The effect of oxidation and Maillard reaction on formation of Nε -carboxymethyllysine and Nε-carboxyethyllysine in prepared chicken breast

ABSTRACT The aim of this work was to investigate the effect and correlation of boiled, deep-fried and roast thermal processing on Nε -carboxymethyllysine and Nε-carboxyethyllysine formation in prepared chicken breast. The three heat processing results indicated that oxidation was one main reason to affect advanced glycation end products (AGEs) formation. For boiled and deep-fried, protein and lipid oxidation could promote the AGEs (P < 0.05), while protein oxidation may inhibit AGEs formation by roast processing (P < 0.05). Furthermore, Maillard reaction showed an important effect on L*, a* and b* values, and the content of AGEs could be easily determined by the changes of meat color. As conclusion, Maillard reaction and oxidation were two key factors to influence the AGEs formation in prepared chicken breast by thermal processing.


Introduction
In recent years, prepared chicken meat consumption has rapidly increased due to their convenient and nutritious characteristics. As one type of ready-to-eat meat products that mainly composed of chicken breast, spices and cornstarch, prepared chicken is recognized as not only delicious but also healthy food. (Bonoli, Caboni, Rodriguezestrada, & Lercker, 2007). The finished products, accomplished with flavoring and a series of steps such as slicing, trimming, pickling and packaging are very convenient for consumers to eat after a quick thermal processing (Alvarado & Sams, 2004). However, some unsolicited and unfavorable consequences are easily formed during meat thermal processing resulting in the amino acids loss and toxic compounds generation, such as heterocyclic amines, 3,4-benzopyrene, cholesterol oxide and acrylamide (Trevisan, Daniele, Geni Rodrigues, Soares, & Deborah Helena, 2016). The formation of harmful substances is closely related to the Maillard reaction in heat processing.
Advanced glycation end products (AGEs) are a series of complex compounds that are mainly formed at the last stage of Maillard reactions, including N ε -carboxymethyllysine (CML), methylglyoxal (MGO), pentosidine, N ε -carboxyethyllysine (CEL), methylglyoxal-lysine dimers (MOLD), pyrraline etc. (Han et al., 2013;Nessar, 2005). As two typical of AGEs, CML and CEL are not only large produced but also stable in processed food. Thus, they are ordinarily used as markers for the occurrence and levels of AGEs in daily consumed food (Gong, Guangwei, Lu, & Mitchell, 2011). Meat shows high potential to generate more AGEs than other foods, such as fruits and vegetables (Sun et al., 2016). So, CML and CEL are easily formed in meat products because of the high contents of protein and fat. Meat types and processing conditions are two key factors influencing the formation and levels of CML and CEL in meat, such as cooking method, temperature range, heating time, sterilization and storage conditions, etc. (Lu, Cheng, & Ying, 2014). Processing temperature has a significant effect on the formation of AGEs. Chen & Smith compared CML generation under frying (204°C), broiling (232°C) and baking (177°C) conditions in beef, pork, chicken and fish (Chen & Smith, 2015). The results showed that higher temperature would produce higher CML. In addition, CML and CEL generated in large quantities not only through Maillard reaction but also through oxidation such as lipid oxidation (Poulsen et al., 2013). High processing temperature promotes the lipid oxidation in meat products and generates a large number of reactive oxygen radicals, which will further promote Maillard reaction to produce active α di-carbonyl compounds, such as methylglyoxal (MGO) and glyoxal (GO) (Degen, Hellwig, & Henle, 2012;Jiang, Hengel, Pan, Seiber, & Shibamoto, 2013;Sheng, Larsen, Le, & Zhao, 2018). Furthermore, Goldberg et al. found that the higher content of protein and fat, the higher levels of CML and CEL in meat (Goldberg et al., 2004).
Commonly speaking, Maillard reaction is the key pathway for AGEs formation. The reaction mechanism of AGEs formation by Maillard reaction pathway is mainly about Schiff's base and Amadori Rearrangement Product (ARP) formation, which will promote the di-carbonyl compound generation (Baskara, Niquet-Leridon, Anton, & Delayre-Orthez, 2017;Thornalley, 2005). Then, the di-carbonyl compound reacts with functional groups of lysine or arginine to generate different AGEs (Smith et al., 1994). Meanwhile, CML and CEL can be generated in meat products not only via the complex Maillard reaction, but also the protein and lipid oxidation (Yu, He, Zeng, Zheng, & Jie, 2016). More importantly, the Maillard reaction and oxidative reaction can take place concomitantly during meat processing, both reactions are closely interconnected, and the reaction products formed each reaction pathway will influence each other (Delgado-Andrade, 2016). The connection of Maillard reaction and oxidation is so inseparable that they should be considered associating with AGEs formation .
So the aim of this work was to investigate the AGEs formation by boiled, deep-fried and roast processing in prepared chicken breast as well as provide a new point of view to reveal the influence of oxidation and Maillard reaction on the formation of AGEs during meat heat processing.

Preparation of chicken breast
Sixty-five frozen chicken breasts were used and manufactured in a food preparation room where the interior temperature was stable at around 25°C. All the frozen chicken breasts were sliced into 1 cm pieces with a food slicer (HB-3D; Nantsune carnivorous Machinery Co., Ltd, Beijing, China). Then, the sliced breasts were mixed with the marinades according to the material proportion: chicken breast 74.39%, NaCl 0.98%, phosphates 0.21%, sugar 3.65%, salad oil 0.07%, cornstarch 1.85% and water 18.85%. The mixed ingredients were tumbled for 40 min at a speed of 40 r/min (125 ESK STL; VAKONA GmbH, Germany). After tumbling, the chicken breast and residual marinades were transferred to a 4°C refrigerator (Compressor-Cooled Incubator ICP260; Memmert, Germany) for heat processing.

Boiled
Three hundred milliliters of water were poured into a cooking pot per 100 g of chicken breast. When the water temperature was heated up to 95 ± 5°C maintaining for 1 min and the chicken breast was immediately placed into the water. Each chicken breast was boiled for 5-55 min in the same way. The chicken breasts were cooled to room temperature after each boiled for 10 min and indicators were measured immediately. Set raw as a control group.

Deep-fried
Three hundred milliliters of soybean oil (Purchased from Suguo Supermarket, the trademark is Fulinmen) were poured into a fry-pan per 100 g of chicken breast. When the oil temperature was heated up to 160 ± 5°C (the oil temperature was maintained for 1 min) and the chicken breast was immediately placed into the oil. Each chicken breast was fried for 1-6 min in the same way. The chicken breasts were cooled to room temperature after each deep-fried for 1 min and indicators were measured immediately. Set raw as a control group.

Roast
The chicken breasts were put into a household oven (Midea MG38CB-AA, Midea Co., Ltd, Guangdong, China). The oven temperature was adjusted to 200°C ± 2°C for heating 5-30 min, and the chicken breast was immediately took out every 5 min. The chicken breasts were cooled to room temperature and indicators were measured immediately. Set raw as a control group.

Color
Markers generated as the Maillard reaction progress are commonly used to evaluate the development of the reaction cascade. In this respect, color is an indirect indicator of the CML and CEL generation (Roldan et al., 2015). The surface color of the prepared chicken breast was evaluated by reflection in four randomly selected zones using a Konica Minolta colorimeter CR-400 (Konica Minolta Sensing, Inc, Osaka, Japan) with a color measuring area 10 mm in diameter. Color parameters include CIELAB chromatic coordinates. L* values refer to the lightness, a* values refer to the yellowness, b* values refer to the redness, all were calibrated against a standard whiteboard before testing the samples.

Composition
Prepared chicken breasts were cut into 1 cm 3 pieces from the heat treatment group and the control, then put them into the pre-cooled homogenate machine (Ultra Turrax T25 BASIS, German IKA Company) 9000 r/min, 20 s for crushing. Crude protein was determined by Kjeltec TM2300 (Denmark FOSS company) according to AOAC Int method 992.15 (King-Brink & Sebranek, 1993). Fat content and moisture were determined by AOAC Int method 2008.06 (Leffler et al., 2008).

A 294 , A 420 , CML and CEL
Absorbance values at 294 nm and 420 nm reflect changes of intermediate and advanced stages in Maillard reaction, respectively (Pei, Xian-Bing, & Shu-Juan, 2015). One gram of control and processing meat samples (with an error of 0.001 g) were accurately added. Samples were mixed with PBS in a ratio of 1:9 (w/v). A total of 200 µL of sample solution were accurately absorbed into a 96-well plate, and then the value was determined by Microplate reader at 294 nm and 420 nm. Chicken ELISA kit was used to determine the CML and CEL. Samples with high concentration were diluted to fall within the linear range of the standard curve (10-320 ng/ml CML, R 2 = 0.9994; 0.25-8 ng/ml CEL, R 2 = 0.9998). In addition to the blank hole, standard and sample holes were set up. Each hole was added horseradish peroxidase (HRP) labeled detection antibody 100 µL, under 37°C dark reaction for 60 min. At the end of the reaction, the liquid was discarded and added 350 µL washing solution into each hole. Kept 1 min and discarded the liquid forcefully. The above steps were repeated 5 times. Then, 50 µL reaction liquid was added to each hole avoiding light for 15 min under 37°C to reaction. In the end, 50 µL terminated reaction liquid were added immediately and the values at 450 nm of each hole were determined by Microplate reader (Spectral Max M2e; Molecular Devices, California, USA).

TBARs
TBARs value was referred to the determination method described by Uchiyama and Chen with slight modification (Uchiyama & Mihara, 1978;. One gram of meat samples and 5 mL of 8% TCA (contained EDTA-Na 2 ) were accurately added into 10 mL centrifuge tube, respectively, and they were mixed at 9000 r/min homogenate under ice bath. The protein concentration was determined by BCA kit. MDA standard curves of different concentrations were prepared by using 1, 1, 3, 3-Tetrathoxypropane, 0.02 mol TBA and 8% TCA contained EDTA-Na 2 . A total of 0.5 mL of protein extraction and 0.5 mL of TBA to were added into 2 mL PE tube for reaction about 30 min under 95°C water bath. After water cooling to room temperature, the OD values at 532 nm of samples were under microplate reader for measuring. The result was expressed as mg MDA/kg meat.

Carbonyl
According to Liu, Xiong, & Butterfields' method described with slight modification (Liu, Xiong, & Butterfield, 2010). Meat samples from different treatments were dissolved in PBS (0.01 mol, pH7.4) and the protein concentration was determined by BCA kit. One milliliter of protein solution was sucked up, and 10 mmol/L DNPH solution was added into 10 mL PE tube (hydrochloric acid concentration is 2 mol/L). After 1 h reaction at room temperature away from light, 2 mL 20% TCA was added and centrifuged 5 min. The supernatant was discarded, precipitation was used by 5 mL ethyl acetate/ethanol (v: v = 1:1) washing three times, then the precipitate was dissolved in 6 mol/L guanidine hydrochloride reacting at room temperature for 30 min. Then, the solution was centrifuged at 10000 × g for 10 min (Avanti J-26S XP; Beckman Coulter, Brea, CA, USA). The supernatant was absorbed and determined by microplate reader at 370 nm, carbonyl was calculated by the formula: Carbonyl content (nmol/mg protein) = (A 370 /ε) * (10 6 /C), where A 370 is absorbance value at 370 nm; ε is Molar extinction coefficient (22400 M −1 cm −1 ) and C is protein concentration (mg/mL).

Total and active sulfhydryl
The sulfhydryl content was determined by the method of Xue with slight modifications (Xue et al., 2017). One gram of minced meat was added to 10 mL of cold PBS (0.01 mol, pH 7.4) and homogenized, then centrifuged at 5,000 × g for 8 min (4°C) and the protein concentration was determined by BCA kit. The active sulfhydryl content was determined by the following steps. A total of 0.4 mL protein sample was added to 4.6 mL PBS and 20 µL DTNB (10 mM DTNB in 20 mM KH 2 PO 4 buffer), incubated at room temperature for 1 h. For total sulfhydryl content determination, 0.4 mL of supernatant were transferred to a 10 mL tube and dissolved in 4.6 mL of 8 M urea buffer (pH 7) with 0.5 mL of DTNB. The mixture was incubated at room temperature for 1 h away from light, and the absorbance value was determined at 412 nm with microplate reader. The results were expressed as umol/mg protein.

Statistical analysis
All experiments were determined with three repetitions and expressed as mean ± SD. SAS analysis software (SAS software research institute, USA, version 8.1) was used for statistical analysis of data, one-way ANOVA method was used for the analysis of variance, and Duncan's multiple range was used to compare the differences between mean values. P < 0.05 indicated a significant difference in the results. Pearson's correlation was used by SPSS analysis software (Version 16; IBM Corp. Armonk, NY).

Color and composition changes
The increase of a* and b* values, and the reduction of L* values were the result of brown pigments formation in the advanced stage of the Maillard reaction (Bosch, Alegría, Farré, & Clemente, 2007; Morales & Boekel, 1998). Furthermore, meat processing may accelerate the Maillard reaction, which is closely related to basic composition variation (Teodorowicz, Neerven, & Savelkoul, 2017). As the result shown in Table 1, for boiled, with the boiled time increase, a* values decreased first and then increased, b* and L* values were not changed a lot, but b* values were reduced in the end comparing with 5 min. The proportion of water was reduced in the first 45 min and then increased, but the proportion of protein was in verse, and the proportion of fat was not changed a lot. The boiled treatment results indicated that Maillard reaction may get inhibited at first and then promoted. For deep-fried, with the deep-fried time increase, a* values were reduced in the first 4 min and then increased gradually, b* values were increased, and L* values were declined slowly. The proportion of water was reduced but protein and fat were increased. The results indicated that Maillard reaction maybe easier to be promoted by deep-fried treatment than boiled. For roast, with the roast time increase, a* and b* values were increased, while L* values were increased in the first 10 min and then decreased. The proportion of protein was increased, but moisture and fat were reduced. The results indicated that Maillard reaction maybe the strongest by roast treatment comparing to boiled and deep-fried treatment. Table 1 3

.2. Maillard reaction and ages
The measurement of fluorescence increase or decrease is an effective procedure to assess the extent of the Maillard reaction in foods (Delgadoandrade, Seiquer, Haro, Castellano, & Navarro, 2010). As shown in Figure 1, boiled, deep-fried and roast processing had different effects on A 294 and A 420 . The value of A 294 not fluctuated under the boiled treatment in 5-55 min, and the trend was relatively stable. However, the value of A 420 was increased in 5-25 min and decreased in 25-55 min. For deep-fried processing, the value of A 294 and A 420 both increased in 1-3 min and decreased in 3-5 min then went up at last 1 min. Meanwhile, for roast processing, the value of A 294 and A 420 was both increased with cooking time. All the results could be summarized that Maillard reaction may get inhibited by boiled processing, especially at the last stage. In addition, deep-fried and roast processing could both promote the Maillard reaction, but roast showed stronger promotion ability than deepfried. This result is similar to Table 1. As shown in Figure 2, boiled, deep-fried and roast processing also showed different effects on CML and CEL. For boiled processing, CML and CEL showed a similar trend. They all gradually declined during the first 35 min, and then the value gradually increased. The content of CML treated by boiled was 1136.17 ± 17.26 to 1451.33 ± 2.92 µg/kg meat, the content of CEL was 18.79 ± 0.17 to 25.94 ± 0.13 µg/kg meat. For deepfried processing, CML and CEL showed different trends with cooking time. CML increased slowly during 1-4 min and then fell, while CEL increased first and decreased during 1-4 min, then increased during 5-6 min. The content of CML treated by deep-fried was 1214.83 ± 12.50 to 1369.83 ± 3.32 µg/kg meat, the content of CEL was 17.77 ± 0.50 to 24.38 ± 0.29 µg/kg meat. For roast processing, CML continuously increased with cooking time, while CEL appeared two peaks in 10 min and 20 min. The content of CML treated by roast was 1014.67 ± 6.02 to 1309.17 ± 6.33 µg/kg meat, the content of CEL was 11.51 ± 0.18 to 20.54 ± 0.54 µg/kg meat. The results in Figure 2 indicated that boiled processing had a similar regularity on the CML and CEL formation, both deepfried and roast processing could promote CML increase. However, there was no obvious regularity for deep-fried and roast on CEL contents. In general, the content of CML was higher than that of CEL during heat treatment (Niu et al., 2017;Sun et al., 2016). Table 1. The value of a*, b*, L*, protein (%), fat (%) and moisture (%) in prepared chicken breast treat by boiled, deep-fried and roast, n = 3, mean ± SD. Tabla 1. Valor de a*, b*, L*, proteína (%), grasa (%) y humedad (%) en la pechuga de pollo preparada en forma hervida, frita y asada, n = 3, media ± desviación estándar.  F2, F3, F4, F5, F6 means cooking during 1 min, 2 min, 3 min, 4 min, 5 min, 6 min). K is for roast (K1, K2, K3, K4, K5, K6 means cooking during 5 min, 10 min, 15 min, 20 min, 25 min, 30 min). C is for raw control group. a-g Los datos dentro del mismo grupo (grupo B, grupo F, grupo K) con diferentes superíndices indican diferencias significativas (P < 0.05). B indica la forma hervida (B1, B2, B3, B4, B5, B6 significan cocinar durante 5 min, 15 min, 25 min, 35 min, 45 min, 55 min). F indica la forma frita (F1, F2, F3, F4, F5, F6 significan cocinar durante 1 min, 2 min, 3 min, 4 min, 5 min, 6 min). K indica la forma asada (K1, K2, K3, K4, K5, K6 significan cocinar durante 5 min, 10 min, 15 min, 20 min, 25 min, 30 min). C indica el grupo de control crudo.
Chen & Smith detected AGEs in cooked chicken, pork, beef and fish (salmon and tilapia) prepared by three common cooking methods: frying, baking and broiling (Chen & Smith, 2015). It was found that broiling and frying at higher cooking temperature (177°C-232°C) produced higher levels of CML, and broiled beef contained the highest CML content (21.8 µg/g) than fried chicken breast (17.16 µg/g). Baked fish (9.71-8.60 µg/g) contained less CML as compared to the beef, pork and chicken samples (12.53-14.31 µg/g). Trevisan et al. also compared the influence of home cooking conditions on AGEs in beef. It was found that boiling in water caused very low AGEs formation but baking was the most severe heat processing at 300°C with high CML (0.8 mg/100 g edible food) (Trevisan et al., 2016). Therefore, processing methods and processing temperatures showed a great influence on the formation of AGEs.

TBARs and carbonyl
The lipid and protein oxidation showed a complicated interaction in Maillard reaction (Delgado-Andrade, 2016). As shown in Figure 3, for boiled processing, TBARs value fluctuated within 5-45 min and then plummeted. Carbonyl value increased continuously in 5-55 min. The results indicated that the effect of boiled processing on lipid oxidation was complicated, and it mainly affected the occurrence of protein oxidation. It has been reported that the occurrence of fat oxidation is superior to protein oxidation and promotes protein oxidation further, while the free radicals produced by protein oxidation will promote fat oxidation in turn (Lorenzo et al., 2018). For deepfried processing, both TBARs and carbonyl value increased continuously with cooking time. The results indicated that the protein and fat oxidation were occurred at the same time and jointly affected the frying. For roast processing, TBARs value increased during 5-20 min and then decreased. There was a peak at 20 min. Carbonyl value increased continuously with cooking time. It was noticed that the highest peak of lipid oxidation corresponding to the peak of CML at 20 min, which indicated fat oxidation may have some correlation with the formation of CML. Meanwhile, protein oxidation could occur continuously with roast time. Corresponding to Figures 2 and 3, protein oxidation may also have some correlation with the CML formation in roast processing.

Sulfhydryl
Total and active sulfhydryl results are shown in Figure 4. For boiled, total and active sulfhydryl increased during 5-45 min, and decreased in the last 10 min. The results indicated that protein oxidation was inhibited during 5-45 min, but when the boiled time was too long (45-55 min), protein oxidation would be promoted. This mainly because the Maillard reaction was inhibited and the protein was in a high water environment so that it was not easy to be oxidized. But when the boiled time was too long, the protein was further degraded, which resulted in the accumulation of AGEs precursors, and promoted more AGEs formation. For deep-fried, total sulfhydryl decreased in the first 2 min. After that, it started to rise and then declined, and peaked at 4 min. Similarly, active sulfhydryl had two peaks at 2 and 4 min. But overall, the sulfhydryl content was decreased. This mainly because the effect of deep-fried on Maillard reaction and oxidation reaction was so complicated that they both affected the content of sulfhydryl. For roast processing, total and active sulfhydryl were both increased during 5-15 min and decreased from 15 to 30 min, and they were both peaked at 15 min. Similarly, one reason was that the concealing sulfhydryl was attacked by free radicals, active and concealing sulfhydryl oxidation reaction was not occurred simultaneously (Benjakul & Bauer, 2010). Another was the high temperature promotes Maillard reaction and oxidation to influence the sulfhydryl (Pischetsrieder, 2010).

Correlation
Oxidation and Maillard reaction occurred simultaneously and interacted with each other . In order to investigate the relationship on Maillard reaction, oxidation and AGEs formation in prepared chicken by the three thermal processing methods more clearly, we did a Pearson's correlation analysis. The correlation of Maillard reaction (A 294 , A 420 ) AGEs (CML, CEL), oxidation (TBARs, total sulfhydryl, active sulfhydryl, carbonyl), color (a* values, b* values, L* values), composition (protein, fat  and moisture) in prepared chicken breast treated by boiled, deep-fried and roast are shown in Figure 5. The results indicated that different heat processing methods were different in the correlation of prepared chicken breast meat indicators. Respectively, for boiled processing, L* values, b* values, protein and lipid oxidation were positively correlated with AGEs, P < 0.05. For deep-fried processing, protein content, L* values, b* values, protein and lipid oxidation were also positively correlated with AGEs, P < 0.05; However, there was a negative correlation between moisture and AGEs, P < 0.05. For roast processing, moisture and fat were both positively correlated with AGEs, P < 0.05; b* values and protein oxidation were negatively correlated with AGEs, P < 0.05. Thus, for boiled and deep-fried processing, protein oxidation and lipid oxidation were two key factors could promote the formation of AGEs. Maillard reaction affected by heat treatment mainly reflects in L* values reduction and b* values  increased which indirectly affected AGEs. For roast processing, the content of moisture and fat in meat would promote the AGEs formation. However, protein oxidation would inhibit the formation of AGEs, b* values were also an important visual indicator to evaluate how much AGEs contained in roast prepared chicken breast.

Conclusion
According to the above research and results analysis, three main conclusions were summarized. Firstly, prepared chicken breasts' basic components, color, Maillard reaction and oxidation degree were influenced a lot by different thermal treatments. Correlation analysis showed that protein and lipid  oxidation are two key factors could promote the AGEs formation by boiled and deep-fried processing, but protein oxidation could inhibit the AGEs formation by roast processing. Secondly, thermal processing could influence the Maillard reaction and showed important effects on L*, a* and b* values, so the content of AGEs could be easily determined by the changes of color. Thirdly, oxidation and Maillard reaction were both important factors on AGEs formation under different heat treatments. In particular, the oxidation reaction occurred during thermal processing played an important role to promote the Maillard reaction on the AGEs formation in our study. However, the interaction between oxidation and Maillard reaction on AGEs formation in meat products under different heat processing needs to be further investigated.