Study On The Formation Regularities Of Heterocyclic Aromatic Amines In The Roasted Meat

Heterocyclic aromatic amines(HAAs) are a group of carcinogenic and mutagenic heterocyclic aroamtic hydrocarbon compounds which could be widely formed in various proteinrich foods. The study on the formation regularity, mechanism and inhibitory pathway of HAAs have become the focus of attention in the scientific field. Up to now, the HAAs formation mechanism have not been completely clarified. The detection strategies currently used usually obtained low recoveries and bad repeatabilities for target HAAs, bringing huge inconvenient to the formation regularity research. The evaluation on the simultaneous changing of HAAs and their precursors, and the effects of process condition on the entire HAAs have been barely reported, mainly due to the limitation from study method. In addition, the effects and mechanisms of some food ingredients and antioxidants on the HAAs formation have not been illustrated, looking forward to the further studies. Therefore, in this study, the formation regularities and influencing mechnisms of HAAs were comprehensive and in-depth investigted by establishing a new detection method, evaluating the changing regularities of HAAs and their precursors in the process, and elucidating the influencing mechanisms of some additives. This study could offer a theoretical foundation for clearly expliciting the formation and inhibition mechanisms of HAAs in complex meat systems in the future.Firstly, a UHPLC-MS/MS detection method for simultaneously detecting seventeen HAAs in meat products were established in this study. By comparison, the source and desolvation gas temperatures were ultmately set at 120 and 350 oC, respectively. The Acquity BEH C18 column was finally chosen to isolate all the 17 HAAs. Based on the explicit UHPLC-MS/MS method, A new sample pretreatment method that could be applied to fast and efficient quantify polar HAAs was developed and optimized. The new method involves an one-step extraction using hydrochloric acid(HCl) and a solid-phase extraction(SPE) cleanup using Oasis MCX cartridges. Significantly higher recoveries from 58.2% to 107.6 were obtained compared with a commonly used reference method based on multiple extraction(P < 0.05). With low detection limits(LODs ranged in 0.026–0.659 ng g-1), good repeatabilities(RSD% <12.03%), and non-significant matrix effects(Rn ranged from 0.89 to 1.18), the new HCl method could be successfully applied to the analysis of five commodity meat products.Secondly, in order to elucidate the simultaneous changing regularities of HAAs and precursors during the heating process, a noval method allowing simultaneous analysis of HAAs and their precursor was established and applied to evaluate the changing regularity of HAAs and precursors based on the HCl method.The method involving a direct 0.1 M HCl extraction, Waters Atlantics d C18 column isolation could successfully applied to simultaneous analysis 5 HAAs and 8 precusors. The recoveries for all 13 targets were ranged from 48.1% and 122.2%, with a wide linear range(r2 > 0.991) and high precision(RSD < 15.7%). Two lyophilized pork model with or without water was set up. And the levels of HAAs and precursor during 0–120 min heating under 200 oC were simultaneously detected. The results showed that all the five HAAs were formed after 5 min heating. Ph IP and 4’-OH-Ph IP were continue to form during the heating time, while a slight degradation was found for the quinoxaline HAAs after 30 min. The HAAs precursors including amino acids, creatine and sugar were dramtically decreased within the initial 5 min, followed with a gentle content variation. The variation of creatinine appear to be a special raise and down trend. The formation of Ph IP and 4’-OH-Ph IP, the cosumption rate of their related precursors amino acids, Phe and Tyr was inhibited by the adding water in pork model, while the formation and degradation of IQx、Me IQx and 4,8-Di Me IQx was promoted with no significant effect on their precursors Gly and Ala.Then, the effects of process condition and changing of precursors on the formation of HAAs in real roasted pork were investigated. The HAAs profiles were established. The effects of temperature and time on such HAAs profiles were then evaluated by principle component analysis(PCA). And the correlation between the precursors changing and HAAs formation were analyzed using Pearson linear dependence model. The results showed that the HAAs profiles were composed of 12 HAAs detected in roasted pork, including DMIP and et al. The principle component analysis(PCA) revealed that the HAAs profiles for different temperatures are sigificant diverse. And the effects of roasted time were more significant in samples roasted under higher temperature. Ph IP, 4’-OH-Ph IP, IQ[4,5-b] and Me IQ, were the core HAAs responsible for the influencing of process condition on the HAAs profiles. The formation of the HAAs belonging to different categories were obviously different. The quinoxalines were obviously different from carbolines, mainly contributed by the HAAs formation levels in sample roasted before 20 min in 175 oC. The correlation analysis showed that creatine and creatinine were signifcantly positive correlated to most of HAAs except DMIP, opposited to creatinine which were negative correlated to HAAs foramtion(P < 0.05). Glc was correlated to only the DMIP and TMIP. And the Thr was related to the TMIP. Phe, Tyr, Gly and Trp were tightly related to all HAAs except IQ.In the next section, the effects of 4 commonly used ingredients, including soy protein isolate(SPI), texture protein(TP), startch and soybean dietary fiber(SDF)(adding levels were 2.5, 5.0, 7.5, 10.0%), plus the influencing mechanism of SPI on the HAAs formation were investigated.. Meanwhile, the SPI promotion effects were further proved by adding SPI into three model systems, including Ph IP system, quinoxalines system and carboline systems. Then, the SPI promotion mechanisms were discussed by detecting the free amino acids and carbonyl changing of SPI after heating, combined with the evaluation of the effects of SPI on the formation of HAAs intermediates(including formaldehyde, acetaldehyde, phenylacetaldehyde and 2,5-dimethylpyrazine). Results revealed that the formation of 9 HAAs detected in the roasted sample were promoted with varying degree when 2.5% above additives were added. Then, the promotion effects gradually decreased along with the increase of doses. Until the dose reached to 10.0%, the adding of SPI, starch and SDF could significantly inhibited the formation of Me IQx and 4,8-Di Me IQx, while TP inhibited 1,5,6-TMIP 、 4’-OH-Ph IP and Me IQx with different degree of inhibition effects. The results obtained from above three model systems showed that the HAAs contents could be obviously increased after adding SPI. After heating, the free amino acids and carbonyl in SPI were significantly increased. At the same times, the SPI could signifcantly promoted the formation of above mentioned HAAs intermediate. Two SPI promotion pathways were therefore speculated: 1) SPI generate more free amino acids that promote the HAAs formation. 2) The increase of carbonyl in SPI during the heating process could accelarated the Strecker degradation reaction(The first step reaction of HAAs formation) to generate more HAAs intermediates.In the last section, the effects of 0.2 mmol 8 different various antioxidants, including quercetin, naringenin, hesperidin, luteolin, EGCG, diallyl disulfide(DAD) and dipropyl disulfide(DD) and Vc, on the HAAs formation were studied.. Meanwhile, the naringenin, DAD and DD were added to the three model systems(same as the system used to test the SPI promotion effect) to verify the influencing effects. Then, the inhibitory effects of naringenin, DAD and DD were further evaluated from the aspect of intermediates. The results revealed the inhibitory effects of antioxidants are obviously differen. The quercetin have the best inhibition effects on DMIP, 1,5,6-TMIP and 4’-OH-Ph IP. And the naringenin could inhibit more Ph IP、Norharman and Harman. DD could signifantly inhibited the formation of polar HAAs, whereas DAD obtained the opposite effect. Compared to luteolin, the excessive glucoside in hesperidin could promote the formation of nonpolar HAAs. In the model systems, the effects of naringenin, DAD and DD were in agree with the real pork. The naringenin and DD could sigificant decrease the levels of phenylacetaldehyde, formaldehyde, acetaldehyde, and 2,5-dimethyl pyrazine, while DAD could only inhibited the formation of formaldehyde. The intermediate scavening test revealed that naringenin and DD have significant scavenging capacity to phenylacetaldehyde, formaldehyde, acetaldehyde, demonstrating that the aldehydes scavenging might be the critical pathway for the inhibitory effects of these two antioxidants. Finally, in our work, two naringeninphenylacetaldehyde adducts and one naringenin-formaldehyde adduct were preliminaryly identified in the roasted pork, further verifying that the naringenin often go through a adduct formation to scavening phenylacetaldehyde and formaldehyde.