Mechanisms Of Hemoglobin-Mediated Lipid Oxidation In Muscle And Its Inhibition

Lipid oxidation is one of primary biochemical reaction during processing and storage in meat products.In most instances,lipid oxidation is a free radical chain reaction.The high reactivity of free radicals can cause body injury,cell damage,aging phenomenon.The peroxides produced from lipid oxidation in muscle food can interact with other food ingredients,which could lead to quality deterioration in flavor,nutritive value,sensory,texture and the production of toxic compounds.Owning to the importance of lipids in meat quality,nutrition and safety,it has been research hotspots in field of meat science during past decades.Transition metals,including iron and copper are thought to be the primary oxidation catalysts in field of food science.Especially,heme proteins have been regarded as most major catalysts for lipid oxidation in meat products.However,many steps related to lipid oxidation all occur in a very short time sequence(and simulaneously)so that it's challenge in understanding the pathway by which heme proteins promote lipid oxidation.It is worth noting that,research the mechanism of Hb or Mb meadiated lipid oxidation in meat and meat products is not only a theoretical breakthrough,is to provide basis for the regulation of lipid peroxidation of meat products.The purpose of this study is to clarify the mechanism of hemoglobin induced lipid oxidation in meat and meat products,explore two kinds of interventions for lipid oxidation,regulation of lipid oxidation,improve meat safety and flavor sensory quality and provide a theoretical basis and accurate experimental guide.The contents and results are as follows:1.Effect of free iron and heme-iron on Hb Induced lipid oxidationEstablishing a lipid oxidation model(WCM)by using high speed homogenate and"washed" techniques to remove pro-oxidation and inhibitor factors in muscle.The different concentrations of Hb and EDTA and free iron ions were added to WCM,the peroxide value(PV)and TBARS were tracked to analyze the role of free iron ions in Hb induced lipid oxidation,to investigate the effects of different iron status on WCM system and on the of lipid oxidation in WCM.The result showed that,during Hb induced lipid oxidation,the highest TBARS values were highly correlated with the amount of Hb added(R2 = 0.98).there is no significant effect on PV and TBARS values in EDTA treatment(P>0.05),and also this is no significant change in PV and TBARS values which content free Fe2+ or Fe3+ in WCM.All of this results indicated that the Hb is a reactant which takes part in the reaction and be consumed other than a stable catalyzer;heme-iron played a decisive role in the Hb-mediated lipid oxidation other than free iron.In addition,the affinity between the heme and Hb.globin can significantly affect the rate of lipid oxidation.2.The effect of autoxidation of Hb and hemin loss on Hb induced lipid oxidation in WCMThe differences of biochemical characteristics of Hb which comes from two different species:pork and turkey,like the autoxidation speed(the formation of MetHb),hemin loss speed were compared.Then the oxyHb and MetHb was added to WCM,the redness,Ferryl Hb content,PV and TBARS values were measured to analyze the relationship between biochemical characteristics of Hb and its capacity in inducing lipid oxidation.Protein crystallography was used to analyze the differences of spatial structure in turkey and porcine Hb.The results showed that,the autoxidation of turkey Hb was significantly higher(p<0.05)than pork Hb at the present of H202;the difference of hemin loss speed between turkey and pork mainly at the first 4 hours,the hemin loss in turkey MetHb was significantly higher(p<0.05)than pork MetHb;the structure differences between turkey and pork mainly lies in the hemin hydrophobic curve in turkey was larger than in pork,which lead to more liquids enter the hemin hydrophobic curve,reducing the affinity of Hb to Hemin.All of this results indicated that,the autoxidation of Hb and hemin loss were two crucial steps,which can significantly affect the speed and extent of lipid oxidation in WCM induced by Hb;structural analysis of protein crystals reveals the underlying reasons for the change in some chemical properties of Hb from the molecular spatial structure level,which can become an effective tool to explore the mechanism of lipid oxidation induced by Hb.3.The effect of pH and NaCl on the biochemical characteristics,molecular structure of Hb and lipid oxidation.The effect of pH(5.7,6.3 and 7.2)and the concentration of NaCl(1%,2%,3%)on the lipid oxidation in WCM was investigared.TBARS values,the affinity of Hb to oxygen,the autoxidation speed of OxyHb,the adsorption capacity between Hb and phospholipid membrane were measured during the lipid oxidation of WCM.The effect of pH on space structure of Hb was analyzed based on above results.These studies indicated that the affinity of Hb to oxygen was weakest at the pH 5.7,however,this was pretty strong when the pH was 7.2;the autoxidation speed of Hb was significantly decreased with the reduce of pH(p<0.05);it needed longest time for TBARS up to maximum at pH 7.2,but there is no significant difference between three treatments(p>0.05).The speed of autoxidation of Hb was significantly increased with the rise of the concentration of NaCl(0%--3.0%);the adsorption capacity between Hb and phospholipid membrane was significantly decreased with the rise of NaCl content(p<0.05),the lowest value of adsorption of Hb on phospholipid was 37.5%,when 3.0%NaCl was added;when the content of NaCl was 1.0%and 2.0%,the starting time of lipid oxidation in WCM was shorted effectively,and also the extent of lipid oxidation was increased.When the content of NaCl was 3.0%,however,the extent of lipid oxidation was inhibited despite of the starting efficiency of lipid oxidation was increased effectively.The results indicated that decreased pH can speed up lipid oxidation in WCM mainly lies in its effect on the speed of autoxidation of Hb,the affinity of Hb to oxygen and hemin loss speed.NaCl showed both peroxidation and inhibition during Hb induced lipid oxidation,which mainly related to the multiple effects of NaCl on Hb.4.Quercetin intervenes lipid oxidation mediated by Hb.The antioxidant capacity of quercetin and its interact capacity with phospholipid membrane was studied,the TBARS values,hydroxyl radical scavenging activity,the transformation of Hb between oxy-and met-as well as interaction extent with cell membrane.The quercetin bind with Hb through covalent bond was studied using ESI-MS based on above results.All of these showed that the highest TBARS values in treatment without quercetin as 4.2 times as the treatment which was added quercetin;the capacity of eliminating free radical up to more than 80%when the concentration of quercetin was 40?g/mL;only 24.7%MetHb was remained after 2 days in the treatment which was added quercetin,however,there was 83.4%MetHb in other treatment without quercetin.ESI-MS results showed that there is 1 molecules of quercetin was adducted on each Hb.The content of quercetin was 102.4?g/10g muscle in the supernatant when the concentration of added quercetin in WCM was 120 ?g/10g muscle.The results indicated that quercetin can inhibit lipid oxidation by reducing MetHb into OxyHb;MetHb can adduct with 1 molecules of quercetin to form adduction,which can decrease its pro-oxidative capacity;eliminating free-radicals as well as interact with phospholipid membrane.5.The effect of PLA2 on Hb-mediated lipid oxidation and the effect of FFA on lipid oxidation in miced turkey meat.We analyzed lipid oxidation using WCM and turkey minced meat,treated with PLA2 and FFA.The TBARS values,free fatty acid production and the change of characteristic spectrum of Hb after interact with FFA as well as TBARS in minced turkey meat was measured in the whole procedure which treated with PLA2.These studies indicated that,lipid hydroperoxide was no significant increase in the whole procedure when the concentration of PLA2 was lOppm in WCM,but also keep a low level(<18.3 LOOH/kg muscle).TBARS values no apparently increase during the 4 days procedure,stay at 2.1-5.4?mol TBARS/kg muscle;the content of free fatty acid(FFA)was kept increasing in the three days procedure,and also up to maximum(7.8 mmol FFA/kg muscle)in the third day.The TBARS values were significantly inhibited when the FFA was added at the concentration is 0.2g/kg muscle.After FFA interact with Hb,we found that the characteristic spectrum of Hb was highly similar with hemichrome.Results showed that,i)PLA2 can inhibit the lipid oxidation induced by Hb effectively in washed muscle;ii)FFA can inhibit lipid oxidation in minced turkey meat;iii)the mechanism of PLA2 and FFA inhibit lipid oxidation may be involved in FFA binds to heme proteins to form a low pre-oxidant named hemichrome.