| Heterocyclic amines(HAs)are a class of carcinogenic and mutagenic compounds formed during the thermal processing of protein-rich foods.Studies have shown that natural products are the important sources of HAs inhibitors.Zanthoxylum bungeanum Maxim.leaves,a by-product of pepper,has high edible and medicinal values,but studies on the effects of the leaves on HAs in meat products have not yet been reported.In this study,Zanthoxylum bungeanum Maxim.leaves extract(ZME)was used to investigate the effects of ZME on the edible quality of roast beef patties and the formation of HAs,and to investigate the inhibition mechanism of HAs formation by three polyphenols mainly contained in ZME.The aim was to provide a theoretical basis for the inhibition of the formation of HAs during the thermal processing of meat products.The details of the study and the results are as follows:1.Effects of ZME on the formation of HAs in roast beef pattiesZME was added to the roast beef patties to investigate the effects on the cooking loss,colour,texture and HAs formation,and also to detect the changing of their precursors contents.The results showed that the addition of ZME significantly reduced the cooking loss of roast beef patties(P<0.05).The L*,a*and b*values tended to decrease(P<0.05),while the hardness and chewiness increased(P<0.05).A total of seven HAs were detected inthecontrolgroupswithoutZMEaddition,namely2-amino-1-methyl-6-phenylimidazo[4,5-b]-pyridine(PhIP),2-amino-3-methyl-imidazo[4,5-f]-quinoline(IQ),2-amino-3,4-dimethylimidazole[4,5-f]quinoline(Me IQ),2-amino-3,8-dimethyl-imidazo[4,5-f]-quinoxaline(8-Me IQx),1-methyl-9H-pyrido[3,4-b]indole(Harman),9H-pyrido[3,4-b]indole(Norharman)and2-amino-9H-pyrido[2,3-b]indole(AαC)with a total HAs content of 6.17 ng/g.The inhibition rates of 0.45 g/kg ZME on PhIP,IQ,Me IQ,Harman and AαC formation were71.76%,78.02%,49.07%,35.82%and 100%respectively,while 0.45 g/kg ZME promoted the formation of 8-Me IQx and Norharman.The addition of ZME significantly inhibited the formation of total HAs(P<0.05),with 0.45 g/kg ZME showing the highest inhibition of total HAs at 39.87%.Therefore,in practical production,when total HAs are used as the evaluation indicator,it is recommended to choose 0.45 g/kg ZME.Further analysis of the effects of ZME on HAs precursors of roast beef patties showed that the consumption of precursors exhibit a decreasing trend with the increasing addition levels of ZME.Correlation analysis showed that the formation of HAs was significantly correlated with a variety of free amino acids(P<0.05),with PhIP formation showing a significant negative correlation with Phe,Ala and His(P<0.05);IQ showed a significant negative correlation with Gly,Ala and His,and Me IQ with Ala(P<0.05).2.Mechanism of major polyphenols from ZME inhibiting the formation of PhIPPhIP is well known as the most common HAs in processed meat,and PhIP is also the largest contributor to the average daily intake of HAs in China.ZME was able to significantly inhibit the formation of PhIP in roast beef patties.Therefore,taken PhIP as the research object,a real meat system(roast beef patties)and a chemical model system(phenylalanine-creatinine-glucose model)were established to investigate the main polyphenols in ZME:chlorogenic acid,hyperoside and quercitrin for their effects on the formation of PhIP and to explore the possible mechanisms of inhibition.The results showed that all three polyphenols were effective in inhibiting the formation of PhIP(except for the chlorogenic acid treatment groups in roast beef patties)(P<0.05).In both systems,the inhibition of PhIP by hyperoside and quercitrin increased gradually with the increasing levels,and the strongest inhibition was achieved by the addition of 45μg/g hyperoside,with the maximum inhibition rates of 76.19%and 63.86%,respectively;chlorogenic acid significantly inhibited the formation of PhIP in the model system(P<0.05),with a maximum inhibition rate of 61.77%;however,higher addition levels showed significant promotion of PhIP formation in roast beef patties(P<0.05).Gas chromatography-mass spectrum(GC-MS)analysis showed that all three additives significantly reduced the amount of phenylethylaldehyde in the model system(P<0.05)and their ability to scavenge phenylethylaldehyde increased with increasing levels.Correlation analysis showed good correlations between the phenylacetaldehyde-scavenging activity of hyperoside and quercitrin and their inhibition of PhIP in both systems,while the correlation was poor for chlorogenic acid.Further Ultra performance liquid chromatography/tandem mass spectrometry(UPLC-MS/MS)and Collision-induced dissociation(CID)analysis revealed that hyperoside and quercitrin were able to undergo electrophilic substitution reactions with phenylethylaldehyde at the C-6 and C-8 positions on their A rings to form four polyphenol-phenylethylaldehyde adducts,namely 8-C-(E-phenylvinyl)hyperoside,6-C-(E-phenylvinyl)hyperoside,8-C-(E-phenylvinyl)quercitrinand6-C-(E-phenylvinyl)quercitrin,thereby scavenging phenylethylaldehyde,preventing its reaction with creatinine and ultimately blocking the production of PhIP.3.Mechanism of major polyphenols from ZME inhibiting the formation of IQ and Me IQIQ-type HAs(mainly including IQ and Me IQ)showed the strongest mutagenic activity in the Salmonella/microsomal in vivo assay.However,at present,the research on IQ-type HAs at home and abroad mostly focused on inhibition,and the mechanism of polyphenols inhibiting the formation of IQ-type HAs remains unclear.Based on this,taking IQ and Me IQ as the research objects,the effects of the three major polyphenols in ZME on the formation of IQ and Me IQ in real meat systems(roast beef patties)and chemical model systems(glycine-creatinine-fructose model)were investigated,and the possible inhibition mechanism was explored.The results showed that the three polyphenols significantly inhibited the formation of IQ and Me IQ in the two systems at all three levels(P<0.05),and the inhibitory activity increased with the addition of chlorogenic acid and quercitrin for both systems(except for the inhibition of Me IQ by quercitrin in the model system).The inhibitory activity of hyperoside on IQ and Me IQ decreased first and then increased with the increasing addition levels.In the roast beef patties,the strongest inhibitory effect on IQ and Me IQ was achieved by the addition of 15μg/g chlorogenic acid,with inhibition rates of 74.58%and 59.83%,respectively.In the model system,the treatment groups with 0.09m M hyperoside showed the highest inhibitory effect on IQ,with inhibition rates of 76.58%;while 0.08 m M quercitrin showed the strongest inhibition of Me IQ,with the ratio of76.73%.The formation of IQ-type HAs involves the participation of free radicals.The present study confirmed the involvement of free radicals in the formation of IQ and Me IQ by the2,2,6,6-tetramethyl-1-piperidinyloxy(TEMPO)assay and the N-tert-butyl-α-phenylnitrone(PBN)spin trapping followed by electronic spin resonance(ESR)techniques.The hyperfine coupling constants a _N=15.0 G and a _H=3.71 G were calculated from the ESR spectra,indicating the formation of carbon-centred radicals during heating.All three polyphenols were able to significantly inhibit the generation of carbon-centred radicals in the model system and roast beef patties,with chlorogenic acid showing the relatively strongest scavenging activity for this radical,with maximum scavenging rates in the two systems of 54.58%and 88.88%,respectively.In addition to free radicals,pyridine and pyrazine formed by the Maillard reaction are also the basis for the production of IQ-type HAs.GC-MS results showed that four pyrazine derivatives,2,5-Dimethylpyrazine(2,5-DMP),2,6-Dimethylpyrazine(2,6-DMP),2,3,5-Trimethylpyrazine(Tr MP)and2,3,5,6-Tetramethylpyrazine(TMP)were detected in the model system,and the inhibition rates were more than 80%.The above results indicated that the inhibition of IQ and Me IQ formation by chlorogenic acid,hyperoside and quercitrin may be achieved through the scavenging of carbon-centered radicals and the inhibition of the formation of pyrazine. |
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