Formation of advanced glycation end products in raw and subsequently boiled broiler muscle: biological variation and effects of postmortem ageing and storage

Suhong Hung, Xioli Dong, Yli Zhng, Yuru Chen, Yjie Yu,Ming Hung,b,*, Yundong Zheng

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

b Nanjing Huangjiaoshou Food Science Technology Co., Ltd., National R&D Center for Poultry Processing Technology, Nanjing, Jiangsu 210095, China

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

Keywords:

Ca rboxymethyllysine

Broiler

Postmortem ageing

Storage

Heating

A B S T R A C T

The study aimed to investigate and compare the contents of carboxymethyllysine (CML) in two kinds of broilers during postmortem ageing and storage. The contents of CML in raw and boiled (100 °C, 30 min)broiler briskets and legs which were from w hite feather broilers (n = 8) and yellow feather broilers (n = 8)with ageing and storage at 4 °C for 0–168 h were determined. Postmortem ageing and storage had a significant(P ＜ 0.05) effect on the color and A420 nm in both boiled broilers meat. In addition, with the ageing and storage time increasing, CML content in raw white feather broiler brisket meat had no significant (P ＞ 0.05) change,while that in boiled brisket meat significantly (P ＜ 0.05) increased during 0–6 h, then decreased during 6–24 h,finally increased again. CML content in leg meat increased significantly (P ＜ 0.05) with the ageing and storage time prolonging. But postmortem ageing and storage had no significant (P ＞ 0.05) effect on the CML contents in raw/boiled yellow feather broilers. Meanwhile, CML contents in white feather broilers were much higher than that in yellow feather broilers. Thus, white feather broilers can be selected as the re search object to st udy the mechanism of ageing and storage on CML content in th e postmortem broiler in the future.

1. Introduction

Advanced glycation end products (AGEs) are sorts of complex compounds produced by Maillard reaction between the carbonyls of reducing sugars and the free amino groups of amino acids.Furthermore, AGEs can also be generated through other reaction pathways, such as oxidation pathways of reducing sugars, fats and amino acids [1-3]. There are more than 20 AGEs compounds that have been successfully identified, including carboxymethyllysine (CML),carboxylethyllysine (CEL), pentagglutinin, glycosylated collagen and pyrrolidine, etc [4,5]. AGEs can trigger oxidative stress and inflammatory responsein vivoto cause various chronic diseases, such as cardiovascular diseases [6,7], atherosclerosis [8], diabetes [9,10],chronic kidney disease, Alzheimer’s disease [11], as well as many others [12]. Although endogenous AGEs generation can be took place, dietary AGEs (dAGEs) are the main portion of AGEs in the body [13]. Therefore, AGEs have attracted extensive interests not only in medical science but also in food science.

Meat, as a kind of food with high protein and/or high fat, generally contains relatively high levels of AGEs [14], and thermal treatment accelerates the formation of AGEs in meat [15-17]. In addition,storage also affects the AGEs contents of meat, because the oxidation and Maillard reaction occur continuously during storage [18,19].Reports on the formation of AGEs during storage are quite limited and have focused on milk powder [20,21]. There are strikingly few studies in the changes of AGEs contents in meats during storage.Typically, Yu et al. [22]found the longer storage time was, the more AGEs were generated in the meat after heat treatment. Niu et al. [18]found that the longer ice storage time was, the more bound CML was generated after the heat processing offish. What’s more, there were biological variation while studying the effect of the freshness offish on the production of AGEs during heat treatment.

Ageing of muscle is a normal procedure [23]. The stages of protein hydrolysis during ageing are related to increases in tenderness and improvement in the taste and aroma of the meat. It is well known that the ageing periods of meat from different animal sources are inconsistent [24]. For example, it takes about 4, 8, 9, 10 days and 7 h respectively for pork, lamb, rabbit, beef and broiler to reach the optimum tenderness after rigor mortis [25]. In addition, muscle foods such as poultry and animal meat are perishable due to bacterial activities. Thus, the raw muscles are generally aged and stored at 0–4 °C to make the meat taste good and slow down rottenness.However, chemical reactions, such as oxidation, still occur in muscle during postmortem ageing and storage. The endogenous antioxidant defenses towards the generation of reactive oxygen species and other reactive compounds lose their original balance, which facilitates oxidative stress in postmortem muscle. Oxidative stress in meat systems leads to oxidative damage to basic muscle components such as proteins and lipids. It is probable to facilitate the formation of some harmful substances in raw and subsequently cooked muscle,such as heterocyclic amines (HA) and AGEs [26]. Polak et al.[23,24]investigated the effects of ageing and the low internal temperature of grilling on the formation of HA in beef and found the total HA content increased with ageing time. However, there is no survey about the content of AGEs in raw meat as affected by postmortem ageing and storage. What’s more, there is no reported study on the formation of AGEs during heating as affected by postmortem ageing and storage. But it is significant to understand the changes of AGEs contents in muscle food as affected by postmortem ageing and storage as well as the formation of AGEs during heating after postmortem ageing and storage, to ultimately reduce the levels of AGEs in muscle foods.

Thus, the goal of this work was to determine and compare the overall amounts of one commonly occurring AGEs calledNɛ-carboxymethyllysine (CML) formed in white and yellow feather broilers during postmortem ageing and storage (0–168 h at 4 °C). In addition, the effects of postmortem ageing and storage time on the formation of CML in boiled broilers were investigated.

2. Materials and methods

2.1 Reagents

96 T broiler CML double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) kit was purchased from Maibo Reagent Co., Ltd (Nanjing, Jiangsu, China). Boric acid, sodium hydroxide, hydrochloric acid, methyl red, bromocresol green, ethanol,blue vitriol and potassium sulfate were analytical purity.

2.2 Sample information

A total of 8 live, male, 45-day-old white feather broilers and 8 live, male, 90-day-old yellow feather broilers were obtained from the Wens Co., Ltd (Huaian, Jiangsu, China). The average weights were about 2.3 kg, and then they were withdrawn feed at about 24 h and water at 4 h before slaughter [27].

Broilers were slaughtered, briskets and legs were rapidly removed.The samples from each brisket and leg were immediately (0 h) snapfrozen in liquid nitrogen (＜ 5 min) and stored at –80 °C until analysis.The remainder of each brisket and leg were stored at 4 °C for 6, 12,24, 72 and 168 h. At the end of each storage period, the meat samples were taken individually and stored at –80 °C until analysis. All procedures were approved by the Animal Care and Use Committee of the College of Food Science and Technology of Nanjing Agricultural University [28,29].

2.3 Muscle pH measurement

The pH of postmortem muscle at 0, 6, 12, 24, 72 and 168 h was determined by a glass electronic pH meter (Testo 205, Testo AG.Company, Germany). Each muscle sample was measured by inserting the glass probe electrode in triplicate, the average value was the result. The pH meter was calibrated by a three-point method against standards buffer of pH 4.01, 7.00 and 10.00 [30].

2.4 Proximate composition

The water, protein and fat contents of brisket and leg from each broiler were determined based on AOAC methods (AOAC 2005): an oven drying method (AOAC 950.46) for water, a Kjeldahl method(AOAC 928.08) for protein, and a solvent extraction method (AOAC 991.36) for fat determination [31].

2.5 Heat treatment

Broiler meat ((6.0 ± 1.0) g) was sealed into tinfoil, heated in boiling water (100 °C) for 30 min, and immersed into the ice/water mixture to cool down immediately [31].

建立定位数学模型,利用该模型对未知节点进行初步定位估计,计算未知节点的坐标,通过距离差判别法获取未知节点坐标。

2.6 Color evaluation

The surface color was measured rapidly after sample cooled down, using a CR-400 colorimeter (Minolta, Osaka, Japan) with illuminant D65, 10 observer, 11 mm aperture for illumination and 8 mm for measurement [32]. The chromaticity coordinates recorded wereL* (black-white, lightness),a* (green to red, redness), andb*(blue to yellow, yellowness). Before measurement, the colorimeter was calibrated by a standard whiteboard.

2.7 Analysis of A420 nm

A420nmwas referred to Zhao et al. [33]with minor modifications.1 g of minced sample was put into a 50 mL centrifuge tube,homogenized with 9 mL phosphate buffer solution (PB, pH 7.2) at 10 000 r/min for 1 min (2 × 30 s with a 10 s interval) in ice bath.Then, the mixture was centrifuged at 10 000 r/min for 10 min at 4 °C.The absorbance of supernate was measured at 420 nm.

2.8 Analysis of CML

The amounts of CML in brisket and leg meat samples were determined according to the method of Zhu et al. [34]and Gómez-Ojeda et al. [35]. 1 g of meat was put into a 50 mL centrifuge tube,homogenized with 9 mL phosphate buffer solution (PB, pH 7.2) at 10 000 r/min for 1 min (2 × 30 s with a 10 s interval) in ice bath.Then, the mixture was centrifuged at 10 000 r/min for 10 min at 4 °C. The supernate was measured by CML ELISA kit (CML linearity range: 10–320 ng/mL,y= 0.004 5x+ 0.001 3,R2= 0.998 9).

2.9 Statistical analysis

Data were evaluated by SAS analysis software (SAS software research institute, USA, version 8), One-way ANOVA method was used for the analysis of variance. Duncan’s multiple range test was used to compare the differences between mean value (P＜ 0.05). The results were expressed as means ± SD.

3. Results and discussion

3.1 Water, fat and protein contents of broiler muscle

Table 1 showed major constituents in white and yellow feather broilers. The contents of water and fat in white feather broiler brisket meat were less than those in leg meat, but the protein in brisket meat was much higher than it in leg meat. Meanwhile, the water contents of yellow feather broiler brisket and leg were similar, but the fat content of the leg was 5.5 times higher than that of the brisket and the protein content of brisket was 1.16 times higher than that of the leg.In addition, the water and fat contents of the white feather broiler and yellow feather broiler were similar, while the protein content of the white feather broiler was higher than it in the yellow feather broiler.

Table 1Major constituents in white feather broilers and yellow feather broilers.

3.2 The pH of broilers after slaughter

The changes of pH in brisket meat and leg meat of white and yellow feather broilers during postmortem ageing and storage were shown in Fig. 1. After slaughter, the initial pH of white feather broiler brisket meat and leg meat were 6.11 and 6.62, respectively, and the initial pH of yellow broiler brisket meat and leg meat were 6.13 and 6.47, respectively. The pH of brisket meat and leg meat in two kinds of broilers decreased rapidly within 0–6 h, and then the changes of pH were not significant (P＞ 0.05). The results showed that the two kinds of broilers meat reached the limit pH value at 6 h after slaughter,which manifested rigor mortis was complete, and then entered the ageing stage. The results were similar to the literature reported by Lee et al. [36]. As can be seen from the Fig. 1, the pH of white feather broiler was higher than that of yellow feather broiler, and the pH of leg meat was higher than that of brisket meat. A possible explanation may be that the content of muscle glycogen in yellow feather broiler was higher than that in white feather broiler, and the content of muscle glycogen in brisket meat is higher than that in leg meat [37,38].Furthermore, it might be also related to the breed, feed, growing environment and transportation of broilers [37,38].

Fig. 1 Variation of pH values during postmortem ageing and storage of broiler brisket and leg (n = 8).

3.3 Effects of postmortem ageing and storage time on the color of boiled broiler meat

Hunter color parameters (L*,a*,b*) are used to describe the quality spoilage of food substrates [39]and indicate the extent of MR [40].The color of broiler has a great relationship with myoglobin content and water content [41]. As displayed in Table 2, postmortem ageing and storage duration had a significant effect on theL*,a*,b* values of boiled white and yellow feather broiler meat. With the postmortem ageing and storage time extending, theL* value of white feather broiler meat decreased significantly (P＜ 0.05) during 0–6 h, then increased significantly (P＜ 0.05) at 6–12 h, and finally showed no significant trend. However, the variation trend ofL* value in yellow feathered broiler was different, which decreased during 0–12 h,and increased thereafter. The initial decline in theL* value for both types of broiler might be due to the muscles that in the stage of rigor mortis tend to lose more water during cooking, resulting in a lowerL* value. TheL* value was positively correlated with the moisture content of meat [42]. In addition, theL* value of brisket meat was significantly (P＜ 0.05) higher than that of leg meat. It might be ascribed to the higher fat content in the leg meat, which is easy to oxidize and reduce itsL* value [43]. Thea* value of white feather broiler brisket increased significantly during 0–12 h, then decreased during 12–72 h, and finally increased markedly, while thea* of leg meat increased significantly (P＜ 0.05). Different from white feather broiler, with the postmortem ageing and storage time extending,thea* value of yellow feather broiler brisket increased significantly(P＜ 0.05), while the leg meat increased firstly, then decreased,and finally increased again. Additionally,a* value of white feather broiler was higher (redder) than yellow feather broiler, and leg meat was higher than brisket meat. This was related to the content of myohemoglobin and the degree of MR in boiled meat. The highera*value is, the more Maillard reaction products are produced [40,44].Except for yellow feather broiler brisket meat, theb* value of the rest changed significantly with the time of postmortem ageing and storage increasing, which may be related to the production system of broilers [45].

Table 2The color of boiled white and yellow feather broiler meat previously postmortem ageing and storage at 4 °C for up to 168 h (n = 8).

3.4 Effects of postmortem ageing and storage time on the A420 nm of boiled broiler meat

The absorbance at 420 nm is always used to indicate the amounts of Maillard reaction late products that include CML [46]. Fig. 2 showed theA420nmvalues of boiled broiler meat after postmortem ageing and storage. Depending on the aged and storage time,A420nmvalue in two kinds of boiled brisket increased during 0–6 h, then decreased during 6–24 h, and finally leveled off, whileA420nmvalues in two kinds of boiled leg increased significantly (P＜ 0.05). Besides,A420nmvalue of white feather broiler meat was higher than that of yellow feather broiler meat,A420nmvalue of broiler leg was higher than that of broiler brisket. These results indicated that more Maillard reaction late products in white feather broiler meat during cooking were generated than in yellow feather broiler meat, and more those in leg meat were generated than in brisket [34], which was consistent with oura* results.

Fig. 2 A420 nm value of boiled white and yellow feather broiler meat after postmortem ageing and storage (n = 8).

3.5 Effects of postmortem ageing and storage time on the contents of CML in raw/boiled broilers

Table 3 showed the contents of CML (data were reported as mg/kg meat) in raw/boiled white feather broiler brisket and leg previously postmortem ageing and storage at 4 °C for up to 168 h. Similar to the results of the study reported by Niu et al. [31], there was a big biological variation among individual broilers. Therefore, to minimize the adverse influence of individual variation, samples from the parts with a minimized sample variance of the same broiler should be aged and stored for a different time [47]. With the extension of ageing and storage time, the content of CML in raw white feather broiler brisket meat had no significant change, while the content of CML in boiled brisket meat significantly increased from 1.75 mg/kg (0 h)to 1.90 mg/kg (6 h) at first, then decreased to 1.76 mg/kg (24 h),and finally increased again (P＜ 0.05). To begin with, there were plentiful postmortem biological reactions in raw broiler because of the presence of enzymes, which probably facilitated the formation of CML. Then a drop in CML after 6 h might due to a protein crosslinking reaction, producing a cross-linking substance similar to CML-lysine [48,49]. Furthermore, the content of CML in leg meat increased significantly with the ageing and storage time prolonging(P＜ 0.05), which might be due to the oxidation reaction. The degree of oxidation was positively correlated with the formation of CML in meat products [19]. In addition, the average content of CML in broiler meat increased about 2 folds after 30 min of heating at 100 °C, which was similar to the studies’ results that thermal treatment gave rise to a significant increase in muscle’s CML [31].

Table 3The amounts of CML in raw and boiled white feather broiler (n = 8) muscle previously postmortem ageing and storage at 4 °C for up to 168 h.

The content of CML in raw broiler brisket meat had no significant difference at each postmortem ageing and storage period, but there were significant differences in boiled broiler brisket meat.This phenomenon could be explained that during postmortem ageing and storage period, biochemical reactions (lipid oxidation,protein oxidation and protein denaturation) in broiler brisket continuously occur due to the functions of endogenous enzymes and microorganisms [50,51]. These reactions might result in exposing some reactive amino acid groups, such as lysine residues,and accumulating some active dicarbonyl compounds like glyoxal(precursor for CML) [1,52]. It might have a mild effect on the contents of CML in raw broiler brisket during postmortem ageing and storage but would greatly accelerate the formation of CML in broiler brisket meat upon heating.

Table 4 showed the contents of CML (data were reported as mg/kg meat) in raw/boiled yellow feather broiler brisket and leg previously postmortem ageing and storage at 4 °C for up to 168 h.With the extension of ageing and storage time, the contents of CML in raw/boiled brisket meat and leg meat had no significant change,which was different from the white feather broiler. While the boiled yellow feather broiler meat contained more CML than raw broiler meat, which was analogous to the white feather broiler.

Table 4The amounts of CML in raw and boiled yellow feather broiler (n = 8) muscle previously postmortem ageing and storage at 4 °C for up to 168 h.

The content of CML in white feather broiler changed significantly during postmortem ageing and storage (P＜ 0.05), while that in yellow feather broiler did not (P＞ 0.05). Meanwhile, the contents of CML in the brisket meat and leg meat of white feather broiler were much higher than those of yellow feather broiler. Raw white feather broiler brisket meat and leg meat contain about 3–4 times as much CML as that in yellow feather broiler. Boiled white feather broiler brisket meat and leg meat contain about twice as much CML as yellow feather broiler, which was consistent with oura* value andA420nmresults. The difference might be related to the basic composition and pH of broiler [53]. A relatively higher pH value is favorable for the MR and increases the formation of AGEs (maximum pH of 10) as protein’s amino groups are in basic form and sugars in the reducing or open-chain form under the condition, thus, increasing their reactivity [54,55]. The pH values of the brisket meat and leg meat in the white feather broilers were higher than in the yellow feather broilers, and the pH values in the two kinds of broilers legs meat were higher than in brisket meat, so it was more conducive to the Maillard reaction to produce CML.

4. Conclusion

There is a large biological variation in CML contents among individual broiler of the same and different species. The color andA420nmvalue in both boiled broilers meat had a significant change with the postmortem ageing and storage time increasing. In addition,postmortem ageing and storage had a significant effect on CML contents in white feather broilers, but had no significant effect on the CML contents in yellow feather broilers. Meanwhile, the contents of CML in white feather broilers were much higher than that in yellow feather broilers. Thus, these findings indicate that the use of nonaged and no storage chicken for cooking is a potentially practical way of reducing human exposure to CML which is produced during the thermal process. Furthermore, white feather broiler can be selected as the research object if the mechanism of ageing and storage on CML in broiler would be studied in the future.

Conflicts of Interest

The authors declared they have no conflicts of interest.

Acknowledgments

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