Dietary Assessment Of Ochratoxin A In Chinese Dark Tea And Inhibitory Effects Of Tea Polyphenols On Ochratoxigenic Aspergillus Niger

Mycotoxin contamination in dark tea has become a topical issue for researchers and consumers in recent years.The presence of mycotoxins in dark tea products that affect people’s health and whether residual mycotoxins can negatively affect the health of consumers are topics of concern.Ochratoxin A（OTA）is a fungal secondary metabolite,mainly produced by Aspergillus and Penicillium fungi,which is nephrotoxic,neurotoxic,hepatotoxic,and teratogenic.Many studies are showing the presence of OTA contamination in tea,which may affect the health of tea consumers.In this study,we detected OTA contamination in dark tea,conducted risk assessment of dark tea,and investigated the inhibitory effect of tea polyphenolic substances on OTA-producing fungi,to provide a theoretical basis for the safe production and storage of dark tea.The main research results are as follows.1.A convenient,rapid,low-cost high performance liquid chromatographic（HPLC）method for the determination of OTA content in dark tea was screened and established.The recoveries of this assay ranged from 78.8%to 103.6%at three different spiked conditions of low,medium,and high concentrations.The limits of detection and limits of quantification were 0.39μg/kg and 1.30μg/kg,respectively,indicating that the present method meets the experimental requirements and is reliable,reproducible,and accurate for the practical determination of dark tea samples.2.A total of 228 commercially available dark tea products from six major dark tea producing areas in China in different years were tested for OTA using the screening established OTA detection method,in which OTA was detected in 21 dark tea samples with a positive rate of 9.2%,and the concentration range of OTA in tea samples was 2.51±0.16-12.62±0.72μg/kg.3.446 random participants were surveyed on tea consumption by questionnaire,then grouped by gender and age group,and then the deterministic risk assessment and probabilistic risk assessment of dark tea consumption were conducted for each group.The maximum HQ values for the large tea consumption group for the deterministic risk assessment and probabilistic risk assessment were 3.257x10^-1 and 2.389x10^-1 respectively.the maximum HQ values for each group were below 1,indicating that dark tea consumption does not pose a potential carcinogenic risk.4.In this experiment,20 strains of Aspergillus and Penicillium spp.were isolated from OTA-containing tea samples,and one strain of Aspergillus niger（AN3）was found to have the ability to produce OTA.Only one strain was isolated from a number of positive tea samples with the ability to synthesize OTA,indicating that the abundance of OTA-producing fungi in commercially available dark teas is extremely low,which may be related to their inhibitory effect by the components contained in the tea leaves.5.OTA-producing Aspergillus niger（AN3）was inhibited using tea polyphenols and EGCG,and it was found that the growth of OTA-producing Aspergillus niger（AN3）decreased with the increase of tea polyphenols and EGCG concentrations,and no OTA was produced by Aspergillus niger（AN3）after the addition of tea polyphenols and EGCG.The results showed that the growth of Aspergillus niger（AN3）decreased with increasing concentrations of tea polyphenols and EGCG,and no OTA production by AN3 was observed in the treatment groups with the addition of tea polyphenols or EGCG for 7 d of observation,compared with 1.55±0.34 ug/kg of OTA in the control.The sequences of related enzyme protein genes in the OTA biosynthesis pathway were screened and the primers were designed for fluorescence quantification.The results showed that both the tea polyphenol-treated and EGCG-treated groups exhibited a highly significant（P<0.01）reduction in the expression of NRPS,a key OTA synthesis gene,leading to the blockage of the OTA biosynthesis pathway and the inhibition of synthesis.In this study,the OTA content of commercially available dark tea from different origins in China was detected and the risk assessment was conducted based on consumer data.The results of dark tea OTA content testing showed that although there were cases of OTA contamination in dark tea commercially available in China,the positive detection rate and detection concentration were very low.The results of the drinking risk assessment indicate that there is no potential carcinogenic risk in drinking dark tea,and consumers can drink dark tea with confidence.Although the open OTA system of black tea can facilitate exogenous harmful fungal infestation,the actual detectable species and number of microorganisms with OTA-producing ability are extremely limited.Moreover,tea polyphenols and EGCG,which are internal components of tea leaves,could significantly（P<0.01）reduce the expression of NRPS,a key OTA synthesis gene,resulting in the blockage of OTA biosynthesis pathway,which may be the main reason for the low OTA detection rate and concentration in dark tea samples.The results clarified the safety of commercially available dark tea for drinking and the intrinsic reasons for low OTA residues,which could provide new insights and ideas for contamination prevention of OTA-producing fungi in food.