Camellia sinensis and 26 herbal teas marketed in Latvia is discussed. The analysis was performed using two-dimensional liquid chromatography with time-of-flight mass spectrometry (2D-LC-TOF-MS) and MALDI-TOF-MS. In total, 87% of the tea samples tested positive for 32 fungal species belonging to 17 genera, with the total enumeration of moulds ranging between 1.00 × 101 and 9.00 × 104 CFU g−1. Moreover, 42% of the teas (n = 70) were contaminated by 1 to 16 mycotoxins, and 37% of these samples were positive for aflatoxins at concentrations ranging between 0.22 and 41.7 µg kg−1. Deoxynivalenol (DON) and its derivatives co-occurred in 63% of the tea samples, with their summary concentrations reaching 81.1 to 17,360 µg kg−1. Ochratoxin A (OTA), enniatins, and two Alternaria toxins were found in 10–37% of the teas at low concentrations. The dietary exposure assessment based on the assumption of a probable full transfer of determined mycotoxins into infusions indicated that the analysed teas are safe for consumers: the probable maximum daily exposure levels to OTA and the combined DON mycotoxins were only 0.88 to 2.05% and 2.50 to 78.9% of the tolerable daily intake levels.In this paper, a study of fungal and multi-mycotoxin contamination in 140  Camellia sinensis (C. sinensis) black and green teas, and teas made from medicinal herbs such as peppermint, chamomile, and dog-rose, are consumed daily as the most favoured beverages in Europe and other countries [C. sinensis and C. assamica varieties, such as oolong (semi-fermented) and Pu-erh (post-fermented) teas, have spread around the world and gained popularity among non-Asian tea users due to their multiple health-protective  properties associated with the high contents of catechins, flavanols, and specific fungal microbiota present in these teas [Traditional ountries ,2. Caffeese teas ,4.2, pests, and insect damage); the thermal resistance of mycotoxins during tea drying, processing, and storage; and poor microclimate conditions can result in contamination of the final tea products [Preharvest and postharvest factors such as environmental issues  Pu-erh tea variety from loosened black tea leaves involves wet-piling fermentation, which can enhance the development of moulds and the potential formation of mycotoxins compared to the aging conditions of raw (sheng) Pu-erh tea, which is produced from nonfermented tea leaves ,7.Recent studies of commercial black and green teas based on routine laboratory analyses using high-performance liquid chromatography (HPLC) with tandem mass spectrometry (MS/MS) and fluorescence detectors (FLD) indicated high rates of storage-related mycotoxins, such as aflatoxins (AFs) and ochratoxin A (OTA), which were found in dry tea leaves and into their infusions ,9,10.n = 76) from a Moroccan tea market were positive for aflatoxin B1 (AFB1) and other AFs [1 and the sum of aflatoxins B1, G1, B2, and G2 exceeded the maximum limits of 5 and 10 µg kg−1 set for these mycotoxins in Morocco [Mannani et al. (2020) reported that more than half of all tested green tea samples , enniatins , etc.) in C. sinensis and herbal teas and infusions, but exposure studies showed that the concentration levels of these contaminants were too low to pose substantial hazards to consumers [Several recent studies also observed onsumers ,14,15,16The spread of mycotoxin-related plant illnesses in different geographic regions has led to the need for screening studies of regulated and nonregulated toxins using sensitive methods for their simultaneous quantification in teas and other products .The aim of the present study was to perform an analysis of the fungal and mycotoxin contaminations in caffeine-rich and herbal teas available on the Latvian market, since most of these commercial teas are imported. The microbial quality was evaluated via mould quantity studies and the identification of fungal species using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS).Aspergillus, Alternaria, Fusarium, and Penicillium species [A recently developed in-house method of online heart-cutting two-dimensional liquid chromatography with time-of-flight mass spectrometry (2D-LC-TOF-MS) was used for the analysis of mycotoxins produced by  species .The dietary exposure assessment assumed that the mycotoxin concentrations determined in the raw dry teas are fully extracted into the beverages during infusion (worst-case scenario). The intake of the upper-bound (UB) level and maximum mycotoxin concentrations determined in the dry samples was evaluated for a conventional tea portion of one cup (200 mL of boiling water and 2 g of dry tea), taking into account survey data of the average intakes of fermented, nonfermented, and herbal teas in Latvia .Two-dimensional liquid chromatography has recently attracted attention as an advanced tool for the multi-contaminant analysis of food products ,20,21. TA mycotoxin-free Pu-erh matrix was used as the blank matrix for validation of the 2D-LC-TOF-MS method. The estimated performance criteria are shown only for the twenty mycotoxins that were determined to be positive in the analysed tea samples. More detailed information on the method of validation is provided in our forthcoming paper dedicated to the development and optimisation of this instrumental method .C. sinensis teas, and an herbal tea blend. The accuracy of QC values for all the compounds ranged between 70% and 124%.The applicability of the method was proved by quality control (QC). QC samples were included in every analysis sequence, as well as being used for quantification matrix-matched calibration on a blank sample, that included a mix of 2) were higher than 0.99, which confirmed the good linear correlation between the detector response and mycotoxin concentrations. The coefficients of determination . The tr1 was 1.8 times lower than the LOQ (0.08 µg kg−1) reported by another recent study [The calculated levels of the detection and quantification (LODs and LOQs) were comparable with the sensitivities reported in studies based on sample preparation using offline SPE methodologies or dispersive SPE (QuEChERS) approaches, and detection methods that primarily use HPLC-MS/MS and other MS techniques ,11,12. Tnt study . The oven = 159), with the exception of seven oolong samples, presented at limited amounts just for mycotoxin analysis, were analysed for their mould content and microbial quality.Almost all the tea samples (w), as the characteristic factor influencing mould growth in products, was evaluated using a standardised method [w value of 0.46 ± 0.05 was determined in most of the C. sinensis and herbal tea samples, except for four oolong and two green tea samples which had aw > 0.60. Typically, aw values exceeding 0.8 will stimulate the growth of fungal species. Common conditions for the generation of AFB1-producing A. flavus and A. parasiticus include 0.90 to 0.98 aw at 27 °C or 0.90 to 0.94 aw at 35 °C [w values (commonly >0.99 aw at 25 °C) are needed for the growth of F. graminearum and other Fusarium species, which produce typical trichothecenes such as deoxynivalenol (DON) and the T-2 toxins present in cereals, as well as other common agricultural products [w < 0.65 contributes to decreasing cellular metabolism and preventing fungal growth under proper storage conditions [Water activity (ad method . The aveat 35 °C . Higher products . Accordi−1) was determined after five days of incubation at 25 ± 0.2 °C, according to the standardised procedure for the determination of yeast and total fungal contents in the products with aw < 0.95 [The total number of the colony-forming units  with yeast counts ranging between 2.00 × 102 and 4.90 × 103 CFU g−1, and four peppermint tea samples, including one blend with green tea, which all presented yeast quantities that were too high to be properly counted, even at the highest dilution ratio used .In addition, yeasts were also determined in some of the samples and counted when possible. These samples included one oolong tea with a 1.00 × 10n = 20) of the analysed teas presented no signs of mould growth. These samples included one black tea, six green teas, eleven Pu-erh teas (one raw (sheng) Pu-erh and 10 ripe (shu) Pu-erh samples), one chamomile sample, and one peppermint sample. Most of the tea samples  presented mould counts of 1.00 × 101–9.00 × 104 CFU g−1.In total, 13%  association and the recommendations of the Regulation No 461 of the Cabinet of Ministers of Republic of Latvia [As indicated in f Latvia  are incl3 CFU g−1, which exceeded the mean counts of the moulds determined in black teas (5.10 × 102 CFU g−1) by 2.1, 14.4, and 15.8 times, especially Pu-erh teas (7.56 × 101 CFU g−1) and oolong teas (6.86 × 101 CFU g−1). The mean mould counts in green teas were around 1.09 × 102 CFU g−1. Mould counts ranged from 6.00 × 101 to 1.6 × 104 CFU g−1 in these samples. Most of these samples were bagged green teas from China, and only three were loose-leaf  tea samples. One was tea from Sri Lanka, with 6.60 × 102 CFU g−1. The other loose-leaf green tea samples were determined to have low quantities of mould between 1.00 × 101 and 4.20 × 102 CFU g−1. The lowest fungal mould levels of 1.00–3.00 × 101 CFU g−1 were determined in two Genmaincha and Bacha weighable tea samples from Japan. Fourteen of the green teas (mainly pure teas or blends with fruits and peppermint) were identified to have mould counts exceeding 5.00 × 10n = 62) contained quantifiable amounts of mould. The mould counts reached at least 5.00 × 102 CFU g−1 in sixteen samples, mostly in black teas from India and Sri Lanka (5.00 × 102–9.60 × 103 CFU g−1). Comparing the bagged (n = 30) and loose-leaf (n = 32) samples, the mean mould counts in the bagged tea samples (6.47 × 102 CFU g−1) were higher than those in the loose-leaf tea samples (3.69 × 102 CFU g−1). The weighable black tea samples mostly presented a lower range of maximum counts ranging between 1.00 × 101 and 2.62 × 103 CFU g−1.Almost all the analysed black teas . The mean values of the mould unity contents counted in chamomile, peppermint, and hibiscus and dog-rose teas were around 1.29 × 104 CFU g−1, 8.52 × 104 CFU g−1, and 2.40 × 104 CFU g−1, respectively.As presented in The fungal identifications were determined using characteristic identification keys reported in the literature ,33. ConfAspergillus spp. and one Penicillium sp. were determined to be the most dominant fungal species and were identified in more than 10% of the analysed tea samples  were found in one to two samples (less than 5% of the teas).The study results indicated 17 fungal genera and 32 individual fungus species observed in the 138 mould-positive tea samples.  samples . Some ofA. niger and A. glaucus isolates and Penicillium sp.  were prevalent in most of the C. sinensis samples (with the exception of Pu-erh) and most of the herbal teas . In comparison, only three Pu-erh samples, three to seven oolong tea samples, and one to seven herbal tea varieties were positive for these two fungus species.As presented in A. niger and A. flavus are the primary fungi of interest as sources that produce OTA and AFs [Aspergillus spp. species, 41 isolates of A. niger were obtained during this study, whereas only 7% of them were able to produce OTA in teas [ and AFs ,37. HoweA. flavus was observed in fourteen black teas but in only two green teas and three herbal teas . The fungi able to produce AFs were not found in the analysed Pu-erh and oolong tea samples.In the current study, P. chrysogenum (n = 9), Lichthemia corymbifera (n = 8), A. versicolor (n = 7), and A. nidulans (n = 7). In other C. sinensis varieties, only A. niger and A. glaucus predominated.The other characteristic fungal species determined in the black tea varieties included Aspergillus (A. niger. and A. glaucus) species, were Mucor, Fusarium, Cladosporium, and Alternaria .Aspergillus. spp. and Penicillium spp. or Rhizopus sp.) fungi were observed in the hibiscus, dog-rose, and linden samples.Only one or two nonfilamentous , peppermint (33%), and fennel (33%). These peppermint teas were produced in Poland, and our results were in good agreement with the data provided by Zimowska (2007), who reported the typical prevalence of both Alternaria and Phoma species in cultivated peppermint plants [Phoma species, whereas the potential risks of Alternaria fungi related to the production of toxic contaminants are an emerging issue [t plants . There ang issue .P. chrysogenum, Lichthemia corymbifera, and Scopulariopsis brevicaulis were common in three of the chamomile teas. Among the other fungi, only two fungi from the Fusarium genus , identified in the four C. sinensis teas , this study also indicated the presence of other mycotoxigenic fungi, which raises the concern of potential mycotoxin production in the tea samples.While a few of the fungal species were common  were mycotoxin-free.n = 70). Almost a third of the tea samples were contaminated with one to three mycotoxins, while in most cases, the co-occurrence of more than four mycotoxins was observed. In total, 15.7% (n =11) of the 70 mycotoxin-positive tea samples were positive for more than 10 mycotoxins: one “gunpowder” green tea contained 13 mycotoxins, nine Pu-erh tea samples contained 10 to 16 mycotoxins, and one supermarket-brand peppermint tea contained 16 mycotoxins.Twenty mycotoxins were determined in 42% of the tea samples (1 (36%), AFB2 (27%), AFG1 (23%), AFG2 (10%), OTA (24%) and its dechlorinated derivate OTB (24%), STC (19%), ENN B (37%), ENN B1 (31%), ENN A (14%), ENN A1 (10%), ALT (24%) and ATX I (19%), ZEN (10%), T-2 toxin (3%), and HT-2 toxin (3%). The following total incidences of the twenty determined mycotoxins were identified in the 70 mycotoxin-positive tea samples: DON (65%) and its derivates 15-AcDON (66%), 3-AcDON (54%), and D3G (37%), AFB−1 (As indicated in −1 .1 (a Class 1 human carcinogen) and the other AFs  were determined in the following order in the analysed C. sinensis teas: Pu-erh tea (n = 8) > green tea (n = 7) > black tea (n = 6). The overall concentrations of AFB1 in these teas ranged between 0.22 and 39.0 µg kg−1 and between 1.37 and 102 µg kg−1 for the sum of the four AFs.AFB−1 of AFB1. The four AFs co-occurred in five of the green tea samples, with the sum of their concentrations reaching up to 7.67 µg kg−1. One sample also contained AFB1, AFB2, and AFG1, with a total concentration of 1.54 µg kg−1. AFB2 and AFG2, which are less toxic, appeared at 1.5- to 3-fold lower concentrations compared to AFB1, while AFG1 presented similar concentrations in most of the samples.The green tea samples contained 0.46 to 2.99 µg kg1 concentrations from 0.39 to 3.45 µg kg−1. The sample with the maximum determined AFB1 contents also contained traces of AFB2 (0.28 µg kg−1). There were only two exceptions among the black tea samples, which both contained elevated concentrations of AFs. The first sample was a loose-leaf tea from Kenya, with 13.9 µg kg−1 of AFB1 and 12.1 µg kg−1 of AFB2. The second tea sample was an Indian spiced black tea blend  that contained 39.0 µg kg−1 of AFB1, 11.1 µg kg−1 of AFB2, 41.7 µg kg−1 of AFG1, and 9.95 µg kg−1 of AFG2. AFB1 and AFG1 were the main toxins affecting the total contamination level (102 µg kg−1) of the sum of the four AFs in this tea sample. These levels may have been caused by the contamination of some spices  used in the production of this tea mix.The black tea samples of the current study also contained relatively low AFB1 (1.14–2.73 µg kg−1), AFB2 (0.27–0.79 µg kg−1), and AFG1 (0.99–1.63 µg kg−1). The sum of their concentrations ranged from 2.04 to 4.45 µg kg−1. AFG2 was not determined in any of the Pu-erh samples, similar to the black tea varieties. This result may be associated with the specifics of the fermentation conditions. One sample of Pu-erh tea (Yunnan Yiwu Spring Shen) presented higher levels of AFB1 (9.17 µg kg−1), AFB2 (2.76 µg kg−1), and AFG1 (7.9 µg kg−1).Seven of the eight aflatoxin-positive Pu-erh tea samples contained relatively low levels of AFB1 concentrations of 0.22 and 0.4 µg kg−1. Relatively low levels of AFB1 at 0.98 µg kg−1 and AFG1 at 0.39 µg kg−1 were determined in the herbal blends of chamomile (27%) with rooibos (25%), peppermint (20%), and other ingredients. The multi-contaminated peppermint sample with 16 mycotoxins was positive for all four AFs at the following concentrations: AFB1: 5.62 µg kg−1, AFB2: 1.71 µg kg−1, AFG1: 6.02 µg kg−1, and AFG2: 2.36 µg kg−1.AFs were observed in only four herbal tea samples. Hibiscus mono tea and the blend formulated with dog-rose and dried fruits contained low AFB1. STC co-occurred at low levels (1.2–1.8 µg kg−1) with the AFs in three of the green tea samples and at 13.0 ug kg−1 in the mycotoxin-rich peppermint tea sample, which contained four AFs. STC also co-occurred at trace to moderate levels from 1.06 to 93.4 µg kg−1 in nine of the Pu-erh samples, whereas the highest concentration was found in the Pu-erh sample that was also contaminated with elevated AF concentrations. These results for different tea matrixes confirmed the potential coincidence between STC and AFs due to the similar parental fungi (Aspergillus spp.) that produce these mycotoxins.Sterigmatocystin (STC), a possible human carcinogen (Class 2B toxin), is a common precursor of AFBOTA, another Class 2B carcinogen, has also been commonly observed in teas, herbs, and their infusions due to its potential formation under improper storage conditions.n = 4) of the green tea samples were positive for OTA at concentration levels between 1.1 and 3.4 µg kg−1. The co-occurrence of OTA with a less-toxic dechlorinated metabolite (OTB) was observed at concentrations ranging between 0.6 and 2.5 µg kg−1 in three of the green tea blends with fruit and natural flavourings.Only 9% (n =4) of the black tea samples were also positive for OTA (1.7–7.7 µg kg−1), whereas OTB was not observed in the black tea varieties.In total, 6%  than those determined in the ripened Pu-erh tea samples of the present study. The DON concentrations in the ripened Pu-erh samples ranged between 145 and 5808 µg kg−1.Comparing the varieties, two were raw Pu-erh teas, which are commonly fermented under hot and humid surroundings, thereby leading to the formation of fungi: these two samples showed DON concentrations 2 to 4 times higher (6255–8946 µg kgn = 10) of the green tea samples, mostly the organic green leaf teas from China and Japan, were positive for DON and one to three of the DON derivatives. DON was observed at concentrations ranging from 97.1 to 859 µg kg−1—up to 10 times lower compared to the maximum concentrations determined for the Pu-erh tea samples. Five of the green tea samples also contained all of the four DON mycotoxins (their combined concentrations ranged between 1446 and 4512 µg kg−1), and three of the samples contained both of the acetylated DON derivatives, 3-AcDON and 15-AcDON, with the combined concentrations ranging from 404 to 3086 µg kg−1. Two of the green samples contained only individual acetylated DON derivatives at relatively low concentrations of 61.7 µg kg−1 (3-AcDON) and 211 µg kg−1 (15-AcDON).Fifty-nine percent  by 2.7, 2.8, and 4.9 times, respectively. The second exception was also a sencha tea mixed with different nuts and sugar-coated pineapple; in this case, the concentrations of D3G, 15-AcDON, and 3-AcDON derivatives exceeded the concentrations of DON (464 µg kg−1) in the tea blend sample by 2.1, 2.7, and 3.9 times, respectively.3-AcDON was the predominant mycotoxin in the analysed green tea samples at concentrations of 213–1792 µg kgAs indicated in n = 7) of the mycotoxin-positive black tea samples at concentrations from 26.5 to 211 µg kg−1. The acetylated derivatives showed similar levels of contamination to those of DON: 15-AcDON was observed in nine of the analysed black tea samples (with concentrations ranging between 25.8 and 189 µg kg−1), whereas 3-AcDON was determined in only three samples of this tea variety (with concentrations ranging between 106 and 428 µg kg−1). Comparing the co-occurrence of DON and its derivatives, only five of the black teas were positive for the co-occurrence of DON and 15-AcDON at summary concentrations from 116 to 290 µg kg−1, and two of the samples were positive for the co-occurrence of 15-AcDON and 3-AcDON at 290 and 537 µg kg−1. In comparison to green teas, 15-AcDON exceeded the levels of DON in only two of the black tea samples, as the derivatives were mostly present at lower or similar concentrations to those of DON.DON was determined in only 39% (n= 9) of the 15 total mycotoxin-positive herbal teas, whereas the contamination levels in most cases were lower than those of the C. sinensis tea samples.DON and its derivatives were also observed in 60% , and four also contained 15-AcDON (with concentrations ranging between 30.6 and 146 µg kg−1). D3G was not found in any of the chamomile tea samples, whereas the combined concentrations of DON and DON acetylated derivatives ranged between 81.1 and 718 µg kg−1, respectively.Six of the chamomile tea samples were positive for DON concentrations ranging between 44.0 and 166 µg kg−1, 3-AcDON ranging between 300 and 2227 µg kg−1, and 15-AcDON ranging between 96.5 and 1140 µg kg−1. The third peppermint sample was noted above for its high contamination prevalence of sixteen mycotoxins, which, besides the four AFs, also included all four DON mycotoxins, with the concentrations of DON, 3-AcDON, 15-AcDON, and D3G reaching 953, 2255, 2227, and 740 µg kg−1, respectively .Three of the peppermint teas were characterised as being the most contaminated with DON and its derivatives compared to other herbal species. Two of the samples contained three DON mycotoxins, with DON ranging between 52.56 and 521.68 µg kg−1 of ZEN.ZEN, a common estrogenic toxin, was not found in most of the analysed tea samples, except for the seven Pu-erh samples, which contained 4.72 to 56.1 µg kg−1 of HT-2 and 8.1 µg kg−1 of T-2, and in one peppermint tea sample, which contained both T-2 and HT-2 toxins at 4.90 and 37.5 µg kg−1 concentrations, respectively.T-2 and HT-2 toxins were identified in only two green tea samples, with 5.58 µg kgFusarium mycotoxins frequently found in grain products; these mycotoxins raise potential concerns due to their chronic toxicity upon long-term exposure [Enniatins (ENNs) and beauvericin (BEA) are emerging exposure ,43. BEA −1 in 15.7% of the ENN-positive samples (n =26) from the 166 analysed teas. ENN B1 was the second most frequently occurring ENN and was present in concentrations between 0.5 and 201 µg kg−1 in twenty-two tea samples.ENN B was the most predominant among this class of toxins, with levels of 0.93 to 143 µg kg1 and ENN A were found in only five and ten samples of Pu-erh teas, respectively (ENN A ranging between 0.69 and 115 µg kg−1). However, 4.33 µg kg−1 of ENN A1 was also observed in one of the green tea samples and at a level of 55.3 µg kg−1 in the multi-contaminated peppermint tea. ENN A−1  in the positive green teas (n = 7), while five of the samples were also positive for the co-occurrence of ENN B1 ranging between 6.34 and 47.5 µg kg−1 .ENN B ranged between 0.93 and 18.7 µg kg1 contents (143 and 125 µg kg−1) was also observed, whereas in the other ten ENN-positive Pu-erh samples, the concentrations of both toxins were similar to those found for green tea varieties. Concentrations of ENN B ranging between 1.28 and 31.4 µg kg−1  were determined in these Pu-erh tea samples, and ENN B1 concentrations between 0.62 and 22.6 µg kg−1  were observed.A sheng Pu-erh sample with exceptionally elevated ENN and ENN B1 ranging between 0.98 and 11.3 µg kg−1. Both ENNs were present at notably higher levels in the two peppermint teas. One was a tea sample from Latvia, which contained 28.3 µg kg−1 of ENN B and 111 µg kg−1 of ENN B1. The multi-contaminated peppermint tea contained 83.9 µg kg−1 of ENN B and 201 µg kg−1 of ENN B1. Three chamomile teas, one peppermint tea, and two dog-rose blends with hibiscus and fruits presented low concentrations of ENN B and ENN B−1. In the six samples, both mycotoxins co-occurred. ALT and ATX I were both observed in only two black teas (ALT ranging between 0.8 and 1.3 µg kg−1) and three green teas .Here, the presence of two emerging Alternaria mycotoxins (ALT and ATX I) was observed for the first time in commercial tea samples available on the Latvian market. Fourteen of the analysed Pu-erh tea samples were positive for both ALT and ATX I at concentrations of 0.10–9.10 and 1.44–28.3 µg kg−1. Moreover, one peppermint tea contained 4.81 µg kg−1 of ATX I, while the multi-contaminated peppermint sample was positive for 7.10 µg kg−1 of ALT and 5.78 µg kg−1 of ATX I.Among the herbal teas, only one formulation of chamomile with rooibos, peppermint, and other ingredients was positive for ALT at 3.9 µg kg−1) from a dry dog-rose sample into its infusion, whereas a 100% extraction of up to 273 µg L−1 of DON was observed for several herbal teas. These results are in good agreement with the data on DON’s water solubility (55 mg mL−1) reported in a comprehensive review [−1) in tea beverages [−1 bw day−1 for both DON and the sum of DON and its derivatives  [A dietary exposure assessment was conducted according to the procedure described in everages . This re5-AcDON) . The resDON and its derivatives were found in green, black, and Pu-erh teas ; ZEN, observed only in the Pu-erh tea samples, and OTA, found in most of the teas, were included in the dietary intake assessment, due to their relatively high prevalence in the commercial teas analysed in this study .As noted in the description of potential acute daily intake calculations , the aveThe approximate average volume 200 mL) and mean dry tea amount (2.00 g) per serving of a hot water infusion  were used  [00 mL andThe probable daily intake (PDI) values were calculated from the average daily intake values of the Latvian population . The aveThe PDI values for acute intake of the maximum mycotoxin concentrations were 1.4 to 16.5 times higher than the calculated values for the mean concentrations as the potential contamination levels of infusions.Considering the low concentrations of OTA found and the similarity of the data to the mean upper-bound and maximum levels, the PDI values reached only 0.17 to 2.05% of the OTA TDI, thus indicating little exposure potential during tea intake.1 and summary AF PDI values were also calculated considering their high prevalence in 30% of the analysed teas. The levels were similar to those reported by other authors [The values of AFB authors . Due to In the case of traditional black and green teas, the PDI values of DON and the summary contents of DON and DON derivatives were only 5.33 to 7.40 times higher in the case of maximum concentrations considered as contamination levels of beverages compared to that of upper-bound levels determined in tea varieties.The PDI values of DON for traditional teas generally reached only 0.96 to 4.23% of the TDI, similarly to herbal teas, where the PDIs for the upper-bound level and maximum determined mycotoxin contents reached only 2.03 to 3.42% of the TDI.The PDI values of the total DON and DON derivatives calculated for traditional teas and peppermint teas were 2.60 to 5.99 times higher than the PDI calculated for DON, which indicates the strong influence of multi-contamination on the overall DON intake. This was notably observed in Pu-erh teas, where the PDI values for the total contamination of DON and DON derivatives reached 27.4% and 78.9% of the TDI. The high rate of DON mycotoxin contents in Pu-erh teas should be considered, as amounts greater than 2 g (8 g approximately) are commonly used during the brewing process, and the first infusion is commonly not used.In this study, the PDI values calculated for the approximate worst-case scenarios remained below the TDI, which indicates a low probability of intoxication via the acute intake of traditional commercial teas and peppermint beverages.C. sinensis and herbal tea varieties that were purchased from Latvian tea stores and supermarkets. The results were compared with recent data from other studies, providing new data on regulated and emerging mycotoxin contamination in teas related to the fungal species explored in the present study.The present study provided an assessment of the microbial quality and mycotoxin prevalence in relatively large datasets of traditional and popular 0 and 4.49× 103 CFU g−1 in green tea varieties and 4.30 × 101 and 3.06 × 103 CFU g−1 in black tea varieties [C. sinensis tea products, although there are several different potential factors [The microbiological data were in good agreement with recently reported results. Carraturo et al. 2018) reported on sixteen black and green teas: the mean total mould and yeast counts ranged between 8.00 × 10arieties . The hig018 repor5 CFU g−1) and yeast counts (≤1.00 × 106 CFU g−1 and ≤1.00 × 105 CFU g−1) set for both dry C. sinensis teas and dry herbal teas in the recommendations by the Tea and Herbal Infusions Europe (THIE) and the American Herbal Product (AHPA) associations [The microbial quality found in the present study was in agreement with the safety recommendations for the total fungal counts , and ≤5.00 × 101 CFU g−1 mould counts for herbal teas consumed by pregnant and breast feeding woman or by children [n = 28) of the tea samples  exceeded the limitations by up to 90 times. The total mould counts in these teas ranged between 1.01 × 103 and 9.00 × 104 CFU g−1.The Technical Regulation CU TR 021/2011, which is used in the Russian Federation, permits fixed mould counts of ≤1.00 × 10children . SeveralC. sinensis and herbal teas  from markets in the Russian Federation was evaluated. This study indicated that only 16.7% of commercial black tea samples from India and Indonesia, and one sample from Sri Lanka were not in agreement with the microbiological limitations. These samples contained mould fungi concentrations up to 1.3 × 103–8.2 × 103 CFU g−1, whereas the levels in tea infusions dropped to nondetectable levels, indicating reduced threats to consumer safety. The authors of this study also reported that 10 out of 18 herbal teas had fungal mould contents ranging between 5.00 × 103 and 1.00 × 106 CFU g−1, which notably exceeded the standard limitations for total fungi, whereas the mould levels dropped to the relevant limitations after tea brewing, unlike the bacterial counts, which were not reduced in herbal teas after preparing the tea infusions [A. niger in C. sinensis teas, noting the multi-contamination of more than five species, including Aspergillus sp., Penicillium sp., Alternaria sp., Fusarium sp., and Cladosporium sp., which were found in all herbal tea samples collected from the markets.Our data for black tea and herbal samples were comparable with the recent results reported by Minaeva et al. (2019) . In theinfusions . The autCladosporium sp. (C. cladosporioides) and A. niger species in these beverages [1–1.80 × 102 CFU g−1), and one raw Pu-erh sample contained a 3.00 × 102 CFU g−1 mould count. Lower levels of mould were observed than those reported by other studies. A. glaucus and A. niger were the predominant species in six of the mould-positive Pu-erh samples, whereas one of the ripened Pu-erh teas was positive for Cladosporium herbarum, Cladosporium chrysogenum, and Lichthemia corymbifera species, which may be due to the aging conditions, as Cladosporium spp. are recognised to be slow-growing fungi [Compared to traditional black and green teas, the oolong and Pu-erh samples showed similarly low mould contents. No data were found from studies on the microbial assessment of dry oolong teas. However, some studies focused on cold green tea and oolong tea infusions have demonstrated the formation of everages . Meanwhieverages ,49,50,51ng fungi .A. niger and Blastobotrys adeninivorans, ultimately yielding a specific stringent taste [4–4.90 × 103 CFU g−1) than in raw loose tea samples (1.6 × 103–4.8 × 103 CFU g−1), due to the fungal production during the wet-piling period [The production of ripe Pu-erh tea includes an additional wet-piling fermentation process, which involves piling and aging stages that lead to the production of mould, bacteria, and fungi species, such as nt taste ,50. Zhao1–2.6 × 106 CFU g−1); the results of the present study coincided with these results [A. niger, A. flavus, and Alternaria alternata, which may affect the production of AFs, OTA, and Alternaria toxins.Haas et al. (2013) reported a relatively high variety of fungi found in raw Pu-erh teas . The low AF prevalence in green teas was in good agreement with the results of a study from Spain, which also indicated a low prevalence of AFB1 in commercial green teas [1, with a concentration of 5.40 µg kg−1.The results for the AFBre found . This stcan teas  comparedeen teas . Only on1 [Considering their potential contamination during aging and wet-milling, Pu-erh teas are commonly evaluated for AF and OTA contamination. Haas et al. (2013) analysed the presence of AFs, fumonisins (FBs), and OTA in 36 Pu-erh samples, where AFs were not found, and only 11% OTA-positive samples were reported . Dong et1 .2 in most of the ripe Pu-erh teas and 10 of the raw Pu-erh varieties [1 concentrations ranged between 0.345 and 8.333 µg kg−1 in the ripe Pu-erh teas and between 1.827 and 15.149 µg kg−1 in the raw Pu-erh varieties; these are comparable data to those reported in the present study. Similar data were also reported by other authors [The present study indicated a low prevalence of AFs in five of the Pu-erh samples. This result agrees with the study data reported by Li et al. (2015) on 14 raw and 14 ripe tea samples tested by HPLC-MS methods, which also indicated the absence of AFGarieties . AFB1 co authors .1 and sum of four AFs are still not regulated in teas in Europe, although Moroccan regulations do define allowable quantities in teas, which coincide with the restrictions of 5 and 10 µg kg−1 of maximum concentrations set for these toxins in spices by European legislative authorities [1 in spices coincide with the regulatory limits for raw tea in the national regulations set by the Customs Union countries [Notably, AFBhorities ,55. The ountries .1 and combined four AF limitations in medical herbs, which are 2.5 times lower (2 μg kg−1 limit for AFB1 and 4 μg kg−1 for combined AFs) than the concentration limitations allowed by the EC for condiments [The European Pharmacopeia has set restrictions for the allowable AFBndiments ,55. Thusndiments ,56.1, whereas most of the teas indicated low contamination with AFs, with the exception of two samples with elevated AF contents , which both had concerning levels of AF exceeding the acceptable limitations set by the relevant worldwide regulations [Comparing fungi and mycotoxin prevalence, only four of the tea samples 16%) showed the co-presence of A. flavus species and AFBulations ,16,55,56% showed −1) in two samples of black tea [−1) in medicinal herbs [However, most of the AF-positive samples also contained detectable levels of carcinogenic STC, mostly at low levels that were in good agreement with other studies. Chaly et al. recently reported STC at trace levels  collected from tea markets in the Russian Federation, whereas no traces of this mycotoxin were found in the green and Pu-erh teas included in this recent study [n =3), Pu-erh teas (n = 3), and mycotoxin-rich peppermint tea raises concerns over their possible cumulative effects, which should be taken into account when evaluating mycotoxins’ impacts on human health. It is well known that several Aspergillus species, including the species indicated in the present study , can produce STC [A. glaucus and A. versicolor was identified in five of the thirteen STC-positive tea samples, these data should be further evaluated using more advanced methods of fungal genus analysis before conclusions can be drawn.Kiseleva et al. (2020) found STC levels below the LOD in one black tea sample and six herbal blends . Additional studies based on PCR and molecular tools will be necessary to genetically determine the isolates able to produce mycotoxins, as reported by a recent study [A. niger. However, only eight cases from a total of 105 tea samples were contaminated with this fungus. Indeed, these data coincided with the low case rates of OTA-producing A. niger isolates found in Reference [OTA raised the most notable concern due to the high prevalence of nt study . The evaeference .It can be concluded that the contamination rate of OTA in the mycotoxin-positive tea samples in this study was comparable to that reported by other researchers, though it must be noted that OTB was not reported in teas in any previous studies. Thus, only the results of OTA content can be compared.−1 in 4 (11%) of the 36 Pu-erh tea samples analysed [The distribution of OTA reported by Haas et al. (2013) ranged between 0.65–94.7 µg kganalysed .−1 in 69% (n =22) of the analysed tea samples [A. niger, with total mould counts of 1.00 × 102 to 2.8 × 105 CFU g−1 in tea. This study indicated a higher transfer rate (54.47 ± 14.48%) for the green tea compared to the OTA in black tea infusions (33.65 ± 4.37%) [Carraturo et al. (2018) studied the OTA levels in dry leaves of black and green tea and their beverages, with the levels in both dry tea varieties ranging between 0.01 and 21.49 µg kg samples . This st± 4.37%) .−1 in medicinal herbs collected from Latvian drug stores [C. sinensis infusions by Pallarés et al.  [The current results for OTA in herbal teas also coincided with our recent report of OTA levels ranging between 2.86 and 30.3 µg kgg stores . However7, 2019) ,15.−1, and the maximum concentration in herbs from Spain reached only 17.3 µg kg−1 [The results for the OTA content in peppermint teas were comparable to those reported by Santos et al. (2009), whose study also indicated a low contamination rate of OTA in chamomile and spearmint. The OTA levels ranged from 0.1 to 1.4 µg kg µg kg−1 .−1 in paprika and other spices, and 5 and 10 µg kg−1 in ground roasted and instant coffee, respectively [−1 were too low to raise concerns from this perspective.In Europe and other countries, OTA is not regulated in teas or herbs. The only available regulations for the maximum OTA levels in plant matrixes include limitations of 15 µg kgectively . Notably−1) [A recent study also noted that coffee samples from a Latvian market did not exceed the maximum permitted levels of OTA in the European Union (5.0 µg kg−1) .n = 12 each) and ground roasted coffee naturally contaminated at 0.92 µg kg−1 [−1 in dry black teas and up to 104 µg kg−1 in dry herbal teas; these are notably higher levels than those reported in the present study. However, the analysis of tea infusions prepared from the samples with the highest OTA contents indicated a low transfer rate of mycotoxins into infusions—34.8 ± 1.3% from black tea and 4.1 ± 0.2% from fruit tea [Malir et al. (2014) reported the results of a survey of OTA in black and herbal teas  reported T-2 contamination in 88% of the herbal species analysed, with concentrations ranging between 0.28 and 260 µg kg−1 .C. sinensis teas and 24 herbal teas [−1), while no traces of DON were found in any other teas tested within this study. While AcDON metabolites cannot be separated using the method reported in this study, two packaged samples of green tea and three samples of black tea were identified to be positive for some of the acetylated DON metabolites found below the LOQ (<250 µg kg−1) [−1 [Kiseleva et al. (2020) also reported different Fusarium fungal metabolites among the 29 total mycotoxins tested in 24 bal teas . This stµg kg−1) . The resg−1) [−1 .C. sinensis and herbal teas or their infusions, whereas 15-AcDON was found at 60.5–61.0 µg L−1 in two green tea blends with peppermint [−1 in one valerian tea sample [Pallarés et al.  reported that neither T-2 nor HT-2 could be found in ppermint  and at 1The dietary risk assessment provided in the current study also indicated a low rate of potential PDI for most traditional and peppermint teas, which agreed with the previous data reported in Reference  and thosMulti-mycotoxin surveys on Pu-erh teas did not indicate DON or DON derivatives, while the current data on the low ZEN contents in Pu-erh tea were in good agreement with the recently reported data in Reference . ConsideThe emerging Fusarium mycotoxins (four ENNs) and Alternaria mycotoxins have been frequently assessed in different agricultural plant species.n = 22) of the 26 teas contaminated with ENNs, mostly in Pu-erh and herbal teas. ENNs were observed together with DON and DON derivatives in 85% (n = 22) of the samples. The co-occurrence of different Fusarium mycotoxins has been commonly described for more frequently evaluated matrixes of grains and cereal products [ENNs presented the second highest frequency of co-occurrence after DON and DON metabolites: at least two ENNs were found together in 85% (products .n =13) of the 24 C. sinensis tea samples at concentrations of <2.5–6.00 µg kg−1. Within this investigation, ENN B was the predominant ENN toxin, which was found in single-herbal teas at concentration levels between <2.5 and 26 µg kg−1, and in six of the herbal blends at concentrations from 13.6 to 55.0 µg kg−1 [While the present study indicated no traces of BEA in the 70 mycotoxin-positive samples, Kiseleva et al. (2020) reported a relatively high co-occurrence of BEA with ENN A and ENN B, mainly in herbal teas . This su µg kg−1 .−1 [These results agree with the contamination levels of ENN B found in the present study and are also in good agreement with our earlier study of medicinal herbs, which indicated a high prevalence of ENN B in 55% of 60 analysed teas, where the concentrations mostly were below 11 µg kg−1 .While we did not find any reports of ENNs in green and Pu-erh teas, a study by Pallarés et al. (2017) indicated one case of a green tea beverage with ENN B levels below the LOQ . A subseAlternaria toxins in C. sinensis and herbal teas. As discussed in the mycotoxin analysis, ALT and ATX I toxins were mainly found in the Pu-erh tea and green tea samples, and in only a few herbal (mainly peppermint) teas. Alternaria alternata species were observed in only five of the chamomile and peppermint teas, whereas in three samples (in one chamomile and two peppermint tea samples), both fungi and their metabolites were found.The present study provided data on the presence of Alternaria toxins commonly co-occur with AFs and emerging ENNs in different condiments [n = 16) of the samples of the 22 teas with both observed Alternaria toxins. These samples mainly included Pu-erh varieties (n= 10), green and black teas (n =3 and n = 2), one chamomile blend, and the peppermint tea sample contaminated with 16 co-occurring mycotoxins.In a recent review, Crudo et al. (2019) suggested that ndiments . Co-occuAlternaria toxins in tea samples were found, except for the interesting results reported in Reference [Alternaria toxins , with the LOQ values for their quantification reaching 8, 750, and 1000 µg kg−1 [No other studies reporting on eference . Besides µg kg−1 . This st µg kg−1 . These d µg kg−1  that a lAlternaria, Aspergillus, Penicillium, and Fusarium strains and their secondary derivatives in 166 commercial teas, including traditional and novel Pu-erh and oolong teas, as well as common herbal teas  available at retail stores and from tea houses. The microbial quality studies indicated that most of the teas were in good agreement with the recommendations of European institutions and with Latvian regulations of tea product quality, while 30% of the cases exceeded the mould count (>500 CFU g−1) stated in Regulation No. 461 of the Cabinet of Ministers of the Latvian Republic. Moreover, the study data on common A. niger and other Aspergillus species raise concerns related to the mycotoxigenic microbiota of commercial teas, which may affect the production of mycotoxins under improper storage conditions. The predominant A. niger contents indicated the potential production of OTA: Fungi were observed in 47% (n = 8) of the tea samples with OTA levels of 0.9–7.7 µg kg−1. The study data for A. flavus and aflatoxin indicated quantities too low to raise concerns. The presence of DON and DON secondary metabolites in commercial teas at high contamination rates suggests notable potential exposure ranging between 2.4 and 78.9% of the TDI values (1 µg kg−1 bw day−1). This study indicates that despite low contamination levels, the co-occurrence of Aspergillus, Fusarium, and Alternaria  toxins should be considered when providing an assessment of these contaminants in tea beverages and evaluating the long-term effects of potential exposure to co-occurring mycotoxins.The current food safety regulations for the European market provide few mandates related to foodborne and environmentally produced toxins in traditional teas and herbal teas used daily. Although there are some regulations available for AFs, multi-contamination should be considered. This study demonstrated advances in the 2D-LC-TOF-MS method for the simultaneous quantification of regulated and emerging mycotoxins produced by n = 63), green (n = 43), oolong (n = 14), and Pu-erh (n = 20) teas imported from China, India, Japan, Taiwan, Vietnam, Sri Lanka, and Kenya, and 26 herbal teas (chamomile (n = 9), peppermint (n = 7), linden (n = 3), hibiscus, and rosehip (dog-rose) fruits (n = 7)) obtained from Ukraine, Lithuania, Poland, the Russian Federation, Latvia, and other countries. More information on these samples is described in ®, Nasco, WI, USA), and stored in a dark and dry location.A total of 166 teas were purchased from tea stores and local supermarkets in Riga, Latvia, including black (w) of the samples was measured at 25.01 ± 0.02 °C in triplicate using an AquaLab 4TE dew-point water activity meter  according to the ISO 18787:2017 standard [The water activity (astandard .−1) in the tea samples were calculated for the number of colonies on all three plates. Different dilution levels  were evaluated to determine the most suitable conditions for each tea variety .The mould colony-forming unit (CFU) counts per 1 g of tea were counted according to ISO standard 21527–2:2009 . A suspePreparation of the obtained fungal isolates for MALDI-TOF MS identification was performed as previously described , accordiAlternaria, Aspergillus, Fusarium, and Penicillium strains , enniatin B (ENN B), enniatin B1 (ENN B1), 15-acetyldeoxynivalenol (15-AcDON), 3-acetyldeoxynivalenol (3-AcDON), tentoxin (TNX), meleagrin (MEL), and citreoviridin (CVD) were purchased from Cayman Chemicals . Deoxynivalenol (DON), aflatoxins , HT-2 toxin (HT-2), T-2 toxin (T-2), ochratoxin A (OTA), zearalenone (ZEN), fumonisins B1 and B2 , sterigmatocystin (STC), citrinin (CIT), fusarenon-X (FUS-X), and deoxynivalenol-3-glucoside (D3G) were all provided by Romer Labs . Penicillic acid (PA), mycophenolic acid (MPA), and roquefortine-C (ROQ-C) were purchased from Santa Cruz Biotechnology . Altenuene (ALT), alternariol (AOH), alternariol monomethyl ether (AME), altertoxin I (ATX I), ochratoxin B (OTB), aflatoxicol (AFL), verruculogen (VCL), penitrem A (PNA), neosolaniol (NEO), T-2 tetraol (T-2TET), 15-acetoxyscirpenol (15-AcS), T-2 triol (T-2TRI), and fumonisin B3 (FB3) were all purchased from Fermentek . Acetonitrile (ACN), methanol (MeOH), dimethyl sulfoxide (DMSO), and ethyl acetate were all of pesticide grade and were purchased from Sigma-Aldrich . N,N-dimethylformamide (DMF), also of pesticide grade, and formic acid of analytical grade were both purchased from Merck Millipore . Ultrapure water was generated using a Milli-Q system . The 6 mL Strata® solid-phase (500 mg) extraction cartridges  based on amine sorbent (NH2-SPE) and silica (C18-SPE) were both purchased from Phenomenex .The 42 tested mycotoxins were of at least 95% purity. Fusaric acid (FA), beauvericin (BEA), enniatin A (ENN A), enniatin Av/v) extraction solution (15 mL) was added to a tea sample (2 g) weighed into a 50 mL centrifuge tube. The mixture was shaken for 15 min and then subjected to centrifugation . The extract was filtered through cellulose paper and divided into two portions. The first portion of the extract (3 mL) was evaporated until dry under a gentle nitrogen stream at 50 °C. The dry residue was then dissolved in a MeOH/water  solution (1.5 mL) and vortexed for 5 min. The second part of the extract (12 mL) was additionally cleaned via an NH2-SPE column that was pre-conditioned with an ethyl acetate/formic acid  mixture (5 mL) followed by loading the extract through the column and collecting the eluate in a 15 mL centrifugal tube. The column was dried by sucking out air under a vacuum for 5 min, and the eluate was evaporated under the same conditions as the first portion of the extract. The residue was dissolved in 5 mL of MeOH/water  and cleaned by passage together with the first portion of the extract through a C18-SPE column that was conditioned with a MeOH/water  solution (10 mL). After passing both parts of the extract through the C18-SPE, the column was dried under a low vacuum followed by the elution of mycotoxins with 5 mL of ACN. The eluent was evaporated, and the dry matter was dissolved in 150 µL of water/methanol  solution containing 0.1% (v/v) formic acid and transferred to autosampler vials for chromatographic analysis. Standard stock solutions of all mycotoxins were prepared in ACN, MeOH, or their compositions with DMSO, except for BEA and ENNs, which were kept in DMF  analytical column packed with fully porous silica particles. The two-dimensional (2D) separation was realised with a Luna® Omega Polar C18  analytical column packed with core–shell silica particles. The column oven temperature was 40 °C. The mobile phase A used in both the 1D and 2D column gradient systems consisted of deionised water with 0.1% (v/v) formic acid. A total of 0.5 mM ammonium acetate in acetonitrile with 0.1% (v/v) formic acid was used in the gradient of the 1D column (phase B1), and acetonitrile supplemented with 0.1% (v/v) formic acid was used as the organic phase in the 2D column gradient programme (phase B2). The mobile phase flow rate was 0.35 mL min−1 for both dimensional columns. The elution programmes for the 1D and 2D separation and valve switching information are summarised in The 2D-LC analysis was performed on an UltiMate 3000 UHPLC system  containing a binary pump, a degasser, a column oven compartment, an autosampler manager fitted with a 500 μL injection loop used to capture the fraction eluted from the one-dimensional (1D) analytical column, and a programmable Rheodyne1, ENN B, ENN B1, CVD, CIT, OTA, OTB, MPA, PNA, STC, T-2, VCL, ZEN) during a time period from 2.0 to 13.1 min. Using the same gradient system, a fraction containing 26 analytes  was collected in the sample storage loop up to 2.0 min and transferred to the 2D column.The gradient programme of the 1D column separation started with 50% of acetonitrile, providing the resolution of 16 analytes  source and a Compact Q-ToF-MS were used for the mass analysis, performed in the positive full-scan mode and operated by the Control 4.0 software . The following TOF-MS source parameters were used: capillary cap voltage of 4.5 kV, spray shield voltage of 0.5 kV, desolvation temperature of 200 °C, nitrogen flow rate of 10 L min2). The precision, repeatability, and recovery studies during intraday tests (two days within a one-week period) were performed by analysing six replicates at each of the three determined spiking levels. The mean values of each replicate were compared to the acceptance criteria specified by the EC regulation No. 519/2014. The signal-to-noise (S/N) approach was used to estimate the LOD and the LOQ. The chromatographic noise and analytical response were estimated using chromatograms of the spiked samples. The LOD and LOQ were defined based on signal (S)-to-noise (N) ratios of S/N > 3 and S/N > 10, respectively. The measurement uncertainties (MU) were calculated according to Eurachem/CITAC Guide CG 4 [u′(wR) is the within-laboratory reproducibility, u′(bias) is the uncertainty component arising from the method and laboratory bias, MU is the expanded measurement uncertainty, and the coverage factor k = 2 is at a 95% confidence level [Validation was performed according to the performance guidelines of the Commission Regulation (EU) No 519/2014 and the “Guidance Document on the Estimation of LOD and LOQ for Measurements in the Field of Contaminants in Feed and Food” . The selide CG 4 :(1)MU=k×ce level .The methodology reported in our previous study was also used here . ConsumpFor the acute risk assessment, the probable daily intake (PDI) values were calculated for two scenarios—exposure to the highest contamination level and the upper-bound (UB) contamination levels. A conservative approach was applied to the occurrence data and all cases of non-detection were replaced with the LOQ values for the particular analytes, in order to obtain the UB values according to EFSA recommendations for the dietary exposure evaluation methodology .−1, according to the data reported in the literature [C is the found mean or highest (maximum) mycotoxin concentration found for the analysed dry tea samples of the selected tea variety, pm is the assumed tea weight per teaspoon (2.00 g), and pV is the volume of a typical tea beverage portion (200 mL), infused for 5 min according to common data and tea manufacturer recommendations; cV is the medium intake volume (mL) of tea beverages by the target group, and B is the median selected body weight of 70 kg for adolescents, adults, and the elderly in Europe, which was adopted based on the EFSA recommendations [As the current study did not provide an evaluation of beverages but only raw tea samples, the worst-case scenario was assessed under the assumption that all of the mycotoxin content found in the dry teas would be transferred to the tea infusions. Our recent study indicated that the DON and ZEN levels in dry herbs and their infusions can be transferred almost fully into beverages . Howeverterature . In the ndations .Camelia sinensis tea. This coincided with results reported by other researchers, namely that 1.5 to 2.0 g of dry tea and 150–250 mL of water are commonly used for the preparation of tea beverages [Most of the bagged tea samples included in the current study contained 1.0 to 2.0 g of tea. The maximum sample weight per portion was chosen to be 2.0 g, which fit the approximate weight of one teaspoon of everages ,15,16.−1 bw day−1 of DON and also the sum of DON and its derivatives , 17.1 µg kg−1 bw day−1 of OTA (which corresponds to the tolerable weekly intake (TWI) of 120 µg kg−1 bw week−1), and 0.25 µg kg−1 bw day−1 of ZEN [The PDIs were compared with the recommended tolerable daily intake (TDI) levels found by the EFSA for certain mycotoxins: 1 µg kg1 of ZEN ,68. The 1 of ZEN  was used1 and 4AFs determined in C. sinensis and peppermint teas, their PDI values were also evaluated. However, due to the carcinogenic effects, AFs have no tolerable levels. To estimate the potential health risk related to the consumption of teas contaminated with AFs, the benchmark dose (BMD) approach of the European Food Safety Authority was used. For dose–response modelling of aflatoxins, the BMD lower confidence limit (BMDL10) for a 10% increase in cancer incidence obtained from animal study data modelling (170 ng kg−1 bw day−1) was considered (European Food Safety Authority [Due to the high prevalence of AFButhority .


FOXG1(*164874), NKX2‐1(*600635), and PAX9(*167416) and a distal region (RO2), including NKX2‐1 and PAX9. We report a 6‐year‐old boy with mild dysmorphic facial features, global developmental delay, and hypoplasia of the corpus callosum.Chromosome 14q11‐q22 deletion syndrome (OMIM 613457) is a rare contiguous gene syndrome. Two regions of overlap (RO) of the 14q12q21.1 deletion have been identified: a proximal region (RO1), including Array‐CGH analysis revealed a 14q12q13.2 microdeletion. We compared the phenotype of our patient with previously published cases in order to establish a genotype–phenotype correlation.SNX6(*606098) and BAZ1A(*605680) as potential candidate genes associated with the corpus callosum abnormalities.The study hypothesizes the presence of a new RO, not including the previously reported candidate genes, and attempt to define the associated molecular and psychomotor/neurobehavioral phenotype. This region encompasses the distal breakpoint of RO1 and the proximal breakpoint of RO2, and seems to be associated with intellectual disability (ID), hypotonia, epilepsy, and corpus callosum abnormalities. Although more cases are needed, we speculated on  We report a 6‐year‐old boy with mild dysmorphic facial features, global developmental delay, and hypoplasia of the corpus callosum and 14q12q13.2 microdeletion. In order to establish a genotype‐phenotype correlation we compared the phenotype of our patient with previously published cases, hypothesize the presence of a new RO and attempt to define the associated molecular and psychomotor/neurobehavioral phenotype. No prenatal/perinatal complications were reported. Motor development was slightly delayed, reaching unaided ambulation at 17 months. Language development delay was also observed; he began saying few words at 12 months, but was able to formulate simple sentences only at 3 years of age. At infant school age, the proband had social impairment due to the motor coordination and language difficulties, and deficits in attention.The patient was referred at age of 5 years. Neurological examination showed generalized hypotonia and severe motor clumsiness. Head circumference was 54 cm (90‒97° centile). He also showed sleep disturbances . Verbal communication skills were severely impaired: his expressive language was characterized by phonological deficits and restricted to simple sentences. Verbal reasoning was limited to simple, contextual, and daily tasks. Nonverbal communication was normal. Routine hematologic and biochemical tests, including complete blood count, liver, and renal function tests, were normal. Awake and asleep EEG monitoring showed a normal brain bioelectrical activity.At the last physical examination, the proband was 6 years and 3 months old, his height was 115 cm (25‒50° centile), his weight 28 kg (>97° centile), body mass index (BMI) 21.2 (99° centile), and the head circumference 55 cm (>97° centile). The parents reported persistent sleep disturbances. Hematologic and biochemical tests  were normal.Physical examination showed wide hands with stubby fingers, short neck, high forehead, low setting ears, regional abdominal fat distribution, small lesion along the right mammary line (possible supernumerary nipple), and phimosis Figure . Skeleta1H‐magnetic resonance spectroscopy (1HMRS) of the frontal lobe white matter did not reveal altered metabolites. Neurocognitive assessment included the administration of the Wechsler Intelligence Scale for Children‐Fourth Edition  showed hypoplasia of the CC including the posterior portion Figure , abnorma3AKAP6(*6046961), NPAS3, EGLN3(*606426), SPTSSA(*613540), EAPP(*609486), RNU1‐27P, RNU1‐28P, SNX6, CFL2, BAZ1A, SRP54(*604857), PROP(*601538), PSMA6, NFKBIA, INSM2(*614027), RALGAPA1. Therefore, the final karyotype of the patient was designed as (ISCN 2016): 46,XY.arr[GRCh37] 14q12q13.2(33147358_36088096)x1dn.Cytogenetic analysis was performed on QFQ‐banded metaphases (550‐band level): in all the examined metaphases, a normal male karyotype  was observed. Genomic DNA was extracted from peripheral blood samples. DNA concentration was measured by fluorimeter  using the Hoechst reagent and adjusted to 400 ng/ml. Array‐CGH analysis was performed using the Cytochip oligo ISCA 4x180K (TechnoGenetics Srl). The analysis revealed an interstitial deletion involving the long arm of chromosome 14: arr[GRCh37] 14q12q13.2(33147358_36088096)x1, size 2.94 Mb Figure . This re4Here, we report a 6‐year‐old patient with mild ID and hypoplasia of the CC due to a de novo 2.94 Mb microdeletion at the 14q12q13.2 chromosomal region.FOXG1, NKX2‐1, and PAX9), spanning from 28,000,000 to 35,000,000, associated to severe developmental delay and poor prognosis, and a distal region , spanning from 35,000,000 to 40,000,000, associated to a milder phenotype with quite normal psychomotor development  locus (HPE8) had been hypothesized by Kamnasaran, Chen, Devriendt, Mehta, and Cox . HoweverFOXG1, NKX1, and PAX9  with SNX6FL haploinsufficiency suggests functional redundancy among the SNX family members  showed that 10 out of these 27 genes are associated to cytoskeleton, integrin and synaptic related pathways, pinpointing the relevance of BAZ1A in neural development as supported by the findings of Zaghlool et al. , encoding a RaI GTPase activating protein, expressed ubiquitously in pre‐ and postnatal human tissues, particularly in the brain, is a candidate gene for many neurological findings reported in 14q deletion patients  including the “proximal” region, characterized by severe ID, CNS malformations and poor prognosis, a more distal microdeletion region (RO2), with variable expressivity, associated with mild/absent ID, and a third small overlapping region (RO3), encompassing the distal breakpoint of RO1 and the proximal breakpoint of RO2, expressing with ID, hypotonia, epilepsy, and CC abnormalities Figure . In thisThe authors declared that they have no conflict of interest.Emanuela Ponzi contributed to conceptualization/design, methodology, investigation, data curation, formal analysis, resources, and participation in writing and revision. Mattia Gentile contributed to conceptualization/design, methodology, investigation, data curation, formal analysis, resources, supervision/oversight, participation in writing and revision. Emanuele Agolini contributed to supervision/oversight and participation in revision. Emilia Matera, Roberto Palumbi, Antonia Peschechera, Alessandra Gabellone contributed to investigation and clinical data curation and Antonia Lucia Buonadonna Maria Fatima Antonucci contributed to methodology, investigation, data curation, formal analysis. Lucia Margari contributed to the study with supervision/oversight and revision. All authors have approved the manuscript and its submission.The authors certify that they have obtained all appropriate patients consent forms. In the form, the patients have given their consent for their images and other clinical information to be reported in the journal.


Echinococcus granulosus sensu lato (s.l.), resulting in large economic losses to livestock husbandry  is one of main features of this parasite, and identifying its genotype would be advantageous for prevention and control of the disease (Cystic echinococcosis (CE) is a parasitic zoonosis caused by the larval stage (hydatid cysts) of usbandry . A high  disease .Macroscopic diagnosis at necropsy alone would resulted in a 15.4% error due to the variability of cystic morphology . If the Compared with visual inspection, molecular techniques, especially PCR assays using mitochondrial markers , are being increasingly applied to CE clinical detection due to the accuracy and specificity . The PCRE. granulosus s.l.Herein, we specifically developed a direct PCR assay for detection and genotyping of hydatid cysts in slaughtered livestock by amplifying the complete mitochondrial NADH dehydrogenase subunit 6 gene  of E. granulosus s.l., were used to amplify the complete ND6 gene (558 bp). PCR reaction volume contained 10 μL 2×PCR Easy Mix (containing D-Taq DNA polymerase), 0.5 μL of each primer (20 mM), 4.0 μL of lysate and 5 μL of double-distilled water (dd H2O). Thermal cycling proceeded within 1 h, and conditions included an initial denaturation at 95 °C for 3 min, followed by 30 cycles of denaturation at 95 °C for 10 s, annealing at 58°C for 20 s, and extension at 72 °C for 20 s, then a final extension at 72 °C for 5 min. All testing were performed and analysed in triplicate.In this study, the lysate of washed hydatid cysts, treated by an Animal Tissue Direct PCR Kit  within 0.5 h, could be directly used as template in our PCR protocol. Then, the forward (5′-TTTCGTGCTGTAGATGGT-3′) and reverse (5′-CACAGATTTCAAAGGGTT-3′) primers designed based on the conserved regions of eight genotypes of E. granulosus (Accession Nos. KJ559023.1). No cross-reactivities were found between hydatid cysts and six other metacestodes commonly found in China including E. alveolaris, Cysticercus tenuicollis, Coenurus cerebralis, C. bovis, C. cellulosa and C. viscerotropica, indicating a high species specificity of this method  samples were positive by the direct PCR. Moreover, the sequence data of ND6 genes showed that 65 cysts belonged to G1 genotype and three cysts belonged to G3 genotype, which were the same as the genotyping results of the COX1 gene (not shown), suggesting the ND6 gene can be also a good genetic marker for the genotyping of In conclusion, the direct PCR assay developed in this study displayed a high specificity, sensitivity and effectiveness. Moreover, a low cost with ∼$0.58 per should make it an ideal tool for large-scale CE clinical detection in future.


P < 0.05) activity, while the mRNA expression (P < 0.05) increased only in the high-dose group. Additionally, CYP2C11 activity was induced and consistent with mRNA expression (P < 0.05). Moreover, ZWD could induce the activity of CYP3A1 (P < 0.05), but the mRNA expression showed no significant differences except in high-dose groups. Additionally, ZWD has no effects on CYP1A2, CYP2B1, CYP2C7, and CYP2D2. In conclusion, the significant inductive effects of ZWD on three CYP450 isoenzymes indicated that when ZWD was coadministrated with drugs mediated by these enzymes, not only should the potential herb-drug interactions (HDIs) be observed, but the dosage adjustment and tissue drug concentration should also be considered. Furthermore, the approach described in this article can be applied to study the importance of gender, age, and disease factors to HDI prediction.This thesis is aimed at shedding light on the effects of the Zhenwu decoction (ZWD) on the activities and mRNA expressions of seven CYP450 isoenzymes. In the first step, we determined the main chemical compounds of ZWD by high-performance liquid chromatography (HPLC). Next, 48 male (SD) rats were randomly divided into the normal saline (NS) group and the ZWD low- (2.1875 g/kg), medium- (4.375 g/kg), and high- (8.75 g/kg) dose groups (12 per group). All rats were gavaged once daily for 28 consecutive days. A mixed solution of seven probe drugs was injected into 24 rats through the caudal vein after the last intragastric administration. Lastly, a validated cocktail method and real-time quantitative reverse-transcription polymerase chain reaction (RT-qPCR) were used to detect pharmacokinetic parameters and mRNA expressions, respectively. Compared with the NS group, ZWD at medium- and high-dose groups could significantly induce CYP2C6 ( Aconitum carmichaelii Debx.), Zingiberis , Atractylodis Macrocephalae Rhizoma , Paeoniae Radix Alba , and Poria (sclerotium of Poria cocos (Schw.) Wolf.), and modern researches have shown that ZWD has therapeutic effects on nephrotic syndrome [The Zhenwu decoction (ZWD), one of the classic prescriptions for the treatment of Yang deficiency, was recorded in the “Treatise on Febrile Diseases” by Zhang Zhongjing. It is composed of Aconiti Lateralis Radix Praeparata  was purchased from Beijing Zhongke Quality Control Biotechnology, Inc. . Paeoniflorin (No. 110736201539) and atractylenolide III (No. 73030-71-4) were obtained from the National Institutes for Food and Drug Control . 6-Gingerol (No. MUST-17120205) was purchased from Chengdu Man Site Pharmaceutical Co., Ltd. . Dehydrotumulosic acid was purchased from Chengdu Chroma-Biotechnology Co., Ltd. . Probe drugs used include phenacetin, bupropion, diclofenac, amodiaquine, omeprazole, dextromethorphan, and midazolam . The standard materials were supplied by the Chinese Food and Drug Administration Research Institute . Glibenclamide  was purchased from Yuanye Biotechnology Co., Ltd. . The purity of the standards was higher than 98%. The following materials were also acquired: TRIzol Reagent , Verso cDNA Synthesis Kit , SYBR Green PCR Kit , PCR primer , and DEPC water .18 Column ; and Millipore Milli-Q purification system .The following instruments were used: Agilent 1290 Infinity ; AB SCIEX 4500 triple-quadrupole mass spectrometer ; real-time quantitative PCR ; Acquity CSH CZingiber officinale Roscoe was homemade . The extraction process was as follows: Aconitum carmichaeli Debx : Poria cocos (Schw.) Wolf : Zingiber officinale Roscoe : Paeonia lactiflora Pall : Atractylodes macrocephala Koidz  at the proportion of 9 : 9 : 9 : 9 : 6, soaked in 10 times distilled water for 30 min and then boiled for 1.5 h, filtered, and the residue boiled with 8 times water for 1 h again. The two filtrates were mixed and concentrated to 2.1 g/mL and stored at 4°C.The dried raw herbs of ZWD were purchased from Anhui Puren Chinese Herbal Medicine Co., Ltd. ; The proper amount of seven probe substrates were dissolved in a certain amount ) of NS and anhydrous ethanol (0.5 mL). Ultrasonically stirred until homogeneous, Tween 80 was slowly dropped into the solution until it was clear and transparent. Finally, the volume with NS was determined. Notably, the solution was prepared when we needed it, and the dose of mixed probe substrates was 1 mL/kg.18 column , a column temperature of 30°C, a flow rate of 1 mL·min−1, a wavelength of 230 nm, and 0.05% phosphoric acid aqueous solution (A)-acetonitrile (B) gradient elution described as follows: 0-10 min, 91%-88% (A); 10-20 min, 88%-85% (A); 20-33 min, 85%-65% (A); 33-38 min, 65%-60% (A); 38-48 min, 60%-52% (A); 48-58 min, 52%-57% (A); and 58-62 min, 57%-91% (A).High-performance liquid chromatography (HPLC) was performed to support the stability and quality of the ZWD extract. The following chromatographic conditions were used: a Shimadzu LC-15C UV HPLC system with a CAll rats were kept at a room temperature of 20 ± 2°C with a 12 h light/dark cycle and 50 ± 5% relative humidity for one week. The use of animals reported here have been approved by the Institutional Animal Care and Use Committee of Anhui Medical University , and the experimental procedures were conducted in accordance with the Guidelines for Proper Conduct of Animal Experiments. 48 male Sprague-Dawley (SD) rats (240~280 g) were randomly divided into the NS group and the ZWD low- (2.1875 g/kg), medium- (4.375 g/kg), and high- (8.75 g/kg) dose groups (12 per group) . After oOn the 29th day, half of the 48 rats received a cocktail substrate solution through the tail vein at a dose of 1 mL/kg, and plasma samples were obtained following the established procedures . MeanwhiA260/A280) was in the range of 1.8-2.0, indicating the excellent quality of RNA [μL) was reversely transcribed into cDNA at 42°C for 60 min, 70°C for 5 min, and 4°C for 5 min. Reverse-transcription products were stored at -80°C until analysis.Based on the manufacturer's protocol, total RNA was isolated from 50 mg liver samples using the TRIzol Reagent. Then, the RNA concentration was determined and the absorbance ratio .The forward and reverse primer sequences are listed in T1/2), clearance (CL), and volume (V). mRNA expressions were performed by 2−ΔΔCT calculation. Results are expressed as mean ± standard deviation , half-life time  were 0.9986, 0.9980, 0.9965, 0.9984, 0.9967, 09977, and 0.9996, respectively.The specificity results  indicateInterday precision and intraday precision of the method were assessed by detecting the low limit of qualification (LLOQ) and the low-, medium-, and high-quantification concentrations  of plasma samples. Relative standard deviation (RSD) values of the precision do not exceed ±15% Tables  and 3.By comparing the different results of analytes added into the blank sample and ultrapure water, the matrix effect was determined at LQC, MQC, and HQC concentrations. The RSD values in The stability of all probe drugs was evaluated by LQC and HQC samples under different experimental conditions, including short-term stability (4 h at room temperature (25°C), 8 h in the automatic sampler, and three freeze and thaw cycles, respectively) and long-term stability (7 d at -80°C). Results showed that the probe substrates tested were within the recommended limits, RSD < 10% .P > 0.05), except for T1/2 changes of bupropion, amodiaquine, and dextromethorphan (P < 0.05).The CYP1A2, CYP2B1, CYP2C7, and CYP2D2 activities were investigated by analyzing the pharmacokinetic parameters of phenacetin, bupropion, amodiaquine, and dextromethorphan, respectively. t0− and AUC0−∞ (P < 0.05) in a dose-dependent manner, which was consistent with the CL and V values (P < 0.05), while no changes were observed between the ZWD-L and NS groups.To describe changes in CYP2C6 activity, t0− and AUC0−∞ were significantly reduced in a dose-dependent manner (P < 0.05), and the CL value was significantly increased (P < 0.05), which means that drug plasma concentration was decreased and drug metabolism was accelerated. Interestingly, the CL value was the lowest in the ZWD-M group, consistent with T1/2. Accordingly, ZWD affects the drug metabolism mediated by CYP2C11.Omeprazole is a specific probe substrate for rat CYP2C11. The corresponding pharmacokinetic profiles and the mean plasma concentration-time curves are presented in t0− and AUC0−∞ decreased and CL increased as the dose increased (P < 0.05); notably, T1/2 was also reduced significantly (P < 0.05) and V was approximately constant. Changes in pharmacokinetic parameters of midazolam in rats suggested that CYP2A1 enzyme activity was induced, leading to an acceleration of the metabolism as well as a reduction of the plasma drug concentration.In rats, midazolam was metabolized by CYP3A1, as shown in As shown in Drug metabolism-induced interactions account for about half of HDIs, and CYP450 enzymes dominate in metabolism. At a basic level, a comprehensive assessment of the effects of ZWD on CYP450 enzymes is therefore critical for predicting HDIs during integrative medicine practice.CYP450 enzyme activity is influenced by species, gender, age, environment, medication, and pathological condition . SimilarFormulaology textbook of the National TCM Industry, “Ancient Classics List (First Batch)” and literature [For the dosage of ZWD, the terature , 21 wereAs mentioned in the literature, 82 volatile components were isolated by GC-MS, and five constituents of ZWD were qualified by HPLC , 23. In In vivo, CYP3A accounts for about 40% of the total liver P450 enzymes and mediates 50-60% clinical drug metabolism such as erythromycin, nimodipine, and lidocaine [Poria cocos aqueous extract can significantly increase the CYP2C6, CYP2B1, and CYP3A1 activities and upregulate their mRNA expressions [Poria cocos and Aconiti Lateralis Radix can induce pregnane X receptor (PXR) activation and further mediate the transcription and expression of CYP3A4 [V of diclofenac, omeprazole, and midazolam exceeded the total fluid volume, indicating that they are easily ingested into tissues. The concentration of drug in tissues, therefore, also needs to be monitored clinically in real time. Notably, T1/2 of midazolam was significantly decreased, CL was consistent with it, V is constant, and the metabolic process of midazolam conforms to the nonlinear pharmacokinetic model.idocaine . CYP2C oidocaine , 30. Stuf CYP3A4 , 32. HowIn summary, this dissertation was undertaken to evaluate the effects of ZWD on the activities and mRNA expressions of seven CYP450 enzymes by using a cocktail method. The method has proven to be sensitive, efficient, and reliable. Pharmacokinetic profile analysis shows that when ZWD is coadministrated with drugs metabolized by CYP2C6, CYP2C11, and CYP3A1, not only should the potential herb-drug interactions (HDIs) be observed but the dosage adjustment and tissue drug concentration should also be considered. Furthermore, the above approach can be applied to study the importance of gender, age, and disease factors to HDI prediction.


Borrelia burgdorferi, has a large number of membrane proteins involved in a complex life cycle, that includes a tick vector and a vertebrate host. Some of these proteins also serve different roles in infection and dissemination of the spirochete in the mammalian host. In this spirochete, a number of proteins have been associated with binding to plasminogen or components of the extracellular matrix, which is important for tissue colonization and dissemination. GroEL is a cytoplasmic chaperone protein that has previously been associated with the outer membrane of Borrelia. A His-tag purified B. burgdorferi GroEL was used to generate a polyclonal rabbit antibody showing that GroEL also localizes in the outer membrane and is surface exposed. GroEL binds plasminogen in a lysine dependent manner. GroEL may be part of the protein repertoire that Borrelia successfully uses to establish infection and disseminate in the host. Importantly, this chaperone is readily recognized by sera from experimentally infected mice and rabbits. In summary, GroEL is an immunogenic protein that in addition to its chaperon role it may contribute to pathogenesis of the spirochete by binding to plasminogen and components of the extra cellular matrix.The spirochete,  Borrelia burgdorferi is the causative agent of Lyme disease [Ixodes ticks and initially infects the host at the site of the tick bite. The spirochete disseminates and invades distant tissues with the help, at least in part, of plasminogen (PLG) [Borrelia can bind to PLG, including OspA [Borrelia dissemination in the host [The spirochete  disease ,2. The sen (PLG) ,4, a zyming OspA , OspC [6ing OspA , BBA70 [ing OspA , and Erping OspA , among othe host ,11,12. Tthe host .B. burgdorferi expresses an array of proteins involved in adhesion to host cells [Borrelia has many proteins involved in binding to ECM components, many of which have redundant activities [Borrelia adhesins bind to laminin, fibronectin, glycosaminoglycans, collagen, and integrins [Colonization of host tissues is vital for extracellular bacteria. During early infection, st cells . These ativities ,15. Borrntegrins . Interesntegrins  while BBntegrins ,18. On tntegrins , whereasntegrins .B. burgdorferi, such as enolase and HtrA, that are present in multiple subcellular compartments [Streptococcus species also have glycolytic enzymes with PLG binding properties [Bacillus anthracix, and Mycoplasma pneumoniae [Canonical outer surface proteins are not the only ones shown to play a role in colonization, dissemination and immune evasion. There are moonlighting proteins in artments ,23,24,25artments , persistartments , PLG [21artments ,23, and artments  binding.operties ,30,31, ioperties . These poperties ,33. GroEeumoniae ,35. GroEeumoniae ,35. The Borrelia GroEL (BbGroEL) was detected in different studies associated with the outer membrane (OM) [ane (OM) ,36,37 anane (OM) . The proane (OM) . This stE. coli Rosetta (DE3) sonicate was subjected to affinity chromatography  by the addition of ε-aminocaproic acid . The results showed that the binding of BbGroEL to PLG, laminin and fibronectin was dose dependent  and was oic acid , a well-B. burgdorferi were tested against Borrelia whole-cell lysates and purified BbGroEL  and its receptor, uPAR, that can facilitate the dissemination of the spirochete by activating the membrane-bound PLG [Mycoplasma pneumoniae has a surface exposed GroEL protein that binds to PLG [There are a large number of proteins associated with the PLG system in Borrelia . Both reembranes ,61,62. Sembranes ,61,63. Iound PLG ,64,65,66s to PLG . Althougs to PLG . GroEL is to PLG ,30,32,68s to PLG ,31, and s to PLG  from difB. burgdorferi tightly regulates gene expression while transitioning from the tick vector to the mammalian host. As a result, the protein composition of the OM changes dramatically. The quantity of BbGroEL changed upon temperature and pH shifts that mimic, with limitations, the host environment [Pseudomonas aeruginosa, Vibrio cholera, and E coli the expression of groEl is controlled positively by RpoH (σ32) [B. burgdorferi does not have a σ32 homolog. Alternatively, other organisms use heat-shock transcriptional regulators that function as repressors, including HrcA, HspR, and CtsR [B. burgdorferi. Interestingly, the quantity of GroEL seems to increased more in the membrane that in the cytoplasm at increasing temperatures [Lastly, ironment ,69,70. Iironment . For exaoH (σ32) ,73,74. Hand CtsR , which aeratures  but the eratures . CollectB. burgdorferi via tick bite and needle, respectively. This result confirms the immunogenicity of BbGroEL, which has also been shown in other spirochetes, including different pathogenic strains of Leptospira spp. [Treponema pallidum [Interestingly, BbGroEL is recognized by sera from a rabbit and mice infected with ira spp.  and Treppallidum . Nonethepallidum ,78,79 anB. burgdorferi strain B31 cultures were grown to mid-log phase in BSK-II medium supplemented with 6% rabbit serum  at 33 °C; except cultures used for the shift protein expression experiment that were grown at 33 °C and pH 6.8 and 23 °C and pH 7.6. Escherichia coli DH5α and Rosetta (DE3)  were grown in LB media  in the presence of kanamycin (50 µg /mL). A New Zealand White rabbit  was inoculated intradermally with 100 μg of recombinant BbGroEL in complete Freund’s adjuvant followed by two boosters of 50 μg of the recombinant BbGroEL in incomplete Freund’s adjuvant. Mouse sera from C3H/HeN mice  infected with 2 × 104 spirochetes were collected 4 weeks after inoculation. B. burgdorferi infected rabbit serum was generously provided by Dr. Jorge Benach.All g for 20 min. After washing with PBS containing 0.1% BSA, spirochetes were incubated in a 25 mM citrate buffer (pH 3.2) in agitation for 2 h at room temperature to separate the OM and the PC. Both fractions were subsequently isolated by using a discontinuous sucrose gradient  followed by a continuous sucrose density gradient . Lastly, the isolated PC fraction was diluted (1:5) in PBS, pelleted at 10,000× g for 20 min, resuspended in PBS and stored at −80 °C. The purified OM fraction was diluted in PBS (1:5), centrifuged at 141,000× g for 4 h, and the pellet was resuspended in a 1 mM phenylmethylsulfonyl fluoride (PMSF) solution and stored at −80 °C.The separation of the OM and IM was carried out as previously described ,80,81. BThe purity of both fractions was evaluated through the use of antibodies that targeted proteins present in the OM, OspA , IM, HflN-terminal Histidine-tagged GroEL was created in a pET-28a(+) vector . Briefly, the GroEL gene (bb0649) was amplified using primers GroELF (5′-ATGGCTAAAGACATATATTTT-3′) and GroELR (5′-TTACATCATTCCCATTCCTGG-3′) followed by digestion with the restriction enzymes Ndel and Xhol .E. coli Rosetta upon induction with 0.3 mM isopropyl-β-d-thiogalactopyranoside (IPTG). Cells were centrifuged at 11,000× g at 4 °C for 10 min, and the pellet was washed with PBS . The pellets were sonicated and the supernatant collected for affinity chromatography.Recombinant BbGroEL was expressed in A 5 mL gravity flow column  was prepared with a Ni-Sepharose resin . The supernatant was run through the column, and the flow-through was collected and stored at 4 °C for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis.An initial wash step was performed by adding a wash-bind buffer  to the column. The wash fraction was collected and stored at 4 °C for SDS-PAGE analysis. Subsequently, the elution buffer  was added to the column. Eluted fractions (1 mL) were collected and kept at 4 °C until analysis by SDS-PAGE. Induced cell extract, flow-through, wash, and elute fractions were all assessed by SDS PAGE analysis followed by staining with coomassie brilliant blue .The elute fractions were subjected to gel filtration chromatography. Briefly, Sephadex G-75 media  was added to a glass chromatography column  and the eluted fractions from the previous purification were run through. Collected fractions were analyzed by SDS-PAGE.g) .Samples containing the purified protein were pooled and subjected to a buffer exchange  using an Amicon Ultra-15 Centrifugal filter unit . Samples were boiled for 5 min and loaded into 12.5% SDS-PAGE gels and separated by electrophoresis. Gels were transferred to a nitrocellulose membrane  and blocked for 1 h with 5% non-fat milk powder in TBST. The primary antibodies used for Western blot include rabbit polyclonal anti-HflC [® PREMIUM Chemiluminescent Substrate  and visualized by LI-COR C-DiGit Blot Scanner . Protein concentration for all samples were calculated using a Bradford protein assay , and equal amounts loaded to a SDS-PAGE gel.Different Western blot assays were conducted to analyze the polyclonal anti-BbGroEL antibody activity, separation of OM and PC fractions, and proteinase K digestion of OM. Briefly, 1 µg of purified BbGroEL, 2 µg of purified OM and PC fractions, 1 µg of nti-HflC , monoclonti-HflC , monoclonti-HflC , polyclonti-HflC . After iB. burgdorferi lysate in bicarbonate-carbonate coating buffer . ELISA plates were blocked with 1% casein  for 1 h at 37 °C and washed with PBS. The plate was incubated with polyclonal rabbit anti-BbGroEL serum or rabbit anti-human PLG at 37 °C for 1 h and washed 3 times with PBS. Different ELISA experiments were carried out as follows: (i) To detect bound BbGroEL to ECM , and PLG rabbit polyclonal anti-BbGroEL was incubated for 1 h at 37 °C, washed 3 times with PBS, and incubated with a goat anti-rabbit IgG-alkaline phosphatase conjugate (Sigma) at 37 °C for 1 h. The experiment was repeated using increasing concentrations of NaCl to address whether PLG binding was mediated by ionic interactions. (ii) To detect PLG bound to BbGroEL in the presence or absence of ε-aminocaproic acid (ACA), rabbit anti-human PLG  was incubated for 1 h at 37 °C, washed 3 times with PBS, and incubated with goat anti-rabbit IgG-alkaline phosphatase conjugate (Sigma) at 37 °C for 1 h. (iii) To test the immunogenicity of BbGroEL, immobilized B. burgdorferi and recombinant BbGroEL were incubated with sera from infected mice and a rabbit for 1 h at 37 °C, washed 3 times with PBS, and incubated with goat anti-mouse IgG and goat anti-rabbit IgG-alkaline phosphatase conjugate at 37 °C for 1 h. (iv) To assess protein expression of BbGroEL upon temperature and pH shift, cell extracts from B. burgdorferi grown at 33 °C pH 6.8 and 23 °C pH 7.6 and probed with rabbit anti-BbGroEL for 1 h at 37 °C, washed 3 times with PBS, were incubated with goat anti-rabbit IgG-alkaline phosphatase conjugate at 37 °C for 1 h. Lastly, the plate was washed three times with PBS, followed by incubation with alkaline phosphatase substrate  for 30 min at room temperature. The absorbance was read at OD405 using a Thermo Scientific Multiskan GO microplate Spectrophotometer . Casein 1% was used as a negative control to detect unspecific binding and the absorbance values used as a blank. A secondary control was included in all experiments  to assess cross-reactivity of the secondary antibody.The 96 well enzyme-linked immunosorbent assay (ELISA) plates  were coated overnight at 4 °C with 1 μg of BbGroEL, PLG, fibronectin, laminin, or B. burgdorferi cells were collected at mid log phase and washed three times with Hank’s balanced salt solution (HBSS) . Spirochetes were incubated in HBSS in the presence of rabbit anti-BbGroEL serum or monoclonal anti-OspA at 33 °C for 1 h. Spirochetes were centrifuged at 10,000× g and washed three times with HBSS. Spirochetes were then fixed onto a teflon microscope slide  with 100% methanol at −20 °C, dried at 33 °C, and washed with HBSS. Fluorescein isothiocyanate (FITC) conjugated Goat anti-rabbit antibody  was added to the wells and incubated at 33 °C in a wet chamber for 1 h. The slide was dried at 33 °C, washed in HBSS, and dried again. Slides were mounted with SlowFade Diamond Antifade Mountant  and viewed with a Zeiss ApoTome.2 microscope .Proteinase K (PK) treatment of OM was done similarly to PK treatments of whole cells as previously described ,88,89. BB. burgdorferi was grown at 35 °C until it reached the midlog phase. This culture was diluted to 106 bacteria/mL and grown at 23 °C pH 7.6 until it reached a density of 1 × 107 bacteria/mL. The culture was diluted to a density of 2.5 × 105 bacteria/mL and separate cultures were incubated at 23 °C pH 7.6 and at 35 °C pH 6.8 until they reached a mid-exponential phase. Cultures at the same density were lysed using BugBuster protein extraction reagent  and subsequently tested by Western blot and ELISA as previously described.t-tests to compare two groups or ANOVA to compare multiple groups.Data was analyzed on GraphPad Prism 8.3.0  using unpaired