Research On The Inhibitory Effects And Pathways Of Pungent Spices And Their Characteristic Ingredients On Heterocyclic Amines

Heterocyclic amines(HAs)are known as a large class of polycyclic aromatic compounds with high carcinogenicity and mutagenicity produced by the reactions of glucose,creati(ni)ne,and amino acids at high temperature,and 2-amino-1-methyl-6-phenyl-imidazo [4,5-b] pyridine(Ph IP)is one of the most abundant HAs.The current control studies are mostly based on blocking key intermediates such as free radicals and aldehydes.Although these methods achieved certain effects,large amounts of HAs still exist in meat products because the formation of HAs is complex.Based on the inhibitory potential of some pungent spices and their characteristic ingredients on HAs in the previous literature,this thesis studied the inhibitory effects of four typical pungent spices and their characteristic ingredients on HAs in roast beef patties,and explored the inhibitory effect of four characteristic components on Ph IP through competitive inhibition of precursors,blocking intermediate,free radical scavenging,and direct elimination of Ph IP in the model systems.It provides the theoretical and experimental basis for the application of pungent spices and their characteristic ingredients to reduce HAs effectively and to develop new inhibitors of HAs.In this thesis,the qualitative and quantitative methods for free bound HAs by UPLCMS/MS in combination with PCA analysis were developed to explore the effects of spices(yellow mustard,garlic,black pepper,and chili pepper)and their characteristic ingredients including allyl-isothiocyanete(AITC),dially-disulfide(DAD),piperine(PP),and capsaicin(CP).The results have shown that six detected free HAs were Ph IP,Me IQx,4,8-Di Me IQx,Me AαC,Harman,and Norharman in control roast beef patties,with the total average amounts of 27.40 ng/g.Meanwhile,five detected bound HAs were Ph IP,IQ[4,5-b],Me AαC,Harman,and Norharman,with the total average amounts of 617.12 ng/g.All the four pungent spices significantly(P < 0.05)inhibited the formation of free HAs(inhibition rate of 13.33%-47.30%)and bound HAs(inhibition rate of 7.69%-72.08%)in roast beef patties.The addition of 1.5% garlic powder had the strongest inhibitory effects on total free HAs and 1.5% chili pepper powder had the strongest inhibitory effects on total bound HAs.The inhibitory ability of adding the characteristic ingredients was stronger than that of adding spices directly.The addition of 0.015%AITC had the strongest inhibitory effects on the total free HAs,with an inhibition rate of 74.43%,while the addition of 0.005%DAD had the strongest inhibitory effects on the total bound HAs,with an inhibition rate of 73.29%.AITC,PP,and CP all inhibited the formation of free Ph IP in a concentration-dependent manner.The inhibition of DAD on Ph IP decreased at first and then increased with the increase of concentration.The highest inhibition rates of AITC,DAD,PP,and CP on free Ph IP were60.20%,52.87%,56.04%,and 53.26%,respectively.Aiming at the high content free Ph IP in roast beef patties,glucose/creati(ni)ne/phenylalanine Ph IP generating model systems were established to explore the possibility of four characteristic ingredients inhibiting Ph IP by competitive inhibition of precursors,blocking intermediate,free radical scavenging,and end product elimination.First of all,it was found that the inhibition trends of four characteristic ingredients on Ph IP in the model system were consistent with that in roast beef patties,and the inhibition rate of Ph IP by adding 0.015%AITC was the highest(64.82%).After that,it was found that amide compounds(PP and CP)showed stronger inhibition in the precursor competitive inhibition pathway,degrading the most glucose(22.25%)and creatine(42.60%),respectively.In addition,AITC also had significant effects on the degradation of precursors of creatinine(25.38%)and phenylalanine(30.92%).In the inhibitory pathway of blocking intermediate of phenylacetaldehyde and free radical capture,sulfur compounds(AITC and DAD)showed stronger ability to scavenge intermediates and free radicals.AITC and DAD scavenged 28.83% and 25.37% phenylacetaldehyde,64.38% and34.81% free radicals in the model systems,respectively.Through correlation analysis,it was found that AITC,PP,and CP had a strong positive correlation with precursors,phenylacetaldehyde,and total amounts of spins when interfering with the formation of Ph IP in the model systems,while there was a negative correlation among various pathways,which showed that the inhibition of Ph IP by the high concentration of DAD decreased.Therefore,it is inferred that the four characteristic ingredients may inhibit Ph IP by competitive inhibition with precursors and blocking intermediate phenylacetaldehyde and free radical capture.Then,the precursor+characteristic ingredients model systems,intermediate+characteristic ingredients model systems,and end product+characteristic ingredients model systems were further established to verify the inhibition of Ph IP by precursors competitive inhibition,blocking intermediate phenylacetaldehyde and end product elimination.The results showed that the four characteristic ingredients could affect the precursors,intermediates,and Ph IP in a single co-thermal system to some extent.In the verification of the precursors competitive pathway,high concentration of DAD consumed the most glucose(43.22%),high concentration of PP consumed the most creatine(42.35%)and creatinine(33.48%),and high concentration of CP consumed the most phenylalanine(35.09%),which was slightly different from glucose/creatin(in)e/phenylalanine model systems.In the verification of the intermediate blocking phenylacetaldehyde pathway,the consumption of phenylacetaldehyde by AITC and DAD was more significant,up to 30.75% and 28.67%,respectively,which was consistent with the results in glucose/creatin(in)e/phenylalanine model system.In the verification of the end product elimination pathway,it was found that CP had the strongest elimination effect on the end product,and the highest elimination rate was 32.45%.