Study On The Inhibitory Pathway Of Non-precursor Amino Acids On Heterocyclic Amines In Meat Products

Heterocyclic amines(HAs)are a class of polycyclic aromatic compounds with carcinogenic and mutagenic effects,and produced by the reaction of carbohydrate,amino acids,creatine and creatinine during the processing of protein-rich ingredients such as meat and fish at high temperatures.Compared with commonly used HAs inhibitors,such as synthetic and natural antioxidants,and natural extracts,amino acids are safe,highly standardized,and inexpensive.The amino and carboxyl groups of amino acids have been reported to have the chemical reactivity of binding to aldehydes intermediates and free amino groups of HAs respectively,and the Maillard reaction product(MRP)of some amino acids has been proven to have strong free radical scavenging ability.These characteristics make amino acids a potential inhibitor and scavenger of HAs.Therefore,this article investigates the inhibitory effect of different levels of non-precursor amino acids-histidine,leucine,proline and methionine on HAs in roast beef patties and chemical model systems,using ultrahigh performance liquid chromatography–tandem mass spectrometry(UPLC–MS/MS).And try to explore the inhibitory mechanism of HAs by amino acids from the perspective of competitive inhibition of precursors,scavenging free radicals,blocking intermediates and scavenging end products.Finally,we tried to combine amino acids with strong inhibitory ability as compound inhibitors,and explore the inhibitory ability of compound inhibitors on free and protein-bound HAs in homemade western sausage.The effects of different levels(0.1%,0.5% and 1.0%,w/w)of histidine,leucine,proline,and methionine on HAs in roast beef patties and glucose/creatine/creatinine /phenylalanine+tryptophan+threonine model systems were studied.The results showed that eight and five HAs were detected and the total amounts(free and protein-bound state HAs)were 620.54 ng/g and 1179.90 ng/mL,in the control group of roast beef patties and model system,respectively.The content of harman was the highest in roast beef patties(384.34 ng/g),followed by norharman(223.72 ng/g),MeIQx(6.06 ng/g),A?C(2.82 ng/g),and PhIP(1.95 ng/g).The content of norharman was the highest in the model system(933.11 ng/mL),followed by harman(259.62 ng/mL),PhIP(2.75 ng/mL)and MeIQx(2.01 ng/mL).All four amino acids have inhibitory effects on HAs in roast beef patties and model systems.1.0% histidine has the strongest inhibitory ability on free and protein-bound heterocyclic amine in roast beef patties,and total HAs in model system,inhibition rates reached 43.84%,49.76% and 66.99% respectively,and all group show dose-depend manner.0.5% and 1.0% leucine have the strongest inhibitory ability to free HAs in roast beef patties(43.10%),and total HAs in model system(27.52%)respectively,and dose-depend manner was show only in model system.0.1% and 1.0% proline have the strongest inhibitory ability on total HAs in beef patties(21.06% for free HAs,10.36% for bound HAs)and in the model system(22.26%)respectively,and dosedepend manner was show only in model system.0.1% and 1.0% methionine have the strongest inhibitory ability to the free(12.81%)and bound(32.30%)HAs of beef patties,respectively.Methionine showed a dose-dependent relationship for HAs in the model system,1.0% methionine had the strongest inhibitory ability on HAs,and the inhibition rate reached 34.04%.For ?-carboline,amino acids can inhibit the production of ?-carboline by scavenging free radicals.Among them,1.0% histidine has the strongest radical scavenging ability(82.59%),followed by 1.0% methionine(61.66%),1.0% leucine(43.93%),and 0.1% proline(33.23%).All amino acids except proline showed a dose-dependent manner.At the same time,amino acids can compete with tryptophan(precursor of ?-carboline)to participate in the reaction to achieve the purpose of inhibiting ?-carboline.Among them,1.0% histidine has the strongest inhibitory effect,with an inhibition rate of 65.60%,followed by 1.0% proline(62.60%),0.1% leucine(57.72%),and 0.1% methionine(39.39%).Histidine and proline show a dose-dependent relationship.For pyridine,amino acids can suppress generation of free radicals,thereby inhibit the production of active carbonyl groups,and then reduce the conversion of phenylalanine to phenylacetaldehyde to inhibit PhIP.Among them,1.0% leucine has the strongest free radicals scavenging ability(71.00%);amino acids may also form adducts by directly condensing with phenylacetaldehyde to inhibit the production of PhIP.Among them,1.0% leucine has the strongest scavenging ability of phenylacetaldehyde(76.60%).At the same time,amino acids may scavenge the generated PhIP by binding with PhIP to form adduct.Among them,proline has the strongest PhIP scavenging ability(22.64%).For quinoline and quinoxaline,amino acids can also inhibit them by eliminating free radicals and condensing with final product.Among them,1.0% proline has the strongest free radical scavenging power(41.48%).Methionine has the strongest scavenging power for MeIQx(44.20%).Leucine has the strongest scavenging power for MeIQ(16.34%).The experimental results of western sausage showed that,for free HAs,the combination of 1.0% histidine+1.0% leucine+1.0% proline had the strongest inhibition ability,the inhibition rate reached 45.53%,followed by 1.0% histidine+1.0% proline combination(32.52%),and 1.0% leucine+1.0% proline combination(31.77%).For the bound HAs,the combination of 1.0% histidine+1.0% leucine+1.0% proline has the strongest inhibition ability,with an inhibition rate of 33.88%,followed by 1.0% leucine+1.0% proline(19.57%).Therefore,the HAs can be suppressed by adding 1.0% histidine+1.0% leucine+1.0% proline during the processing of western sausages.