Changes In The Levels Of Advanced Glycation End-products In Muscle Food During Heat Treatments And Storage

Advanced glycation end-products(AGEs)are a group of compounds generated from non-enzymatic reactions between carbonyl groups of reducing sugars or sugar derivatives with free amino groups of protein and amino acids.Typical AGEs in foods include N?-carboxymethyl-lysine(CML),N?-carboxyethyl-lysine(CEL),pyrraline,and others,which present in foods as either free form(glycated amino acids)or protein-bound form(protein glycation adducts).Detary AGEs have been linked to aging and increased oxidative stress,as well as increased risks for kidney diseases,diabetes,and obesity.Although some reported studies show that the tormation of AGEs in foods is influenced by food ingredients,processing methods,and storage conditions,there is still a lack of systematic studies on the levels of AGEs in muscle foods as affected by various factors.The objectives of this study are to use freshwater fish including grass carp and catfish as representive muscle food systems to(1)understand the effect of biological variance on the levels of AGEs in the muscle of individual animals from the same species;(2)investigate the effect of freshness on the AGEs levels in raw and heated muscle foods.Grass carp and catfish were chosen as a model system for muscle food animals since these were widely cultivated fishes that could be obtained alive,providing the opportunity to closely control the storage conditions of a large number of individual fish upon slaughter in a laboratory setting.We chose these cultivated fishes also because they are big enough to provide adequate samples for conducting the storage and heat treatment experiments.The other objectives of this study were to(3)use pork to verify the relevant conclusions from the study results of freshwater fish,and to further investigate the influences of commonly used meat additives(including NaCl and NaNO2)on AGEs contents in pork during cold storage and heating;and(4)investigate the mechanism of AGEs formation in muscle foods as affected by NaCl and NaNO2 during storage and heating by using a meat-like model system(myofibrillar protein-reducing sugar-fatty acid)and a solution model system(lysine-reducing sugar-fatty acid).The main methods and results were as follows:1.Live and immediately killed grass carp and catfish were used in the study.An HPLC-MS/MS method was used to analyze free and protein-bound CML and CEL in raw and heat treated(100 °C,5,10,30 min)white muscle above the lateral line of fish.The recovery results of AGEs in grass carp and catfish muscle were: free CML 100.6–121.9%,CEL 103.4–121.3%,and protein-bound CML 80.6–101.1%,CEL79.3–105.3%.The average amounts of free CML and CEL in grass carp(n = 15)muscle were 1.3 ± 1.2 and 0.2 ± 0.1 mg/kg(based on sample weight),respectively,and that of protein-bound CML and CEL were 1.3 ± 0.5 and 2.8 ± 1.0 mg/kg,respectively.AGEs content in the muscle of individual grass carp varied greatly,especially for free CML.The relative standard deviation(RSD)of free CML in different grass carp was as high as 89%.Free CML and CEL in catfish(n = 21)muscle were 0.11 ± 0.09 and 6.5 ± 7.2 mg/kg,respectively and protein-bound CML,CEL were 0.7 ± 0.3 and 3.1 ± 2.7 mg/kg,respectively.The RSD of AGEs contents were greater than 80% except for protein-bound CML,indicating a large variance of AGEs contents among individual catfish,especially for free CEL(RSD = 111%).A longer heating time led to higher levels of protein-bound CML and CEL formation.After 30 min of heat treatment,the protein-bound CML in grass carp muscle increased by 2.4–10.8 folds and protein-bound CEL increased by 27–242%;the protein-bound CML and CEL in catfish muscle increased by 2.1–8.5 folds and 28–224%,respectively.However,heating has little or no effect on the levels of free CML and CEL in grass carp and catfishfish muscle.2.Freshly killed grass carp and catfish were buried in ice and stored for 0–21 days.The free and protein-bound CML,CEL contents in raw and heat treated(100 °C,5,30 min)fish muscle during different storage time were investigated.The levels of free and protein-bound AGEs in raw grass carp and catfish during 3 weeks of ice storage didn't change significantly.But with longer storage time,the formation of protein-bound AGEs of fish muscle due to heat treatment became larger.The average amounts of AGEs formed in the muscle of 12 catfish during 5 min of heat treatment(100 °C)were as following(in mg/kg protein): CML,31.0 ± 10.7(0 day),39.0 ± 16.3(10 days),64.4 ± 33.6(21 days);CEL,2.9 ± 5.9(0 day),5.9 ± 6.8(10 days),16.1 ± 10.6(21 days).The average amounts of AGEs formed after 30 min of heat treatment were(in mg/kg protein): CML,54.0 ± 13.7(0 day),91.8 ± 43.3(10 days),154.0 ± 79.2(21 days);CEL,17.2 ± 8.2(0 day),27.3 ± 7.6(10 days),50.0 ± 20.3(21 days).As compared with fresh samples,after 3 weeks ice storage,the formation of protein-bound CML in catfish muscle heated at 100 °C for 5 min increased from 214% to 318%,and in catfish muscle heated for 30 min increased from 302% to 614%;formation of protein-bound CEL in catfish muscle increased from 123% to 162%(5 min),and from 184% to 292%(30 min).Similarly,the average amounts of AGEs in grass carp muscle formed after 5 min of heat treatment were(in mg/kg protein): CML,5.6 ± 2.3(0 week),8.4 ± 3.5(1 week),10.3 ± 2.9(2 weeks),13.6 ± 3.3(3 weeks);CEL,3.9 ± 6.6(0 week),5.2 ± 6.3(1 week),5.0 ± 3.8(2 weeks),9.1 ± 3.6(3 weeks);after 30 min of heating were: CML,21.3 ± 5.3(0 week),27.5 ± 5.7(1 week),35.6 ± 10.4(2 weeks),49.3 ± 8.9(3 weeks);CEL,18.6 ± 6.1(0 week),19.3 ± 9.2(1 week),26.7 ± 6.2(2 weeks),36.3 ± 8.7(3 weeks).After storage for 3 weeks,the increase of heat induced formation of AGEs in grass carp were: CML 167–233%(5 min),215–580%(30 min);CEL 135–163%(5 min)and 237–348%(30 min),respectively.The longer storage time led to more formation of AGEs in muscle food during heat treatment.3.NaCl(0?5%),NaNO2(0?150 mg/kg meat)and conjunction use of NaCl-NaNO2(2.5%-50/150 mg/kg meat)were added to fresh pork tenderloin.The ground samples were stored in ice for 8 days and then heated at commercial sterilization conditions(121 °C,10 min).Lpid oxidation and color changes of pork due to the addition of salts during storage time were measured.Protein-bound N?-(carboxymethyl)lysine(CML)and N?-(carboxyethyl)lysine(CEL)in pork samples were analyzed with HPLC-MS/MS.TBARS of pork samples increased during cold storage time and became larger with the addition of NaCl.On the other hand,the addition of NaNO2 reduced lipid oxidation of pork samples,but didn't influence TBARS values during the storage time.Protein-bound CML and CEL contents in raw pork were not affected by NaCl or NaNO2 during 8-day ice storage.Levels of CML and CEL in fresh samples(with no salt addition)after heated increased by 198% and 246 %,respectively,and continued to increase during storage time.NaCl accelerated AGEs formation rate when stored samples were heated,while NaNO2 had the opposite effect.With addition of 5% NaCl,heat induced formation of CML increased by 278% and CEL increased by 293%.However,the addition of 150 mg/kg NaNO2 reduced the formation of CML in fresh pork by 53%,and CEL 31%.The conjunction use of NaCl-NaNO2 reduced AGEs concentrations to the similar level with control samples,and far smaller than pork added with 2.5% NaCl.4.Myofibrillar proteins(MFP)were extracted from pork tenderloin.Meat-like model systems were formed by mixing proteins with glucose(G),ribose(R)or oleic acid(OA).Solution model systems were formed by mixing lysine(L)with glucose,ribose or oleic acid.The changes of AGEs contents in model systems heated at 121 °C for 10 min with different salts addition(NaCl 1%,2%;NaNO2 50,150 mg/L)were analyzed.The results showed that oleic acid increased the formation of CML and CEL in protein model systems by 72.5% and 11.5%,respectively,but decreased their formation in lysine model systems.In both of these model systems,ribose had much more reactivity than glucose or oleic acid.CML contents in ribose systems were 9 times higher than in glucose systems and 50 times higher than in oleic acid systems.CEL contents were 3–5 times higher in glucose systems and 20 times higher than in oleic acid systems.The addition of NaCl didn't influence CML and CEL levels in MFP,MFP/G,and MFP/R systems.CML and CEL contents in MFP/OA,MFP/G/OA and MFP/R/OA systems decreased by 20–60% with addition of NaCl,but the increase of NaCl concentration didn't further inhibit the formation of CML or CEL.In the lysine systems with oleic acid,the addition of NaCl reduced CML levels by 10–30%,while didn't influence the levels of CEL in the systems.NaNO2 improved the formation of CML and CEL in all the protein systems by 20–40%,except for MFP/R and MFP/R/OA,but didn't change the levels of CML and CEL in lysine systems.This study investigated the levels of free and protein-bound AGEs(CML and CEL)in muscle food as influenced by different storage conditions,different heat treatments and different salts with HPLC-MS/MS methods.Although the mechanisms of the above factors on AGEs formation in complicated food systems were analyzed based on the results of model systems,the results showed that simple model systems could not fully represent food systems and further work is needed to improve the study.Studying the influence mechanisms of these factors to AGEs formation in food systems could help us inhibit the formation of AGEs in food and improve food safety.