 ng/L (Figure 5).

Mycotoxin Detection
The set of isolates (Figure 5), representing the different locations from where the various teas were bought, were tested for potential toxigenicity (Table 5). A. niger strains were shown to produce OTA with values ranging between 0.328 and 1.660 ng/L, while A chevalieri strains produced OTA in the range between 0.663 and 39.182 ng/L (Figure 5).

Discussion
In this study, 137 isolates were obtained from herbal teas and teas from local markets in northern Thailand.Eleven isolates, of which six isolates belonged to A.
Environmental factors such as temperature, air wetness, and water activity play a significant role in influencing mycotoxin production and contamination levels in pre-and post-harvest products.Various studies have highlighted the impact of these factors [89][90][91][92][93][94].Mycotoxin production is influenced not only by the genetic makeup but also by environmental conditions, such as those from northern Thailand, and potential host effects.In our study, all isolates of A. niger produced low amounts of OTA, which was probably due to environmental factors.The European Union specifies the maximum limits for ochratoxin A (OTA) in dried herbs of 10 µg/kg [95].OTA production from A. chevalieri has not previously been reported [53,89,96].OTA production was detected in six isolates of A. chevalieri in this study (Figure 5).This finding raises concerns about OTA contamination in herbal teas and highlights the need for further research and monitoring to ensure consumer safety.
In conclusion, the increasing concern over Aspergillus contaminants and mycotoxin production in teas necessitates further research and analysis of OTA to ensure consumer health and safety.Analyzing and monitoring fungal contamination in teas is essential to meet consumer expectations and demands.The discovery of OTA production by A. chevalieri underscores the importance of continued vigilance in this area.

Diversity 2023 , 18 Figure 1 .
Figure 1.Phylogram generated from RAxML analysis based on combined BenA, CaM, ITS, and RPB2 sequence data of Aspergillus section Aspergillus taxa. A. osmophilus (CBS 134258) and A. xerophilus (CBS 938.73,NRRL 6132) are selected as the outgroup taxa.Bootstrap support values for ML values equal to or >60% and BYPP values equal to or >0.90 are shown as MLBS/BYPP above the nodes.Newly generated sequences in this study are in blue.Type strains are indicated in bold.

Figure 1 .
Figure 1.Phylogram generated from RAxML analysis based on combined BenA, CaM, ITS, and RPB2 sequence data of Aspergillus section Aspergillus taxa. A. osmophilus (CBS 134258) and A. xerophilus (CBS 938.73,NRRL 6132) are selected as the outgroup taxa .Bootstrap support values for ML values equal to or >60% and BYPP values equal to or >0.90 are shown as MLBS/BYPP above the nodes.Newly generated sequences in this study are in blue.Type strains are indicated in bold.

Figure 2 .
Figure 2. Phylogram generated from RAxML analysis based on combined BenA, CaM, ITS, and RPB2 sequence data of Aspergillus section Nigri taxa. A. flavus isolates (CBS 100927 and NRRL 447) are selected as the outgroup taxa.Bootstrap support values for ML values equal to or >60% and BYPP values equal to or >0.90 are shown as MLBS/BYPP above the nodes.Newly generated sequences in this study are in blue.Type strains are indicated in bold.

Figure 2 .
Figure 2. Phylogram generated from RAxML analysis based on combined BenA, CaM, ITS, and RPB2 sequence data of Aspergillus section Nigri taxa. A. flavus isolates (CBS 100927 and NRRL 447) are selected as the outgroup taxa.Bootstrap support values for ML values equal to or >60% and BYPP values equal to or >0.90 are shown as MLBS/BYPP above the nodes.Newly generated sequences in this study are in blue.Type strains are indicated in bold.

Figure 5 .
Figure 5. Analysis of OTA production by isolates of A. chevalieri and A. niger on YES at 25 °C for 14 days.(A,D) A. chevalieri (MFLUCC 23-0184) isolated from bael fruit and A. niger isolates from green tea (MFLUCC 23-0195), respectively.(B,E) UV absorbance traces of extracts from MFLUCC 23-0184 and MFLUCC 23-0195.(C,F) Amount of OTA production by different isolates of A. chevalieri and A. niger.

Figure 5 .
Figure 5. Analysis of OTA production by isolates of A. chevalieri and A. niger on YES at 25 • C for 14 days.(A,D) A. chevalieri (MFLUCC 23-0184) isolated from bael fruit and A. niger isolates from green tea (MFLUCC 23-0195), respectively.(B,E) UV absorbance traces of extracts from MFLUCC 23-0184 and MFLUCC 23-0195.(C,F) Amount of OTA production by different isolates of A. chevalieri and A. niger.

Table 1 .
Tea and herbal teas used in this study obtained from local markets in northern Thailand.

Table 2 .
PCR amplification conditions used in the thermal cycler of each gene.

Table 3 .
GenBank and culture collection numbers of Aspergillus section Aspergillus used in the phylogenetic analysis.The newly generated sequences are indicated in blue.
Note: The ex-type cultures are marked as " T ", and "N/A" indicates sequence is unavailable.

Table 4 .
GenBank and culture collection numbers of Aspergillus section Nigri used in the phylogenetic analysis.The newly generated sequences are indicated in blue.

Table 5 .
Fungal identification of 11 isolates from seven herbal tea samples.