| Heterocyclic amines(HAs)are a class of heterocyclic aromatic compounds with carcinogenic and mutagenic effects that are generated during high-temperature cooking of raw materials that rich in animal-derived proteins.The current research on HAs is mostly based on the free HAs produced by the reaction of glucose,creati(ni)ne and free amino acids.However,the protein content of meat(approximately 20%)is much higher than the content of glucose,creati(ni)ne and free amino acids(0.1%-0.5%),muscle protein is likely to participate in Maillard reaction to produce HAs in the form of muscle protein-HAs conjugates(muscle protein-bound HAs).Previous studies have shown that their levels in beef patties were up to?g/g.In addition,IQ?Me IQ?A?C and other HAs with high toxicity are easy to exist in the bound state.After in vitro digestion,it can be partially transformed into free HAs.Therefore,the existence of protein-bound HAs cannot be ignored,but it is currently rarely reported.In order to specifically inhibit the formation of protein-bound HAs during processing and reduce its toxic effects in the body,it is important to elucidate its generation pathways and digestion rules.In this study,two major muscle proteins,sarcoplasmic protein(SP)and myofibrillar protein(MP),were used to establish SP/MP+HAs and SP/MP+glucose+creati(ni)ne chemical model systems,which were the research objects together with roast beef patties,and 17 kinds of HAs was determined by ultra-high performance liquid chromatography triple quadrupole mass spectrometry synchronously.The possible generation pathways of muscle protein-bound HAs,the effect of heating temperature and duration on its production,the release rules in different digestion stages,and the effects of some common meat matching dietary ingredients(seasonings,fruits and vegetables)on the release of protein-bound HAs during in vitro digestion were explored.Two generation pathways of muscle protein-bound HAs were confirmed by the establishment of SP/MP+HAs and SP/MP+glucose+creati(ni)ne model systems,respectively:muscle protein is directly adducted with free HAs to produce protein-bound HAs(pathway 1);muscle protein replaces free amino acids in the Maillard reaction to produce protein-bound HAs(pathway 2).SP-bound HAs are easier to generate via pathway 1,and the binding rate of most free HAs to SP were>80%;MP-bound HAs are easier to generate via pathway 2,and 8 types,up to 206.28±13.97 ng/100 mg MP were generated after heating at 90?for 10 min,the types and content were higher than SP.The main categories of protein-bound HAs in two systems were almost the same:?-carbolines(?62.99%),?-carbolines,pyridines and quinolines.The effects of different heating temperatures(90?,120?,150?and 180?)and durations(10 min,20 min,30 min and 40 min)on the formation of SP/MP-bound HAs in the SP/MP+creati(ni)ne+glucose model system were explored.The results showed that most of the bound HAs can be formed by heating at 90?-120?for a short time.Principal component analysis revealed no significant difference in protein-bound HAs at 90°C-120°C(P>0.05),some HAs increased significantly or began to form at about 150°C(P<0.05),and increased most significantly(P<0.05)and changed fastest with the extension of heating time at 180°C.The protein-bound HAs were always higher than the free HAs.The content was the highest at180°C/40 min,the SP system produced 452.06±33.32 ng/100 mg SP,while the MP system produced 387.01±37.50 ng/100 mg MP.?-Amylase,pepsin,trypsin and bile salt were used to simulate in vitro digestion in the oral,gastric and intestinal phase,respectively.The transformation rules of the protein-boud HAs in the SP/MP+creati(ni)ne+glucose model systems(heating at 180~oC/40 min)after different digestive stages(oral phase,oral+gastric phase and oral+gastric+intestinal phase)were investigated.The results showed that the initial total free HAs was 97.40±7.75 ng/100 mg SP and 15.12±1.33 ng/100 mg MP,respectively.After oral and gastric digestion,there was no significant change(P>0.05).Most of the SP/MP-bound HAs experienced enzymatic release after intestinal digestion,resulting in a significant increase in total free HAs(P<0.05).The total release rate of bound state HAs was 36.67%in SP system and 68.09%in MP system.?-carbolines had the highest release rate(34.41%-88.20%),indicating that they were easily hydrolyzed by digestive enzymes and converted into free HAs.In order to investigate the effect of co-digestion of chili pepper powder,apple and onion with protein-bound HAs during the in vitro digestion process,these ingredients were added in different amounts to the SP/MP+creati(ni)ne+glucose model system(heating at 180~oC/40 min),then in vitro digestion was performed simultaneously with protein-bound HAs.The results showed that they can all promote the conversion of SP/MP-bound HAs to free HAs to a certain extent.In the MP system,the total free HAs in the control group was 4.57±0.61 ng/100 mg MP,which was significantly increased to 20.44 times that of the control group by 1.0%pepper powder.The apple and onion groups showed a concentration dependent manner,with the highest levels increased to 41.56 times and 33.28 times,respectively(P<0.05).In the SP system,the control total free HAs was 147.72±28.47 ng/100 mg SP,1.5%chili pepper powder made it a significant increase of 43.43%(P<0.05).Both the apple and onion groups showed a concentration-dependent,the highest increased the free HAs significantly by 54.45%and56.06%,respectively(P<0.05).Compared with the MP system,the growth-promoting effect of additives in the SP system was significantly smaller and there are fewer types of HAs that were influenced.In beef patty systems with complex matrix,the effect of simultaneous intake of different amounts of pepper powder,apple,onion with protein-bound HAs on the release rate of protein-bound HAs in the in vitro digestion process was further explored.The total free HAs in the control group was 111.54±9.54 ng/g,1.5%chili pepper powder made it a significant increase of 28.80%(P<0.05).Both the apple and onion groups showed a concentration-dependent,the highest increased the free HAs significantly by 104.17%and 124.78%,respectively(P<0.05).The promotion rates of the free HAs in the beef patties by the three additives were generally between SP system and MP system,but closer to SP system. |
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