Study On The Determination Method And Control Strategy Of Potential Hazardous 3-MCPD Esters In Beef Flavors

3-chloro-1,2-propanediol?3-MCPD?esters are the contaminants generated during food processing,which were widely found in oils,fats and fatty foods.Due to their potential toxicity,3-MCPD esters have become a worldwide safety issue in food processing.During the last decade,researchers have mostly paid attention to the formation of 3-MCPD esters during oil processing.However,less work was focused on the relationship between 3-MCPD esters and the meat flavors,which have favorable conditions for 3-MCPD esters formation during their preparation.With its increasing application in food industry,the consumers have paid more attention to the safety of meat flavors.Therefore,the studies on the formation of 3-MCPD esters during meat flavor preparation is significant for controlling the safety of meat flavor and promoting the healthy development of meat flavor industry.In this work,we take beef flavor as an example to develop a direct method for the determination of 3-MCPD esters in meat flavors.The influential factors of the formation of3-MCPD esters during beef flavor preparation were investigated.And the potential strategies are proposed to reduce 3-MCPD esters concentration without changing the flavor quality of beef flavors.Based on the above study,the optimal conditions for thermal reaction beef flavor preparation were proposed.The main contents and results are as follows:1.A sensitive ultra-performance liquid chromatography tandem quadrupole mass spectrometer?UPLC-TQ-MS?method was developed for the direct determination of 3-MCPD esters in thermal reaction beef flavors.The purification of samples was carried out on the amino solid-phase extraction?SPE?column with the elution solvent of 8 m L methylene chloride-isopropanol?1:1,v/v?.The quantitative determination of 3-MCPD esters was performed on UPLC-TQ-MS with reversed phase ACQUITY UPLC?BEH C₈ column.The mobile phase was methanol+acetic acid?3 mmol/L?at a flow rate of 0.3 mL/min.The data validation indicated that the determination coefficients?R²?were over 0.99 for nine 3-MCPD esters.The spiked recoveries were between 80.5 and 113.7%.The intra-day precision and the inter-day precision were 0.4-9.9%and 2.4-13.8%,respectively.The limit of detection?LOD?and the limit of quantification?LOQ?were found to be in the range of 1.0-5.0?g/kg and 4-16?g/kg for3-MCPD monoesters,in the range of 0.04-1.0?g/kg and 0.1-4.0?g/kg for 3-MCPD diesters,separately.2.The effects of enzymatic hydrolysis condition of tallow were evaluated on the formation of 3-MCPD esters and sensory characteristic of beef flavors.The results indicated that the enzymatic hydrolysis condition had significant effects on the content of 3-MCPD mono/di-esters in beef flavors.Considering the 3-MCPD esters concentration?especially the content of3-MCPD monoesters?and sensory characteristics of beef flavors,the optimal enzymatic hydrolysis conditions were selected through the single factor experiment.And enzymatic hydrolysis conditions were finally set as:lipase concentration 75 U/g tallow,tallow concentration 80%?w/w?and pH 7.0 for 9.5 h.The beef flavor prepared with the optimal selected conditions exhibited noticeable beef flavor and strong simulate characteristics.The concentration of 3-MCPD diesters was 269.10?g/kg and no 3-MCPD monoesters were detected.3.The effects of enzymatic hydrolysis conditions of bovine bone protein were evaluated on the formation of 3-MCPD esters and sensory characteristic of beef flavors.The beef flavors were prepared with the obtained bovine bone hydrolysate at different enzymatic hydrolysis conditions and enzymatic hydrolyzed tallow which was prepared with the optimal enzymatic hydrolysis conditions.The results showed that no 3-MCPD monoesters were detected.And compared to their effects on the content of 3-MCPD diesters,enzymatic hydrolysis conditions of bovine bone protein had a much greater effect on the flavor quality of beef flavors.Therefore,enzymatic conditions of bovine bone were finally chosen as:substrate concentration 40%?w/w,wet?,papain concentration 6.3 U/mg protein,pH 6.0,reaction temperature 60oC and reaction time 3.0 h,in consideration of sensory characteristics of beef flavors.4.Based on the above work,the changes of 3-MCPD esters were investigated in beef flavors derived from various flavor precursors,kinetic parameters,sodium chloride?NaCl?addition and its different addition methods?before or after thermal reaction?.The results showed that:only the 3-MCPD diesters were detected and their concentration had a significant positive correlation with the addition of enzymatically hydrolyzed tallow.The addition of amino acids,reducing sugars,hydrolyzed vegetable protein?HVP?and yeast extracts would affect the concentration of 3-MCPD diesters,but the effects were not significant.The 3-MCPD diesters'concentration was negatively correlated with thermal reaction time in the range of 60-180 min and the concentration of 3-MCPD diesters decreased with the increasing reaction time.In addition,3-MCPD diesters'concentration was strongly dependent on NaCl concentration and its addition moment?before or after thermal reaction?at different temperature.3-MCPD diesters'concentration showed a significant positive correlation with NaCl addition?added before thermal reaction?and it could be significantly reduced by adding NaCl after thermal reaction.And 3-MCPD diesters'concentration decreased with the decreasing addition temperature.Both 3-MCPD esters concentration and flavor quality of beef flavors were taken into consideration;the beef flavor was prepared at 110oC with initial pH 7.0 for 100 min,with the reaction temperature controlled by high pressure device.The composition of flavor precursors of the thermal reaction system was as follows:enzymatically hydrolyzed tallow?10%,w/w?,bovine bone enzymolysis hydrolysate?50%?,L-cysteine?1.2%?,DL-methionine?1.2%?,HVP?6%?,yeast extract?6%?,glucose?1%?and D-xylose?0.5%?.And 10%NaCl was added when the system was cooled to 60oC.The total concentration of 3-MCPD esters was reduced by 97.3%?from825.73 to 22.47?g/kg?through optimizing the beef flavors preparation conditions during this study.In addition,the beef flavor prepared with the optimal selected conditions exhibited noticeable beef flavor and strong simulate characteristics5.The thermal reaction models between NaCl,triglyceride,amino acids and deionized water were established and the influential factors of 3-MCPD esters formation were evaluated.The results indicated that only 3-MCPD diesters were detected in tristearin and triolein thermal reaction model systems,and their concentrations showed similar trend with the changing triglyceride addition,NaCl addition,reaction temperature and reaction time.The concentration of 3-MCPD diesters increased with the increasing addition of triglyceride.With the increasing NaCl addition,the concentration of 3-MCPD diesters increased,reaching the highest level at the addition of 21.43%NaCl,and decreased afterward.The content of 3-MCPD diesters showed a similar trend with the changing reaction temperature and time.3-MCPD esters'concentration increased with increasing reaction temperature and time,reaching the highest level at 110oC and 45 min,respectively,and then decreased.DL-methionine,L-cysteine and L-glutamic acid had similar effects on the content of 3-MCPD diesters,promoting their formation at low concentration,whereas,reducing 3-MCPD diesters'concentration when added at high concentration.In contrast,the concentration of 3-MCPD diesters increased with increasing addition of L-proline.Based on the results of 3-MCPD esters formation in the thermal reaction models and beef flavors,it can be found that the concentration of 3-MCPD esters could be reduced to improve the safety of meat flavor by selecting an appropriate pretreatment process for fat,decreasing the fat addition and adding NaCl at low temperature to the meat flavors which were prepared at neutral pH and without NaCl before thermal reaction.