Aspergillus flavus and aflatoxins control in long-term storage of food ingredients of Puerh tea, peanut and polished rice.

Aflatoxins are a group of high toxic mycotoxins in food chain. Recent studies showed that aflatoxins might contaminate post-fermented tea, but the result remains controversial. Here, Aspgergillus flavus growth and aflatoxin production were characterized in Puerh tea, peanut and polished rice at different initial water activity (aw) values for long-term storage. As a result, food initial aw value was the critical factor for A. flavus growth and aflatoxin production, and A. flavus almost not grew on foods at aw value lower than 0.8. A. flavus grew best in peanut, followed by rice, but growth on Puerh tea was limited. A. flavus growth was inhibited significantly by adding tea to Potato Dextrose Agar (PDA). Accordingly, aflatoxins produced dramatically in peanut, followed by rice at the first 90 days storage. However, aflatoxin neither produced in Puerh tea nor on tea modified PDA, indicating tea components inhibited A. flavus growth and aflatoxins synthesis.

Comprehensive and cumulative risk evaluation of dietary exposure to aflatoxins and ochratoxin A on fermented teas worldwide by a new assessment model.

Recently, mycotoxin risks in fermented tea have received high attention, but mycotoxin transfer rates from tealeaf to infusion during brewing were rarely considered. In addition, the assessment data (i.e., mycotoxin occurrences and tea consumption) in previous assessments were usually limited. Here, a comprehensive and cumulative risk assessment of aflatoxins and ochratoxin A was performed using a tea assessment model, by which mycotoxin transfer rates were included and the assessment data were collected worldwide. By 10 times of brewing, the aflatoxin transfer rate was only 2.94% and OTA was 63.65%. Besides the extreme case, hazard quotients (HQs) from all consumers were lower than the threshold of 1.0, indicating no noncarcinogenic risk; the P95 cumulative margin of exposure (1/MoET) values were 2.52E-04 (30-39 years of age) and 2.42E-04 (≥50 years of age) for two high exposure groups under the upper bound scenario, which a little higher than the carcinogenic risk threshold of 1.00E-04. Notably, the P95 cumulative 1/MoET values (3.24E-03 -7.95E-03) by food assessment model were ten times higher than those of by tea assessment model. The comparative results showed that mycotoxin dietary risks on tea consumption by food assessment model were much overestimated. The result of this study indicated that the contaminants transfer rates should be considered for risk assessment on tea consumption in future work.

Determination of four aflatoxins on dark tea infusions and aflatoxin transfers evaluation during tea brewing.

In daily tea drinking, little is known on aflatoxin transfers from tea-leaf to infusion during brewing and the actual intake. A verified aflatoxins analytical method on tea infusion is significant to the exposure assessment. Here, an optimal method 3 (M3) was screened and validated on four aflatoxins (B1, B2, G1 and G2) simultaneous determination. Recoveries of AFG1 and AFB1 were 87.37 %±1.99 %-102.03 %±8.62 %, and AFG2 and AFB2 were 83.99 %±8.65 %-100.14 %±1.69 %. The correlation coefficient on the fortified samples determination was > 0.99 for each aflatoxin. In accuracy and precision validation, aflatoxins recoveries in high and low fortified samples were 85.94 %-103.83 %, and RSDs were 2.20 %-9.15 %. Method applicability test showed that the M3 was qualified for six different dark tea types. Extraction rate of 30-days stored AFB1 fortified tea-leaf by acetonitrile-water (84:16, v/v) was < 50 %; meanwhile, only 2.94 % spiked AFB1 was released by ten times tea brewing which indicated that Puerh tea have strong adsorbability to aflatoxin.

Plant Growth-Promoting Activity of Herbaspirillum aquaticum ZXN111 on the Zijuan Tea Plant (Camellia sinensis var. assamica).

Herbaspirillum aquaticum ZXN111 which was isolated from the tea plant Zijuan can produce indole-3-acetic acid (IAA) and contain abiotic-stress tolerance gene 1-aminocyclopropane-1-carboxylate deaminase (accd). In this study, ZXN111 PGP activity and the molecular mechanism were investigated. The result showed that ACCD activity of wild-type ZXN111 was 0.4505 mM α-KB/mg·Pro·h, but mutants Δacc and Δacc-tyrb did not showed ACCD activity. IAA production by ZXN111 within 48 hrs was 20.4 µg/mL, while mutants of Δtyrb and Δacc-tyrb were lower than 3.6 µg/mL, indicating that indole-3-pyruvic acid is the primary IAA synthesis pathway. Potting tests found that ZXN111 displayed significant PGP activity to the tea plant Zijuan, but Δtyrb and Δacc-tyrb did not show PGP activity, indicating that IAA is critical to PGP activity. In a salt-stress test, ZXN111 did not enhance the tea plant NaCl tolerance by gene accd. The results of this study indicated that strain ZXN111 has potential for biofertilizer development on tea plantation.

Determination and Comprehensive Risk Assessment of Dietary Exposure to Ochratoxin A on Fermented Teas.

A specialized method for ochratoxin A (OTA) determination on fermented teas was developed and validated using ultraperformance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Methodology results showed that recovery, relative standard deviation, accuracy, and precision were qualified. The limits of detection and quantification were 0.32 and 0.96 µg/kg, respectively. Two of 158 collected samples were screened for OTA contamination. Comprehensive risk assessment based on OTA contaminations of this study and other peer-reviewed publications was performed. The highest hazard quotient (HQ) value (8.86 × 10-2) and the highest 1/MoE value (8.61 × 10-5) in probabilistic assessment were equally below the recommended non-neoplastic and neoplastic thresholds, indicating no health risks. However, the HQ and 1/MoE values of the 95th percentiles in 20-39 and ≥50 years of age were close to thresholds of 1.0 and 1.0 × 10-4, respectively. Under the extreme case, there were only a few scenarios (e.g., 40-49 years of age) of HQ values below the non-neoplastic threshold, but the 1/MoE value of each group exceeded the neoplastic threshold. This is the first extensive risk assessment on OTA from fermented teas worldwide, but the sample size is still limited, and a large number of samples is encouraged in a future study for a more accurate assessment.

Quantitative analysis and dietary risk assessment of aflatoxins in Chinese post-fermented dark tea.

Recently, mycotoxins safety in Chinese post-fermented teas has attracted much attention because it is still controversial whether environmental fungi in the teas are able to produce aflatoxins. In this study, a rapid and selective analytical method based on immunoaffinity column (IAC) cleaning coupled with liquid chromatography-mass spectrometry (LC-MS) was developed to quantify four aflatoxins (B1, B2, G1 and G2) in post-fermented teas. Recoveries ranged from 80.8 to 92.2% with relative standard deviation less than 3%. The limits of detection and quantification were 0.109-0.348 µg kg-1 and 0.364-1.159 µg kg-1, respectively. Two out of 158 samples were positive for aflatoxins examination (occurrence rate 1.27%). The deterministic assessment for the maximal aflatoxins exposure under upper bound was 9.19 × 10-6 µg kg-1 day-1 from heavy exposure consumers (≥50 year age group), but the exposure was lower than the JECFA acceptable value of 1.0 ng kg-1 day-1 on liver risk. Probabilistic assessment showed that the upper bound 95th percentile carcinogenic risk value for the 318 consumers (Yunnan China and Ulan Bator Mongolia) was 1.75 × 10-7, and for heavy exposure consumers was 2.4 × 10-7, and the values equally below the acceptable carcinogenic risk level.

Determination of six groups of mycotoxins in Chinese dark tea and the associated risk assessment.

Chinese dark tea is widely enjoyed for its multiple health-promoting effects and pleasant taste. However, its production involves fermentation by microbiota in raw tea, some of which are filamentous fungi and thus potential mycotoxin producers. Accordingly, whether mycotoxins pose health risk on dark tea consumption has become a public concern. In this study, a cleaning method of multi-functional column (MFC) and immunoaffinity column (IAC) in tandem combined to HPLC detection was developed and validated for determining ten mycotoxins of six groups (i.e., aflatoxins of B1, B2, G1 and G2, ochratoxin A, zearalenone, deoxynivalenol, fumonisins of B1, B2, and T-2) in dark teas. The interferences from secondary metabolites were effectively reduced, and the sensitivities and recoveries of the method were qualified for tea matrices. Six groups mycotoxins were determined in 108 samples representing the major Chinese dark teas by using the new method. Subsequently, the dietary exposure and health risks were evaluated for different age and gender groups in Kunming and Pu'er in China and Ulan Bator in Mongolia. The occurrence of zearalenone was 4.63% and that of ochratoxin A was 1.85%, with the other four groups mycotoxins were below the limits of quantification. The hazard index values for the five groups' non-carcinogenic mycotoxins were far below 1.0. The deterministic risk assessment indicated no non-carcinogenic risks for dark tea consumption in the three areas. Probabilistic estimation showed that the maximum value of 95th percentile carcinogenic risk value for the aflatoxins was 2.12 × 10-8, which is far below the acceptable carcinogenic risk level (10-6). Hereby, six groups mycotoxins in Chinese dark tea showed no observed risk concern to consumers.

Simultaneous Determination of Four Aflatoxins in Dark Tea by Multifunctional Purification Column and Immunoaffinity Column Coupled to Liquid Chromatography Tandem Mass Spectrometry.

Dry tea matrix contains an abundance of caffeine and polyphenols which are different from the food matrix (e.g., protein, lipid, and carbohydrates), and only a few studies have tried aflatoxins determination with tea samples. Here, a specific, accurate, and sensitive method was developed and validated for the simultaneous determination of aflatoxin B1, B2, G1, and G2 in dark teas. Aflatoxins were extracted by acetonitrile/water, press-filtered, and cleaned by multifunctional purification column (MFC) and immunoaffinity column (IAC) in tandem. The cleaned extract was analyzed by liquid chromatography tandem mass spectrometry. The matrix interference was effectively reduced by MFC-IAC cleaning method. Recoveries at the spiking concentrations of 5-60 µg/kg ranged from 77.5 to 93%, with relative standard deviations <11.0%. The correlation coefficients of aflatoxins standard were >0.998. The limits of detection were 0.024-0.21 µg/kg and the limits of quantification were 0.08-0.74 µg/kg. The intra- and interday accuracy ranged from 74 to 87%, and the intra- and interday precisions ranged from 0.4 to 3.1%. After the method validation, the aflatoxins contaminations in 100 collected dark teas were detected, and the results were compared with those of other methods.