Suhong Hung, Xioli Dong, Yulong Zhng, Ming Hung,*,Yundong Zheng

a Jiangsu Collaborative Innovation Center of Meat Production and Processing, Quality and Safety Control, College of Food Science and Technology,Nanjing Agricultural University, Nanjing 210095, China

b Jiangsu Province Research Engineering Center for Livestock and Poultry Meat Processing, National R&D Center for Poultry Processing Technology,Nanjing Huangjiaoshou Food Science and Technology Co., Ltd., Nanjing 210095, China

c Henan Province Qi County Yongda Food Industry Co., Ltd., Hebi 458000, China

Keywords:

Nε-carboxymethyl-lysine

Chicken

Post-mortem ageing

Storage

Oxidation

Precursors

A B S T R A C T

Advanced glycation end products (AGEs) might pose health risks, and processing and storage could accelerate the generation of AGEs in meat. However, limited few reports indicated the changes of AGEs contents in meat during storage. In this study, the aim is to investigate the oxidation and precursors and their roles in the formation of Nε-carboxymethyl-lysine (CML) in raw and cooked chicken meat after post-mortem ageing and storage. As post-mortem ageing and storage time increased, the CML content in cooked chicken breast significantly increased from 1.81 mg/kg to 2.00 mg/kg during 0-6 h, and then decreased from 2.00 mg/kg to 1.80 mg/kg during 6 h-1 day, finally increased again during 1-7 days, while the CML contents of raw and cooked leg significantly and continuously increased from 1.78 mg/kg to 2.08 mg/kg. Furthermore, CML was extremely positively correlated with fat oxidation (R2 = 0.793, P < 0.01), protein oxidation (R2 = 0.917,P < 0.01) and glyoxal (R2 = 0.678, P < 0.05), and was negatively correlated with lysine (R2 = 0.536, P < 0.05).No significant correlation was observed between the Schiff base and CML.

1. Introduction

Advanced glycation end products (AGEs) are a series of heterogeneous compounds formed by the non-enzymatic reaction between a carbonyl compound of reducing sugar and an amine group of protein [1-3]. AGEs have been study objects of drastic debates because they are pathogenic factors resulting in diabetes, obesity and other chronic diseases [4]. Although the endogenous AGEs can generatein vivo, the dietary AGEs (dAGEs) are the main exogenous source of AGEs in the body [5]. Thus, AGEs have attracted extensive interests not only in medical science but also in food science.

Nε-carboxymethyl-lysine (CML) is a kind of stable AGEs in food system, and is usually recognized as a typical marker of AGEs [6]. In food systems, studies have shown that there are mainly four pathways to the formation of CML. First, Schiff base, an intermediate product of the Maillard reaction, is rearranged by Amadori to form rearranged products and then these products are decomposed by self-oxidize to form CML (Hodge path) [7]. Second, glucose reacts with lysine to directly form Schiff base, and the glyoxal formed by the cleavage of the Schiff base reacts with lysine to form CML (Namiki pathway) [8].Third, glucose self-oxidizes to produce glyoxal, and then the glyoxal reacts with lysine to form CML (Wolf pathway) [9]. Fourth, lipid selfoxidize to produce the glyoxal, and then the glyoxal reacts with lysine to generate CML (Fu pathway) [10]. Therefore, the major precursor substances of CML are glyoxal, lysine and Schiff base. With regard to analytical methods, a series of analytical methods are performed to detect CML, such as instrumental analysis and immunochemical methods (ELISA). The instrumental analysis has high sensitivity and high selectivity, but the sample preparation procedures are timeconsuming, while ELISA is fast, convenient as well as large-scale in application, especially for determining a large number of samples [11].Gómez-Ojeda et al. [12]compared 3 different ELISA assays and HPLC-ESI-ITMS/MS for the analysis of CML in food system and demonstrated a reasonable relationship between CML determined by ELISA and HPLC-ESI-ITMS/MS, supporting the implementation of ELISA in food CML/AGEs screening. Subsequently, more and more researchers are using ELISA method for CML [13-15].

Meat is one of the most popular foods all over the world.Meanwhile, meat is a rich source of CML due to high protein and/or high fat contents [16,17]. During processing and storage, oxidation of protein and lipid is unavoidable in meat, which affects the formation of CML [6,18,19]. Up to now, reports about the effect of storage time of raw meat on the generation of CML in meat and meat products are still quite limited. Typically, Niu et al. [20]found that the longer the ice storage time was, the higher CML content was generated in heat-processed fish, and there were biological differences while studying the effect of fish freshness on the production of advanced glycation end products. Niu et al. [18]also investigated the effects of cold storage (0 °C, 0-8 days) on the formation of CML in raw and subsequently commercially sterilized pork, and the authors found storage time did not significantly affect on the amounts of CML in raw pork, but had a significantly effect on the contents of CML in stored pork. Afterwards, Yu et al. [21]investigated the impact of frozen storage of raw pork on the formation of AGEs in meat products, and they observed an increase in the contents of AGEs in meat products with the increasing frozen storage time of raw pork.These reports mainly focused on pork and fish. However, poultry meat, as the world’s largest type of animal protein, has exceeded pork production and consumption in recent years, but few relevant studies have reported on CML formation in poultry meat during storage.

Ageing of poultry meat is a normal procedure. The protein unfolding, proteolysis and aggregation during ageing promote the increase in tenderness and improvement in the flavor [22]. During post-mortem ageing and storage, protein and lipid oxidation is inevitable, which may produce some CML’s precursors, affecting the CML formation during meat processing. Therefore, the aim of this work was to investigate the changes of CML contents in raw and subsequent cooked chicken meat after different post-mortem ageing and storage time by ELISA. At the same time, we also explored the effects of oxidation and precursor substances on CML contents in raw and subsequent cooked chicken meat after different post-mortem ageing and storage time, thereby providing a valuable reference on the influence mechanism of CML in chicken meat during post-mortem ageing and storage.

2. Materials and methods

2.1 Reagents

96 T Chicken CML double-antibody sandwich enzymelinked immunosorbent assay (ELISA) kits were all purchased from Maibo Reagent Co., Ltd. (Nanjing, Jiangsu, China). Trichloroacetic acid (TCA), thiobarbituric acid (TBA), malondialdehyde (MDA),2,4-dinitrophenylhydrazine (DNPH), hydrochloric acid (HCl), guanidine hydrochloride, ethyl acetate, absolute ethanol, 1,1,3,3-tetraethoxypropane(TEP), bovine serum albumin (BSA), sodium hydroxide, blue vitriol, potassium sodium tartrate, dichloromethane, sodium acetate,hydroxylamine hydrochloride,O-phthaladehyde (OPA), methanol,sodium tetraborate, sodium dodecyl sulfate (SDS),β-mercaptoethanol and ethylenediaminetetraacetic acid disodium salt (EDTA-Na2) were analytical reagents.

2.2 Sample information

All procedures were approved by the Animal Care and Use Committee of the College of Food Science and Technology of Nanjing Agricultural University. The average market age of 8 live white feather chickens was 45-day-old (average weight between 2.0 to 2.3 kg). All chickens were slaughtered according to the commercial slaughter process. They were bled by severing the jugular vein and carotid artery using a hand-held knife on one side of the neck to allow bleeding for 150 s [23,24]. Samples from each breast and leg were immediately (0 h) snap-frozen in liquid nitrogen and stored at-80 °C until subsequent analyses. The remainders of each breast and leg was rapidly buried in ice and transported to the laboratory within 30 min. Then, the samples were stored at 4 °C for 6 h, 12 h, 1 day,3 days and 7 days. At each time point, samples were immediately taken from the breast and leg muscles of each group, snap-frozen in liquid nitrogen and stored at -80 °C until analyses [25].

2.3 Measurement

The pH values of post-mortem chicken breast and leg at 0 h,6 h, 12 h, 1 day, 3 days and 7 days were determined using a glass electronic pH meter (Testo 205, Testo AG. Company, Germany).Each muscle sample was measured in triplicate by inserting the glass probe electrode. The pH meter was calibrated by a three-point method against standard buffer solutions of pH 4.01, 7.00 and 10.00 [26].

2.4 Heat treatment

Chicken muscles ((6.0 ± 1.0) g) were sealed into tinfoil, heated in boiling water (100 °C) for 30 min, and immediately immersed into the ice/water mixture to cool down [20].

2.5 CML measurement

The amounts of CML in cooked chicken breast and leg meat samples were determined by Chicken CML ELISA kit according to our previous methods [27]. The samples with high CML concentration were diluted to the limited range of the standard curve (CML linearity range: 10-320 ng/mL,y= 0.004 1x+ 0.122 3,R2= 0.998 8). The content of CML was expressed as mg/kg meat.

2.6 Thiobarbituric acid-reactive substances (TBARs)

TBARs indicate the fat oxidation changes in meat foods during post-mortem ageing. The amounts of TBARs in raw breast and leg meat samples were determined according to the method of Guo et al. [28].Briefly, 0.5 g minced sample was put into a 10 mL centrifuge tube, and homogenized (Ultra Turrax T25, IKA, Germany) with 3.5 mL TCA (7.5%, m/V) at 10 000 ×gfor 1 min (2 × 30 s with a 10 s interval) in an ice bath. The mixture was then centrifuged at 13 000 ×gand 4 °C for 10 min. Then, 2 mL supernatant was aspirate into another 10 mL centrifuge tube, mixed with 2 mL TBA(0.02 mol/L) and incubated in a 95 °C water bath for 30 min. After cooling in tap water, the absorbance of supernatant was measured at 532 nm. The TBA-reactive substances content was calculated according to a standard curve of TEP solution (linearity range:0-1 mg/mL,y= 0.643 3x- 0.029,R2= 0.999 6). Results were expressed as mg MDA/ kg sample.

2.7 Protein carbonyl

Protein carbonyl assay was performed referring to Fu et al. [29]with minor modifications. 0.5 g raw ground meat was put into a 10 mL centrifuge tube, and homogenized with 3.5 mL phosphate buffer (20 mmol/L sodium phosphat, 0.6 mol/L NaCl, pH 6.50) at 9 000 ×gfor 1 min (2 × 30 s with a 10 s interval) in an ice bath(Ultra Turrax T25, IKA, Germany). Two aliquots of 0.2 mL 0.1 mg/mL protein were reacted with 1 mL DNPH (10 mmol/L DNPH dissolved in 2 mol/L HCl) and 1 mL HCl (2 mol/L) as a blank. The reaction was kept away from light for 1 h and oscillated for 10 s every 10 min. After the reaction, the 2 fractions were then precipitated with 2 mL 20% TCA and centrifuged at 10 000 ×gfor 5 min at 4 °C.The precipitate was washed 3 times with 2 mL ethyl acetate/ethanol solution (1/1,V/V). The protein precipitate was then incubated with 2 mL guanidine hydrochloride (6 mol/L, pH 2.30) in a water bath at 37 °C for 30 min. The absorbance was measured at 370 nm.The protein concentration was measured by the Biuret method,using bovine serum albumin (BSA) as a standard (linearity range:0-10 mg/mL,y= 0.030 1x+ 0.067 8,R2= 0.999 9) [30]. Carbonyl was calculated by the fomula:

whereA370nmis absorbance value at 370 nm;?is molar extinction coefficient (22 400 L/mol·cm) andcis protein concentration (mg/mL).

2.8 Lysine measurement

The determination of lysine referred to the method reported by Guan et al. [31]with slight modifications. 1 g raw minced sample was put into a 50 mL centrifuge tube, homogenized with 9 mL phosphate buffer solution (pH 7.20) at 10 000 ×gfor 1 min (2 × 30 s with a 10 s interval) in an ice bath. Then, the mixture was centrifuged at 10 000 ×gfor 10 min at 4 °C. 200 μL supernatant, 0.3 mL A solution(0.004 g OPA + 1 mL methanol + 3 mL distilled water) and 3.7 mL B solution (25 mL sodium borate of 100 mmol/L + 2.5 mL SDS of 20% + 100 μLβ-mercaptoethanol) were mixed in a 10 mL centrifuge tube. Then, the mixture was kept at room temperature for 2 min. The absorbance at 340 nm was measured and the content of lysine was calculated by lysine standard curve (linearity range: 0.25-2 mmol/L,y= 0.152 3x+ 0.038,R2= 0.999 4).

2.9 Glyoxal measurement

The determination of glyoxal based upon the studies of Gilbert et al. [32]with modifications. The absorbance at 233 nm was used to indicate the level of glyoxal. 1.0 g raw minced sample was put into a 50 mL centrifuge tube, homogenized with 9 mL phosphate buffer solution (pH 7.20) at 10 000 ×gfor 1 min (2 × 30 s with a 10 s interval) in an ice bath. Then, the mixture was centrifuged at 10 000 ×gfor 10 min at 4 °C. 0.5 mL supernatant mixed with 1 mL sodium acetate solution (15 g/L) and 2 mL hydroxylamine hydrochloride (2 g/L) was added to a 50 mL volumetric flask. And the volumetric flask was put into 50 °C for 20 min. After cooling, the volume was fixed with distilled water. The absorbance of supernatant was measured at 233 nm.

2.10 Schiff base measurement

The method proposed by Gatellier et al. [33]with some modifications was used to analyze Schiff base in chicken breast and leg. 1 g raw meat was put into a 50 mL centrifuge tube, homogenized with 9 mL phosphate buffer solution (pH 7.20) at 10 000 ×gfor 1 min (2 × 30 s with a 10 s interval) in an ice bath. Then, the mixture was centrifuged at 10 000 ×gfor 10 min at 4 °C. 1 mL supernatant was incubated with 4 mL dichloromethane:ethanol (2:1,V/V)solution and agitated for 15 min. After centrifugation at 4 000 ×gfor 15 min at 4 °C, 2 phases were observed. The fluorescence intensity of the upper phase was measured at the excitation wavelength of 360 nm and emission wavelength of 430 nm. All measurements were performed at room temperature, and the fluorescence intensity values were shown in arbitrary units.

2.11 Statistical analysis

All results were evaluated using one-way analysis of variance(ANOVA) with SAS analysis software (SAS Institute Inc., Cary,NC, USA). Duncan’s multiple range test was used to compare the differences of indexes at different post-mortem ageing and storage time (P< 0.05). Pearson’s correlation was performed using SPSS analysis software (Version 18; IBM Corp. Armonk, NY). The results are expressed as mean ± SD. All graphics were drawn by Origin 9.64(OriginLab Corporation, Northampton, MA, USA).

3. Results and discussion

3.1 pH of chicken meat during post-mortem ageing and storage

The changes of pH in chicken breast and leg during post-mortem ageing and storage are shown in Fig. 1. The initial pH of chicken breast and leg was 6.09 and 6.63, respectively. With the ageing and storage time increasing, the pH of chicken breast and leg significantly decreased to 5.81 (6 h) and 6.41 (6 h) (P< 0.05), respectively,indicating that the pH of chicken meat reached ultimate pH;subsequently, no significant difference was found, which is in good accordance with the result of Lee et al. [34]. Due to the termination of oxygen supply in muscles after slaughter, the lactic acid from anaerobic respiration and phosphate ions from ATP decomposition are accumulated, which leads to a rapid decrease in the pH value of muscles. When glycogen is depleted, the pH value reaches the lowest called ultimate pH, at which the muscle enters rigor. Afterwards,the pH will rise to a certain extent because of the decomposition of lactic acid and the degradation of partial protein during post-mortem ageing [34]. Additionally, the pH of the leg was much higher than breast’s. This might be explained by faster glycogen utilization and muscle metabolism in chicken breast muscle [34,35].

Fig. 1 The change of pH in chicken during post-mortem ageing and storage at 4 °C for up to 7 days. Different letters indicate significant difference (P < 0.05).

3.2 CML content of chicken during post-mortem ageing and storage

Fig. 2 shows the changes of CML contents in raw and cooked chicken breast and leg during post-mortem ageing and storage(0-7 days). Similar results were reported by Niu et al. [20], who found that there was a big biological variation among individual chicken. To minimize the adverse influence of individual variation when determining whether the post-mortem ageing and storage affects the contents of CML in raw and cooked chicken, samples from the parts with minimized sample variance of the same individual chicken should be aged and stored for different durations and immediately analyzed for CML contents [36]. The initial contents of CML in raw chicken breast and leg were 0.95 and 1.19 mg/kg, respectively. With the extension of ageing and storage time, the CML content in raw chicken breast had no significant change. For subsequent cooked chicken breast, the content of CML had a significant change, which indicated that CML content firstly significantly increased from 1.82 mg/kg (0 h) to 2.00 mg/kg (6 h), then decreased to 1.80 mg/kg(1 day), and finally increased again (P< 0.05). Previous studies indicated that cold storage did not significantly influence the CML content in raw fish muscle or raw pork, but longer storage time of raw fish or raw pork could result in more CML in subsequent cooked meat [20,37]. This phenomenon could be explained that during postmortem ageing and storage period, biochemical reactions (lipid oxidation, protein oxidation and protein denaturation) in chicken breast continuously occur due to the activities of endogenous enzymes and microorganisms [38]. These reactions could lead to the exposure of some amino acids’ reactive groups, such as lysine residues, and the accumulation of some active dicarbonyl compounds, such as glyoxal [10], which might have a mild effect on the CML content in raw chicken breast during post-mortem ageing and storage, but would greatly accelerate the formation of CML in chicken breast upon heating. Validation studies indicated that the chicken meat took about 6 h to achieve rigor mortis and reach ultimate pH [39]. Due to the presence of enzymes, there may be numerous biochemical reactions to promote the formation of CML in rigor mortis. A drop of CML after 6 h might result from protein cross-linking reaction that produces a cross-linking substance similar to CML-lysine. The final increase in CML might be due to oxidation. Furthermore, the CML contents of raw leg significantly increased from 1.19 mg/kg to 1.58 mg/kg, and the CML contents of cooked leg greatly increased from 1.78 mg/kg to 2.08 mg/kg during 0-7 days (P< 0.05). These results may be due to the oxidation that is proportional to the CML content [18]. Whether raw or cooked, the changes of CML contents in breast and leg meat during ageing and storage were different.The results also indicated that both raw leg had more CML when compared with breast meat. It might be attributed to different basic constitutions, the oxidation degree, the different pH as well as others. Yu et al. [40]studied the effect of pH and amino acids on the formation of methylglyoxal in glucose-amino acid model system, and the author and co-workers observed that lower pH could inhibit the formation of CML. This is one reason why the CML content in the leg is higher than that of breast. In addition, the average content of CML in cook meat increased after boiling, which was similar to the studies’ results that heating treatment promote a significant increase in the content CML of meat [18,20].

Fig. 2 CML levels in raw and cooked chicken breast and leg during postmortem ageing and storage at 4 °C for up to 7 days. Different letters indicate significant difference (P < 0.05).

Consequently, post-mortem ageing and storage time had a significant effect on the CML in chicken meat. In general, CML in food system can be formed by the direct reactions of lysine and glyoxal that can be formed by degradation of protein, oxidation of Schiff base, sugar and lipid, etc. [41,42]. Considering all above,lipid oxidation, protein oxidation and precursors of CML should be investigated to study the reason for the changes of CML during chicken meat post-mortem ageing and storage.

3.3 Lipid and protein oxidation

The TBARs and carbonyl values of chicken meat during postmortem ageing and storage are shown in Fig. 3. With the prolongation of post-mortem ageing and storage time (0-7 days), the MDA and carbonyl contents of raw chicken breast and leg meat significantly increased (P< 0.05), indicating that the oxidation of fat and protein existed simultaneously during the post-mortem ageing and storage of chicken meat. The oxidation degree of leg meat was higher than that of breast meat, which was similar to the results of Soyer et al. [38].But our results of the carbonyl group (breast: 0.48-0.65 nmol/mg protein, leg: 0.67-0.84 nmol/mg protein) were lower when compared to those in chicken meat (breast: 1.3-1.8 nmol/mg protein, leg:1.7-2.1 nmol/mg protein) reported by Soyer et al. [38]. This difference might be ascribed to different kinds of chicken meat.Poultry meats are easy to oxidize because of their high content of oxidation catalysts (such as iron and myoglobin) and lipids [43].Additionally, the oxidation reaction could induce the protein to unfold, and enable the cleavage of peptide bonds and the exposure of amino acids’ reactive residues, which might contribute to the formation of CML through Maillard reaction during high temperature processing (100 °C, 30 min) [44]. Han et al. [45]reported that the hydroxyl produced by oxidation facilitated the change from fructose lysine (FL) and glyoxal to CML. Yu et al. [37]found a linear correlation between TBARs/carbonyl values and CML in sterilized meat. Thus, these results could explain that CML content in the leg meat was higher than that in the breast meat, and the CML content in the chicken breast and leg meat increased at a certain stage during post-mortem ageing and storage in our study.

Fig. 3 Oxidation levels in raw chicken breast and leg during post-mortem ageing and storage at 4 °C for up to 7 days. Different letters indicate significant difference (P < 0.05). (A) TBARs value; (B) carbonyl value.

3.4 Precursors content (lysine, glyoxal, Schiff base)

It is well known that the breakdown of protein and the formation of peptide occur during the post-mortem aging and storage process [22].As can be seen from Fig. 4A, with the time prolonging of postmortem ageing and storage, lysine contents in raw breast and leg meat significantly increased from 0.14 mol/L to 0.18 mol/L, 0.11 mol/L to 0.15 mol/L (P< 0.05), respectively. The results can be explained that the protein-bound lysine may be gradually released and exposed due to the degradation of the protein by endogenous enzymes and microorganisms during postmortem ageing and storage. The content in breast meat was higher than that in leg meat, possibly because more lysine in leg meat has participated in the formation of CML during post-mortem ageing and storage. Chicken meat has remarkably higher contents of lysine, which makes chicken meat more easily produce CML during processing [46]. According to Fig. 4B, with the time extension of post-mortem ageing and storage, the glyoxal content in raw breast meat significantly increased during 0-12 h, then decreased during 12 h-1 day, and finally increased, while the glyoxal content in raw leg meat significantly increased (P< 0.05). Liu and Li [47]investigated the changes in glyoxal content in the fried dough twist during storage, and the authors found that the glyoxal could be formed and reduced during short storage periods. The result is consistent with our result, but the change trend is different from ours. This difference may depend on whether the sample has been processed or not. As for Schiff base, the content in raw chicken breast notably increased during 0-6 h, then decreased during 6-12 h, and finally increased again,while the content in raw chicken leg significantly increased (P< 0.05)(Fig. 4C). The post-mortem ageing and storage time prominently affected these precursors. And the changing trends of glyoxal and Schiff base were similar to CML’s. A possible explanation was that glyoxal and Schiff base were accumulated during post-mortem ageing and storage, which could facilitate the production of CML during subsequent heat processing.

Fig. 4 Precursor substance levels in raw chicken breast and leg during postmortem ageing and storage at 4 °C for up to 7 days. Different letters indicate significant difference (P < 0.05). (A) Lysine value; (B) glyoxal value;(C) Schiff base value.

3.5 Correlation analysis

It should be noted that a high correlation does not mean that causation really exists, but that a relationship becomes more likely [46].The results of Pearson correlation analysis are shown in Table 1.The CML was extremely positively correlated with TBARs(R2= 0.793,P< 0.01) and carbonyl (R2= 0.917,P< 0.01). These were consistent with our previous results that protein and lipid oxidation played an important role in the correlation of AGEs [27].As for precursor substances, CML was positively correlated with glyoxal (R2= 0.678,P< 0.05), and negatively correlated with lysine(R2= 0.536,P< 0.05). No significant correlation was observed between CML and Schiff base. Zhu et al. [15]found that the CML was significantly positively correlated with lysine during frying,which was contrary to our results. The phenomenon may be caused by different processing methods. In addition, the glyoxal was prominently positively correlated with TBARs (R2= 0.801,P< 0.01) and carbonyl(R2= 0.680,P＜ 0.05). Significant positive correlations existed between Schiff base and TBARs contents (R2= 0.613,P< 0.05).

Table 1The correlation of CML, TBARs, carbonyl, glyoxal, lysine and Schiff bases in chicken during post-mortem ageing and storage at 4 °C for up to 7 days.a

4. Conclusions

The CML contents in cooked breast, raw and cooked leg meat significantly changed during post-mortem ageing and storage time except for raw chicken breast, but there were different changes in breast and leg meat. With the extension of post-mortem ageing and storage time, the CML content in cooked chicken breast increased during 0-6 h, then decreased during 6 h-1 day, and finally increased again, while the CML contents of raw and cooked leg significantly increased. Furthermore, CML was extremely positively correlated with fat oxidation, protein oxidation and glyoxal, and was negatively correlated with lysine. These results could confirm our hypothesis that during post-mortem ageing and storage, fat oxidation and protein oxidation are inescapable in chicken meat, which could produce precursors of CML (glyoxal, lysine) and affect the formation of CML.From a health perspective, it is the most suitable way to eat chicken meat at 1 d after the slaughter. It is also concluded that the use of non-aged and no storage chicken for cooking is a potential practical way of reducing human’s exposure to CML produced during the thermal process.

Declaration of competing interest

The authors declared that there was no conflict of interest.

Acknowledgments

This work was supported by China Agriculture Research System(CARS-41-Z06).