Detection And Control Of 3-MCPD Esters In Thermo-Reaction Chicken Flavorings

3-chloro-1,2-propanediol esters(3-MCPDE) are internationally recognized food contaminants in recent years, which were found metabolized to free 3-chloro-1,2-propandiol(3-MCPD) in vivo thereby exerting toxic effects indirectly. 3-MCPD has reproductive toxicity, kidney toxicity and potential carcinogenicity, and exists mostly in the form of 3-MCPDE achieving diversity from hydroxy groups on 3-MCPD replaced by different types and numbers of fatty acids. In this paper, a new direct UPLC-TQ-MS method was studied for the determination of various 3-MCPDE separately in chicken flavorings which is significant for researching the contamination status in meat flavorings as well as promoting the development of flavorings industry. The main contents of this paper are as follows:1. A direct UPLC-TQ-MS method for the synchronous determination of 9 kinds of 3-MCPDE in chicken flavorings was established. Firstly, the flavorings samples were processed with two-steps solvent extraction and purified with an injection of 200 μL of extract into SPE NH2(activated by 10 mL of hexane) for UPLC-TQ-MS detection. A comparation of the response values between two modes indicated that the formation of ammonium adduct was superior to sodium adduct. The appearance time of 9 kinds of 3-MCPDE standards differs from each other in extract ion chromatogram obtained by UPLC-TQ-MS under MRM operation. Quantitative ions were identified as [CH2·Cl-CH-O-R2-CH+H]+ for mono-esters and [R1+H]+ for diesters(R=acyl group).2. The feasibility of the method was investigated. The LOD were 0.44~0.48 μg/kg for mono-esters and 0.35~6.45 ng/kg for diesters and LOQ were 0.63~1.18 μg/kg for 3-MCPD mono-esters and 3.46~14.39 ng/kg for diesters in the UPLC-TQ-MS method. The intraday precision ranged between 1.4% and 3.4%, and the interday precision ranged from 2.0% to 4.8%. The recoveries of mixed 3-MCPDE standards spiked reference sample before solvent extraction at the concentrations of 10, 150 and 250 mg/kg were ranged from 79.5% to 115.8% with RSDs ranged between 7.4% and 12.8%.3. The formation regularity for both the 3-chloro-1,2-propanediol mono-esters and diesters as well as its total content were investigated in chicken flavorings derived from various enzymatic hydrolysis parameters of chicken fat, kinetic parameters and thermo-reaction formulas. The results showed that the mono-esters and diesters reached up to 2214.04 μmol/kg and 4236.83 μmol/kg,respectively at initial conditions(105℃, 60 min) and formulas and after optimizing the process this two indexs were reduced to the range of 0~80.28 μmol/kg and 47.83~307.84 μmol/kg, respectively. It turned out that 1-oleoyl-2-stearoyl-3-chloropropanediol had the highest content among diesters and 1-palmitoyl-3-chloro-1,2-propanediol was highest among mono-esters. To achieve a low 3-MCPDE amount considering good flavorings simultaneously, the best enzymatic conditions for chicken fat were enzymolyzing for 7.5 h at p H 7.00 with 70% of substrate concentration and 0.55% of lipase addition; the optimized thermo-reaction kinetic parameters were reacting for 60 min at 105℃ and pH 6.52; the final formulas were confirmed as 10% of chicken fat, 55% of chicken bone enzymolysis hydrolysate, more than 1.8% of L-cysteine, 1.8% of methionine, 8% to 10% content of glucose, 0.6% of VB1 and 5% or 15% of NaCl. It was proved that adding Na Cl in the process of lowing temperature after completion of the Maillard reaction could reduce the formation of 3-MCPDE, for instance, the total content of 3-MCPDE was reduced to 102.65 μmol/kg, equivalent to 11.34 mg/kg(~3-MCPD), at 60℃ with 10% addition of NaCl.4. Correlation analysis by using PLSR method showed a significant negative correlation between substrate concentration of chicken fat enzymolysis and variable Y, and an obvious positive correlation between time of thermo-reaction and variable Y for PLS2 analysis. Furthermore, PLS1 analysis revealed that enzymatic pH had a significant positive correlation with total content of 3-MCPDE, while the reaction temperature had no significant correlation which resulted from complicated effects of temperature on the generation of 3-MCPDE. The Maillard reaction materials all had no significant correlation. L-cysteine, methionine and glucose could possibly play a dual role by inhibiting or promoting the content of 3-MCPDE which depended on both the esterification rate and cracking rate. Moreover, these reaction rates were influenced by temperature. L-cysteine was proved more important than methionine in forming 3-MCPDE and the content of 3-MCPDE would have an upward trend at high concentration of VB1 that increase precursor substances. NaCl could accelerate nucleophilic reaction of Cl- at low concentration and decrease the formation of 3-MCPDE at high concentration. Besides, adding NaCl at low temperature after the Maillard reaction could effectively cut down the amount of 3-MCPDE.