Dietary Risk Assessment Of Cyanogenic Glycosides In Cassava Roots And Its Mitigation Strategy Based On Cassava Linamarase

As a staple food for more than 800 million people worldwide,cassava play a very importantrole in the food supply.Cassava contain a large amount of cyanogenic glycosides,which are the main source of dietary exposure to cyanogenic glycosides.After ingestion,cyanogenic glycosides cause potential health risks to the human body.In recent years,there are still reports of deaths due to improperly processed cassava food in China.But there are no relevant studies on the dietary exposure of cyanogenic glycosides yet.China is also the largest importer of the cassava,so the effective control methods of cassava cyanogenic glycosides is extremely important.The specific degradation of cyanogenic glycosides by cassava linamarase during processing is the key to the removal of cyanogenic glucosides from cassava.However,cassava linamarase is easily inactivated during cassava processing,resulting in a decrease in the degradation efficiency of cyanogenic glucosides.Based on the above problems,this study developed a method for the detection of cyanogenic glycosides based on liquid chromatography tandem mass spectrometry,to calculate the dietary exposure of cassava cyanogenic glycosides of Chinese population and evaluate health risks of cyanogenic glucosides;the key enzyme cassava linamarase gene for cassava cyanogenic glycoside degradation was recombined and expressed,combined with rational design and protein engineering to improve the activity and stability of linamarase,and finally enhanced the degradation of cyanogenic glucosides by adding cassava linamarase.The synergistic ultrasonic treatment of cassava linamarase improved the degradation efficiency of cyanogenic glycosides during cassava flour processing and reduces the risk of eating cassava cyanogenic glycosides.This study laied a theoretical and practical foundation for promoting the food safety of cassava food and cyanogenic glycosides.The main results are as follows:1.A quantitative detection and analysis method for cyanogenic glucosides in agricultural products was established by ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry,and the method validation and application analysis were carried out.The results showed that the cyanogenic glucosides were extracted,and pretreated by solid-phase extraction column,and C18 chromatographic column separated,and acetonitrile/water mobile phase gradient eluted.After that,linamarin,linustatin,lotaustralin,neolinustatin,taxiphyllin,dhurrin,amygdalin and prunasin were determined.The limits of quantification of the eight cyanogenic glycosides ranged from 5-100μg/kg,and the average recoveries ranged from89.88%to 125.69%with the relative standard deviations of intra-day and inter-day precision less than 10.38%and 11.33%,respectively.This method was used to analyze the content of cyanogenic glycosides in foods such as flaxseed,cassava,bamboo shoots,sorghum,and almonds,and achieved more accurate quantification of cyanogenic glycosides than the hydrolysis method.2.Integrate accurate detection methods,Monte Carlo simulation probability assessment and cooking factors to calculate the dietary exposure of cassava cyanogenic glycosides for Chinese residents,and assess the health risks of cassava cyanogenic glycosides dietary exposure.The results showed that the concentration of cyanogenic glycosides in cassava varieties wasdistributed from less than 100 mg/kg to 1000 mg/kg.After cassava was boiled,39%of the hydrocyanic acid and 89%of the cyanogenic glycosides were eliminated.There was an acute dietary exposure risk to cassava and cassava cyanogenic glycosides among Chinese residents,and the dietary exposure to cassava and cassava products was 5.0 and 1.7 times that of the acute reference dose（ARf D）（900μg/kg bw）,but the risk of chronic dietary exposure to cassava cyanogenic glycosides among Chinese residents was low.The maximum residue limit of cyanogenic glycosides in cassava and cassava products was recomanded as 200 mg/kg.3.Using E.coli Rosetta-gami2（DE3）as the expression vector,the recombinant expression system of cassava linamarinase gene was constructed.The active expression and the kinetics and crystallographic properties of recombinant linamarinase were were explored.The results showed that the cassava linamarase gene was only soluble and active expressed on the p MAL-C2X vector.The expression conditions were optimized as follows:induction with 0.1m M isopropyl-β-D-thiogalactoside at 37°C for 3 h.After purification,the the specific activity of the recombinant linamarase was 42.1 U/mg.Under the crystallization conditions of LFS A6crystallization/45%MPD precipitant/p H 8.2/18℃/100 m M Na I additive,cassava linamarase protein crystals with good single crystals of about 300μm was obtained,and the diffraction resolution was up to 2.8（?）.The optimum temperature for the enzyme to catalyze the linamarin substrate was 35°C,the optimum p H was 6.0-6.5,and the relative activity after treatment at 55°C for 90 minutes was 24.1%,with a large loss of enzyme activity.4.Through computer-aided protein engineering strategy to screening for mutant libraries with potentially high catalytic activity and thermal stability.The structure and activity mechanism of linamarase were explored.The best mutants were verified by site-directed mutagenesis experiments.The results showed that the optimal temperature of the optimal mutant K263P-T53F-S366R-V335C-F339C(CAS^mut)was 10℃higher than that of the wild type,the specific activity of CAS^mutwas 2.02 times that of the wild type,and the catalytic constant K_cat/K_m increased by 1.96 fold.The molecular dynamics simulation showed that the root mean square fluctuation value of the residues in the mutation region was reduced,and the flexible docking of CAS^mut and linamarin molecules found that CAS^mut and linamarin formed more hydrogen bond interactions,and it was observed that the aliphatic aglycone part of the linamarin ligand was surrounded by hydrophobic residues,making the substrate conformation more stablized.These factors revealed the mechanism of the specific activity and stability improvement of CAS^mut.5.The degradation effect of cassava cyanogenic glycosides by adding CAS^mutduring cassava flour processing and the combination of adding CAS^mut and ultrasonic treatment were explored.The reaction conditions for the degradation of cassava flour cyanogenic glucosides was optimized.The results showed that the content of cyanogenic glucosides in cassava flour decreased by62.6%after 90 min of degradation,when CAS^mut was added at 47 mg/L and the reaction temperature was 45°C at p H 6.0.The addition of CAS^mut combined with ultrasonic treatment reduced the cyanogenic glycosides in cassava flour by 81.9%,and further improved the degradation effect.In contrast,short-term soaking was not effective in removing cyanogenic glycosides,with the cyanogenic glycosides in cassava flour reduced by 7.9%for 12 h soaking.The synergistic mechanism of CAS^mut combined with ultrasonic treatment was related to the increase in CAS^mut activity due to the movement of hydrophobic residues of the enzyme after ultrasonic treatment.In summary,this study revealed the health risks of dietary exposure to cassava cyanogenic glycosides of Chinese residents,and improved the catalytic activity and thermal stability,and finally,the degradation efficiency of cyanogenic glycosides in cassava flour was promoted by adding CAS^mutand the ultrasonic treatment,which reduced the food safety risk of cassava cyanogenic glycosides.It lays the foundation for the development and promotion of cassava food.