Establishment Of Non-Targeted Screening Method For Estrogenic Disrupting Compounds And Its Application In Dairy Product Detection

Estrogenic disrupting compounds?EDCs?are a class of exogenous compounds with estrogen effects,which are widely found in food,cosmetics,metal products and packaging materials.At present,there are many kinds of EDCs with complex structure and they are not easy to degrade.They tend to accumulate in organisms and have the characteristics of causing high toxicity in low doses.In addition,the harm caused by EDCs will not only disrupt the endocrine system,but also affect the immune system and nervous system of the body.Therefore,more and more attention and researches have been paid to EDCs in recent years.At present,research on EDCs is mainly divided into two categories.One is the detection of known EDCs,such as 17?-estradiol?17?-E₂?and diethylstilbestrol?DES?.The other is screening of unknown EDCs in the environment or end products.However,screening studies of unknown EDCs during processing have not yet been reported.Due to the presence of EDCs such as estrone?E₁?,estradiol and estriol in raw milk and the complex composition of dairy products,heat treatment during processing and other conditions may lead to the generation of unknown EDCs.Therefore,this paper explored the screening of unknown EDCs during liquid milk processing.In order to screen unknown EDCs during liquid milk processing,the paper first established two kinds of simple,sensitive and high-throughput screening methods for EDCs based on MCF-7 cell proliferation and estrogen receptors??ER??-gold nanoparticles?AuNPs?colorimetry.Next,11 brands of liquid milk on the market were selected to screen whether there were unknown EDCs in finished product compared with raw milk.Finally,the processing of liquid milk was simulated to explore whether unknown EDCs were generated under different heating temperatures and heating times due to operational errors or uncontrolled of machine.The paper consisted of five parts,as follows:The first chapter mainly summarized the physicochemical properties and hazards of EDCs.The advantages and disadvantages of the existing screening methods were further compared.Then,the structural characteristics,mechanism of action of estrogen receptor?ER?and the research progress of screening methods for EDCs based on ER were introduced.Finally,the research status and progress of unknown EDCs during dairy processing were summarized.In the second chapter,an EDCs screening method based on MCF-7 cell proliferation was established.By calculating the cell proliferation rate of cells with different concentrations of EDCs to determine whether the test substance had estrogen activity.The paper first optimized the time of EDCs on MCF-7 cells and the optimal time was 48 h.Next,17?-E₂,DES,hexestrol?HES?,17?-estradiol?17?-E₂?,E₁,2,2-bis?4-hydroxyphenyl?-1,1,1-trichloroethane?HPTE?,genistein,bisphenol A?BPA?and bisphenol B?BPB?were tested respectively.When the concentration of 17?-E₂,DES,HES were 10^-88 mol·L^-1,17?-E₂,E₁,HPTE were 10^-77 mol·L^-11 and Genistein,BPA,BPB were 10^-6mol·L^-1,the cell proliferation rate was the largest?P<0.01?.As the concentration of EDCs continued to increase,it showed a cytotoxic effect and the cell proliferation rate decreased significantly.And the estradiol equivalents of nine EDCs were compared:17?-E₂>DES>HES>17?-E₂>E₁>HPTE>Genistein>BPA>BPB.This method was sensitive,easy to operate and can screen multiple EDCs simultaneously.In the third chapter,a screening method for EDCs based on ER?and AuNPs colorimetry was established.ER?protein can prevent the salt-induced aggregation of AuNPs by recruiting Na⁺.When there were EDCs as targets in the sample,the specific binding of ER?and EDCs resulted in a reduction in the binding between Na⁺and ER?.Then most Na⁺bound to the AuNPs and induced their aggregation.Based on this principle,standard curve of 17?-E₂,DES,HES,17?-E₂,E₁,HPTE,Genistein,BPA and BPB were established by calculating the absorbance ratio at 650 nm and 520 nm(A₆₅₀/A₅₂₀)and the concentration of EDCs.And estrogen activity of nine EDCs was compared:DES>17?-E₂>HES>17?-E₂>E₁>HPTE>Genistein>BPA>BPB.Moreover,the recoveries of the nine kinds of EDCs spiked in tap water and milk samples were all between 80.0%and 120.0%,which can be used for the detection of actual samples.The fourth chapter screened unknown EDCs during liquid milk processing.Firstly,11brands of liquid milk on the market were selected to investigate whether there were unknown EDCs in processed finished products compared with raw milk.There was no significant difference between experimental results and control group?P>0.05?,indicating that no unknown EDCs were produced in the 11 brands of liquid milk.Next,the processing of liquid milk was simulated to investigate whether unknown EDCs were generated under different heating temperatures and heating times due to operational errors or uncontrolled of machine during the processing of dairy products.The heating temperature and heating time were designed as follows:65?:26 min,28 min,30 min,32 min,34 min;75?:11 s,13 s,15 s,17 s,19 s;135?:1 s,3 s,5 s,7 s,9 s;30 min:61?,63?,65?,67?,69?;15 s:71?,73?,75?,77?,79?;5 s:131?,133?,135?,137?,139?.After the above different heating temperatures and heating time,there was no significant difference between experimental results and control group?P>0.05?,indicating that no unknown EDCs were produced during the processing of liquid milk under this processing condition.The fifth chapter summarized two kinds of screening methods for EDCs based on MCF-7cell proliferation and ER?-AuNPs colorimetry,and the screening of unknown EDCs during the processing of liquid milk.The two kinds of EDCs screening methods established in this paper have the advantages of simple operation,high throughput and low cost.The screening of unknown EDCs during the processing of liquid milk provided ideas and laid theoretical and technical foundation for further research on the aspects of unknown EDCs during processing.