Variations of the fungal microbiome in Corydalis Rhizoma with different collection areas, processing methods, and storage conditions.

Corydalis Rhizoma (CR, Yanhusuo in Chinese) has been widely used as an analgesic in herbal medicine and functional food. Cases of fungal and mycotoxin contamination in CR have been reported. In this study, the composition and diversity of fungal microbiome in CR samples from four herbal markets and two processing methods were investigated by DNA metabarcoding. Variations of the fungal microbiome in CR during cold and conventional storage were monitored. Results showed that Aspergillus was the dominant genus and saprotroph was the dominant trophic mode. Six potential toxigenic fungi, namely, Aspergillus fumigatus, Aspergillus ostianus, Aspergillus terreus, Penicillium citrinum, Penicillium oxalicum, and Trichothecium roseum, were detected. Differences in fungal composition and diversity among various groups based on collection areas and processing methods were also observed. Moreover, the relative abundance of dominant genera in CR samples stored at different temperatures was significantly different and changed with storage time. This study is the first to reveal the influence of collection areas, processing methods, and storage conditions on the fungal microbiome in CR, which was expected to provide a basis for control strategies of fungal contamination in the industrial chain of CR.

Comprehensive analysis of neonicotinoids in Chinese commercial honey and pollen: A corresponding health risk assessment for non-targeted organisms.

Neonicotinoids are broad-spectrum and highly effective insecticides that work by affecting neural activity in insects. Neonicotinoids are systemic pesticides that are absorbed by plants, transported, and accumulated in plant tissues, including nectar and pollen. Currently, there is a lack of a comprehensive assessment of the level of neonicotinoid contamination and the associated health risks to non-targeted organisms in commercial honey and pollen produced in China. This study collected 160 batches of honey and 26 batches of pollen from different regions and plant sources in China, analyzed the residue patterns of neonicotinoid pesticides, and comprehensively evaluated the exposure risks to non-targeted organisms including bees (adults and larvae) and humans. Furthermore, this study addresses this imperative by establishing a high-throughput, rapid, and ultra-sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) based on broad-spectrum monoclonal antibodies to detect and quantify neonicotinoids, with validation conducted using the LC-MS/MS method. The findings indicated that 59.4 % of honey samples contained at least one of eight neonicotinoids, and the ic-ELISA rapid detection and calculation method could detect all the samples containing neonicotinoids. Additionally, the dietary risk assessment for humans and honeybees indicates that the consumption of a specific quantity of honey may not pose a health risk to human due to neonicotinoid intake. However, the Risk Quotient values for imidacloprid to adult bees and bee larvae, as well as clothianidin to bee larvae, were determined to be 2.22, 5.03, and 1.01, respectively-each exceeding 1. This highlights the elevated risk of acute toxicity posed by imidacloprid and clothianidin residues to honey bees. The study bears significant implications for the safety evaluation of non-targeted organisms in the natural food chain. Moreover, it provides scientific guidance for protecting the diversity and health of the ecosystem.

Determination of mycobiota and aflatoxin contamination in commercial bee pollen from eight provinces and one autonomous region of China.

The co-occurrence of fungi and mycotoxins in various foods has been frequently reported in many countries, posing a serious threat to the health and safety of consumers. In this study, the mycobiota in five types of commercial bee pollen samples from China were first revealed by DNA metabarcoding. Meanwhile, the content of total aflatoxins in each sample was investigated by high-performance liquid chromatography with fluorescence detection. The results demonstrated that Cladosporium (0.16 %-89.29 %) was the most prevalent genus in bee pollen, followed by Metschnikowia (0-81.12 %), unclassified genus in the phylum Ascomycota (0-81.13 %), Kodamaea (0-73.57 %), and Penicillium (0-36.13 %). Meanwhile, none of the assayed aflatoxins were determined in the 18 batches of bee pollen samples. In addition, the fungal diversity, community composition, and trophic mode varied significantly among five groups. This study provides comprehensive information for better understanding the fungal communities and aflatoxin residues in bee pollen from different floral origins in China.

Broad-specificity monoclonal antibody against neonicotinoid insecticides via a multi-immunogen strategy and development of a highly sensitive GNP-based multi-residue immunoassay in ginseng and tomato.

Neonicotinoid insecticides (NNIs) are extensively used across the agricultural products and foods. In order to meet the rapid detection requirements, a novel broad-specificity monoclonal antibody against NNIs was developed for the first time using a multi-immunogen strategy. The antibody's high affinity and its ability to bind target molecules were verified by ic-ELISA. Furthermore, molecular docking was used to evaluate the pivotal forces affecting binding affinity and to determine binding sites. Subsequently, a highly sensitive gold nanoparticle-based immunochromatographic assay was established for the rapid detection of eight NNIs and the IC50 values were 0.03-1.61 ng/mL. The limits of detection for ginseng and tomato ranged from 0.76 to 30.19 µg/kg and 0.87 to 31.57 µg/kg, respectively. The spiked recovery ranged from 72.04% to 120.74%, and the coefficient of variation were less than 9.0%. This study provides a new direction for the development of multiple NNIs residue immunoassays.

Analysis of Fungal Microbiomes in Edible Medicinal Morindae Officinalis Radix and Alpiniae Oxyphyllae Fructus Using DNA Metabarcoding.

Morindae Officinalis Radix (MOR) and Alpiniae Oxyphyllae Fructus (AOF) have been widely used as dietary supplements and traditional herbal medicines for centuries. Fungal and mycotoxin contamination in MOR and AOF has been reported recently. In this study, fungi in MOR and AOF are first investigated using DNA metabarcoding, and the differences in fungal microbiome between moldy and non-moldy samples are analyzed. The results show that Ascomycota is the most prevailing fungus at the phylum level in MOR and AOF with relative abundances of 49.53-94.32% and 14.81-81.85%, respectively. Penicillium (1.86-76.14%), Cladosporium (1.82-56.65%), and Trichoderma (0.12-19.71%) are the dominant genera in MOR. Penicillium (0.27-56.06%), Papiliotrema (0.04-51.71%), and Cladosporium (3.08-44.41%) are the dominant genera in AOF. Two potential toxigenic fungi were detected, namely, Trichoderma atroviride and Fusarium equiseti. Moreover, the differences in fungal communities between moldy and non-moldy samples were monitored. In conclusion, DNA metabarcoding can be used to assess the fungal microbiome in edible medicinal herbs, thereby providing a basis for ensuring food safety and drug efficacy.

[Domestic and international research progress of edible and medicinal Arecae Semen].

Arecae Semen, as the first place among "Four South Medicines" in China, has great dual-use value of medicine and food. The research of Arecae Semen was mainly focused on the active ingredients and efficacy value, and its potential safety hazards were also concerned. Until now, there is still a lack of clear boundaries between medicine and food, resulting in its safety cannot be guaranteed. Therefore, it is of great significance to establish clear boundaries of medicine and food use and health risk assessment. In this paper, the differences of pretreatment and application methods of Arecae Semen were analyzed, and the research progress of Arecae Semen in chemical composition identification and toxicology research and safety evaluation were reviewed emphatically. Finally, the differences of quality control and safety evaluation of Arecae Semen in pharmacopoeias or standards were analyzed at home and abroad. It was expected to provide reference value for quality control, safety evaluation and international standardization research of Arecae Semen.

Assessment of the Microbiome and Potential Aflatoxin Associated With the Medicinal Herb Platycladus orientalis.

Platycladi Semen, which is derived from the dried ripe seed of Platycladus orientalis, has been used for the treatment of insomnia and constipation in China for 2000 years. However, it is susceptible to fungal and aflatoxin contamination under proper humidity and temperature during storage. Although aflatoxin contamination in Platycladi Semen has been reported preliminarily, few studies have been conducted on fungal infection and aflatoxin contamination simultaneously. Thus, this work aims to provide an in-depth understanding of fungal contamination in Platycladi Semen, and information on aflatoxin contamination. We focused on a comparison of the difference in fungal diversity between aflatoxin-contaminated and aflatoxin-free Platycladi Semen samples. First, aflatoxin levels in 11 Platycladi Semen samples, which were collected from local herbal markets in Shandong, Anhui, and Hebei provinces throughout China, were determined by IAC-HPLC-FLD, and positive confirmation of detected samples was performed by LC-MS/MS. The samples were divided into two groups, based on production or non-production of aflatoxin. We then used the Illumina MiSeq PE250 platform, and targeted the internal transcribed spacer two sequences to analyze the diversity and composition of the fungal microbiome, as well as to assess the presence of potential mycotoxin-producing fungi. Results showed that five samples were contaminated with aflatoxins, one of which exceeded the legal limits of Chinese Pharmacopeia Commission (2015). At the phylum level, the Ascomycota was the most dominant in all tested samples, with a relative abundance of 83.04-99.46%. Aspergillus (27.88-97.28%), Xerochrysium (0-28.49%), and Xeromyces (0-22.24%) were the three predominant genera. Furthermore, differences in fungal composition between the aflatoxin-contaminated and aflatoxin-free groups, as well as between different provinces were observed. A total of 74 species were identified, and four potential mycotoxin-producing fungi were detected in all samples, namely Aspergillus flavus, Aspergillus fumigatus, Fusarium poae, and Penicillium steckii. In conclusion, we report the great diversity of fungi associated with Platycladi Semen, highlight the risk to consumers of ingesting potent aflatoxin, and provide a reference for the safe application and quality improvement of Platycladi Semen.

Investigating the authenticity of Ophiopogonis Radix and its Chinese patent medicines by using a nucleotide signature.

ETHNOPHARMACOLOGICAL RELEVANCE: Ophiopogonis Radix (Maidong), derived from the dried root tuber of Ophiopogon japonicus (Thunb.) Ker Gawl., has been widely used in the treatment of chronic inflammatory and cardiovascular diseases. However, Ophiopogonis Radix is often adulterated with some species because of morphological similarities. Adulterants circulating in herbal markets are a latent threat to the clinical safety and consumers' interest. AIM OF THE STUDY: We aimed to develop a nucleotide signature for identification of Ophiopogonis Radix and its Chinese patent medicines. MATERIALS AND METHODS: A total of 255 ITS2 sequences representing 39 species and 4 varieties were used to develop a nucleotide signature of Ophiopogonis Radix. The nucleotide signature was used to investigate 17 commercial crude drugs and eight batches of Chinese patent medicines. RESULTS: A 69 bp nucleotide signature unique to Ophiopogonis Radix was found. The survey revealed that 2 of 17 crude drug samples were adulterants detected as Liriopes Radix (Shanmaidong). Fortunately, no adulterants were detected in the eight batches of Chinese patent medicines. CONCLUSIONS: The newly developed nucleotide signature could be efficiently applied to identify Ophiopogonis Radix and its Chinese patent medicines, aiding in the authentication, quality control, and supervision of processed products in herbal markets.

Characterizing fungal communities in medicinal and edible Cassiae Semen using high-throughput sequencing.

Cassiae Semen (CS) has been widely used as roasted tea and traditional Chinese medicine for decades. However, CS is easily contaminated by fungi and mycotoxins during pre-harvest and post-harvest process, thus posing a potential threat to consumer health. In this study, we used the Illumina MiSeq PE300 platform and targeted the internal transcribed spacer 2 sequences to survey the occurrence of fungi in raw and roasted CS samples. Results showed the fungal contamination in all 12 test samples. Ascomycota was the prevailing fungus at the phylum level, with the relative abundance of 66.50%-99.42%. At the genus level, Aspergillus, Cladosporium, and Penicillium were the most dominant genera, accounting for 0.66%-85.51%, 0.20%-29.11%, and 0.11%-32.92% of the fungal reads, respectively. A total of 68 species were identified, among which six potential toxigenic fungi belonging to Aspergillus, Penicillium, Candida, and Schizophyllum genera were detected. Moreover, differences in fungal communities were observed in raw and roasted CS samples. In conclusion, amplicon sequencing is feasible for analyzing fungal communities in CS samples, which provides a new approach to investigate the fungal contamination in edible-medicinal herb, thereby ensuring food safety and drug efficacy.

Detecting Schisandrae Chinensis Fructus and Its Chinese Patent Medicines with a Nucleotide Signature.

Schisandrae Chinensis Fructus (Wuweizi) is often adulterated with Schisandrae Sphenantherae Fructus (Nanwuweizi) in the herbal market. This adulteration is a threat to clinical treatment and safety. In this study, we aimed to develop a nucleotide signature for the identification of Wuweizi and its Chinese patent medicines based on the mini-DNA barcoding technique. We collected 49 samples to obtain internal transcribed spacer 2 (ITS2) sequences and developed a 26-bp nucleotide signature (5'-CGCTTTGCGACGCTCCCCTCCCTCCC-3') on the basis of a single nucleotide polymorphism (SNP) site within the ITS2 region that is unique to Wuweizi. Then, using the nucleotide signature, we investigated 27 batches of commercial crude drug samples labeled as Wuweizi and eight batches of Chinese patent medicines containing Wuweizi. Results showed that eight commercial crude drug samples were adulterants and one of the Chinese patent medicines contained adulterants. The nucleotide signature can serve as an effective tool for identifying Wuweizi and its Chinese patent medicines and can thus be used to ensure clinical drug safety.

Analysis of the Fungal Community in Ziziphi Spinosae Semen through High-Throughput Sequencing.

Ziziphi Spinosae Semen (ZSS) has been widely used in traditional Chinese medicine system for decades. Under proper humidity and temperature, ZSS is easily contaminated by fungi and mycotoxins during harvest, storage, and transport, thereby posing a considerable threat to consumer health. In this study, we first used the Illumina MiSeq PE250 platform and targeted the internal transcribed spacer 2 sequences to investigate the presence of fungi in moldy and normal ZSS samples collected from five producing areas in China. Results showed that all 14 samples tested were contaminated by fungi. Ascomycota was the dominant fungus at the phylum level, accounting for 64.36⁻99.74% of the fungal reads. At the genus level, Aspergillus, Candida, and Wallemia were the most predominant genera, with the relative abundances of 13.52⁻87.87%, 0.42⁻64.56%, and 0.06⁻34.31%, respectively. Meanwhile, 70 fungal taxa were identified at the species level. Among these taxa, three potential mycotoxin-producing fungi, namely, Aspergillusflavus, A. fumigatus, and Penicillium citrinum that account for 0.30⁻36.29%, 0.04⁻7.37%, and 0.01⁻0.80% of the fungal reads, respectively, were detected in all ZSS samples. Moreover, significant differences in fungal communities were observed in the moldy and normal ZSS samples. In conclusion, our results indicated that amplicon sequencing is feasible for the detection and analysis of the fungal community in the ZSS samples. This study used a new approach to survey the fungal contamination in herbal materials. This new approach can provide early warning for mycotoxin contamination in herbal materials, thereby ensuring drug efficacy and safety.

Inspecting the True Identity of Herbal Materials from Cynanchum Using ITS2 Barcode.

Cynanchum is a large genus with some important medicinal species in China. The medicinal species in Cynanchum are easily confused, leading to potential safety risks. In this study, the internal transcribed spacer 2 (ITS2) barcode was used to discriminate the medicinal plants in Cynanchum. The identifying capability of ITS2 was assessed using the specific genetic divergence, BLAST1, neighbor-joining (NJ) tree, maximum-likelihood (ML) tree, and single-nucleotide polymorphism (SNP) methods. Results indicated that the intra-specific genetic divergences of Cynanchum species were lower than their inter-specific genetic divergences. Of the 87 samples from 17 species, ITS2 showed a high identification efficiency of 90.8 and 87.4% at the species level through BLAST1 and the nearest distance methods. NJ tree and ML tree also demonstrated the suitability of ITS2 to differentiate Cynanchum species. Meanwhile, a stable SNP was found, and it could accurately authenticate Cynanchum paniculatum and Cynanchum atratum. Furthermore, we collected 64 commercial samples from three commonly used herbal medicines and evaluated the capability of ITS2 to survey their authentication. Of these samples, Cynanchi Atrati Radix et Rhizoma (Baiwei) showed a potential safety problem, and all the 11 test samples were adulterants. In conclusion, ITS2 can distinguish medicinal species in Cynanchum effectively, and its application could greatly improve the identification efficiency and accuracy of commercial herbal medicines in this genus.