**1. Introduction**

Chinese herbal medicines (CHMs), which are widely used for clinical treatment and daily healthcare, have undoubtedly played a significant role in medical and healthcare industries. About 70–80% of the global population depends on herbal medicinal products in their primary healthcare [1]. The global market of medicinal plants is growing and expected to reach USD 5 trillion by 2050 [2]. The popularity of herbal products is increasing as they are considered natural or harmless [2]. Unfortunately, fungal contamination in CHMs is a concern. Since the sources of CHMs are diversified and the planting and processing are decentralized, CHMs may be contaminated by fungi at any link of the complex traditional Chinese medicine industry chain if good manufacturing procedures are not followed, and even mycotoxins may be produced by potential toxigenic fungi under suitable conditions. Cases of fungal and mycotoxin contamination associated with CHMs have been constantly reported worldwide and have attracted considerable global attention. A study on microbial contamination in 132 herbal medicines and 18 water samples used in the preparation of herbal medicines from northern Brazil showed that 31% of tested samples exceeded the safety limits for fungal growth [3]. Keter et al. assessed the risk of fungi in 100 herbal

**Citation:** Jiang, W.; Chen, X.; Guo, M.; Yu, J.; Yang, M.; Pang, X. Analysis of Fungal Microbiomes in Edible Medicinal Morindae Officinalis Radix and Alpiniae Oxyphyllae Fructus Using DNA Metabarcoding. *Foods* **2022**, *11*, 1748. https://doi.org/ 10.3390/foods11121748

Academic Editors: Dapeng Peng and Yongzhong Qian

Received: 14 May 2022 Accepted: 10 June 2022 Published: 14 June 2022

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products from the Kenyan market, and the results revealed that 69% of the samples did not meet the requirements about microbial limits shown in Pharmacopoeia. Among these, *Aspergillus* and *Penicillium* were dominant [4]. In Latvia, Reinholds et al. investigated the contamination profile of multi−mycotoxin and fungi in 140 *Camellia sinensis* and 26 herbal teas; 87% of the tea samples were positive for fungi and 42% had 1 to 16 mycotoxins, for instance, ochratoxin A (OTA), deoxynivalenol, and aflatoxins (AFs) [5]. A Poland investigation on the occurrence of OTA and fumonisins in 79 herbs and spices showed that 31% of the samples were positive for fumonisins and 49% for OTA [6]. Another investigation on the fungal and multi−mycotoxin contamination in 48 root herbs marketed in China determined that all samples were affected by fungal contamination; 37.5% were positive for AFs and 16.67% for OTA [7]. Moldy CHMs affect drug efficacy, pose threats to consumer health, affect the export trade, and cause economic losses.

Morindae Officinalis Radix (MOR, Bajitian in Chinese), namely, the dried root of *Morinda officinalis* How. (Rubiaceae), has long been used as a tonic herbal medicine for tonifying the kidney, strengthening sinew and bone, and dispelling wind−dampness [8]. MOR is also a popular dietary supplement for daily healthcare (e.g., bone protection and gynecological and andrological healthcare) [9]. The constituents extracted from MOR, e.g., anthraquinones, oligosaccharides, polysaccharides, and iridoid glycosides, have various bioactive activities, including anti-osteoporosis, anti-depressant, pro-fertility, immuneregulatory, anti-inflammation, and antioxidant effects [10–14]. Alpiniae Oxyphyllae Fructus (AOF, Yizhi in Chinese), a famed edible medicinal herb, is from the dried, ripe fruit of *Alpinia oxyphylla* Miq. AOF has the functions of warming the kidney to secure essence to reduce urination, and warming the spleen to check diarrhea and constrain spittle [8]. Contemporary research shows the presence of sesquiterpenes, polysaccharides, diarylheptanoids, flavonoids, and volatile oils in AOF [15]. AOF and its extracts exhibit neuroprotective, anti-ulcer, anti-inflammatory, and anti-hyperuricemic effects, and have been used for the treatment of dementia, ulceration, and tumors [16–18]. The consumption and demand for MOR and AOF are high, owing to their outstanding pharmaceutical properties and edible values. MOR and AOF are mainly produced in Hainan, Guangdong, and Guangxi provinces, as well as in other tropical and subtropical regions of China [19], in which the climate situation contributes to the development of fungi and the production of mycotoxins [20]. MOR and AOF are easily affected by the contamination of fungi without obeying proper harvest, processing, transportation, and storage procedures. Therefore, it is necessary to comprehensively and efficiently investigate the fungal contamination before the use of MOR and AOF. Contamination of fungi is challenging to identify because of their complex morphological and taxonomic characteristics. Thus, a method that simultaneously and effectively analyzes fungal microbiomes in MOR and AOF is urgently desired.

DNA metabarcoding, an emerging culture-independent technique, refers to highthroughput multispecies (or higher-level taxon) identification using the total and typically degraded DNA extracted from an environmental sample (i.e., soil, water, and feces) [21]. It has a wide range of applications in the study of fungal ecology and provides new insights into fungal microbiomes in different environments. The internal transcribed spacer (ITS) region of rDNA was recommended as a universal DNA barcode marker for fungi [22]. Most amplicon sequencing studies of fungal diversity have focused on ITS1 or ITS2 sublocus [23]. In this study, we first use DNA metabarcoding to characterize the fungal microbiomes in MOR and AOF, and compare the differences between moldy and non−moldy samples.
