The Mechanism Of Low-dose Sodium Nitrite Inhibiting The Oxidation Of Yak Meat Myoglobin

Nitrite is a commonly used additive in meat products,and currently its maximum limit in meat products is around 150 mg/kg at the domestic and international.Although it is recognized that there is a carcinogenic risk of excessive use of nitrite,it is difficult to replace its role in meat products at safe doses.At present,studies on the role of nitrite in meat products are mainly focused on coloring and preserving,but few studies on antioxidant have been reported,especially the inhibition of myoglobin(Mb)oxidation at low doses have not been reported.Besides,the differences in myoglobin content,amino acid composition and structure between yak meat(red meat)and chicken meat(white meat)can lead to differences in the stability of myoglobin.And the differences have already existed in the application and presentation of nitrite between yak meat and chicken meat.In this study,we set up sodium nitrite treatment and non-sodium nitrite treatment to investigate the mechanism of inhibition of myoglobin oxidation in yak meat with high myoglobin and chicken meat with low myoglobin by low dose of sodium nitrite(<60 mg/kg)during the curing process.Through endogenous antioxidant capacity,myoglobin structure,intermolecular forces and mitochondrial electron respiratory chain pathway,we explored the mechanism of myoglobin stabilization,analyzed the key role of low dose sodium nitrite on myoglobin and mitochondrial stabilization in in situ and in vitro models,elucidated the mechanism of action of low dose sodium nitrite to inhibit myoglobin oxidation in yak meat,clarified the positive effect of low dose sodium nitrite in meat products,and then provide theoretical basis for proper application and control of nitrite in meat enterprises.The main research results are as follows.1.Low dose of sodium nitrite inhibited the oxidation of myoglobin in yak meat significantly and promote myoglobin oxidation in chicken meat.Compared to the non-sodium nitrite group,sodium nitrite increased the redness values and nitrosomyoglobin(NOMb)content in yak and chicken meat(P<0.01)and decreased oxymyoglobin(OMb)(P<0.05),with metmyoglobin(MMb)first increasing(P<0.05)and then stabilizing;turbidity,dimeric tyrosine and surface hydrophobicity of yak meat myoglobin decreased(P<0.05),indicating the aggregation of yak meat myoglobin was inhibited,and chicken meat was the exact opposite of yak meat;carbonyl content of myoglobin was reduced in yak meat and elevated in chicken meat(P<0.05),showing that sodium nitrite inhibited myoglobin oxidation in yak meat and promoted myoglobin oxidation in chicken meat.When compared to chicken meat,the degree of myoglobin oxidation and aggregation was lower in yak meat(P<0.05),indicating that low dose of sodium nitrite inhibited myoglobin oxidation in yak meat more significantly than in chicken meat.This is related to the different myoglobin content of yak and chicken meat and the sites of sodium nitrite action.2.Low dose of sodium nitrite improved the endogenous antioxidant capacity and reduced the production of reactive oxygen species of yak meat,similar change trends were found in chicken meat.Compared to non-sodium nitrite group,low dose of sodium nitrite decreased superoxide dismutase(SOD)activity(P<0.01)and led a progressive production of superoxide anion(O₂^-)in both yak and chicken meat;increased activity of catalase(CAT)and decreased hydrogen peroxide(H₂O₂)content in meat(P<0.01);improved activity of nitric oxide synthase(NOS)and increased NO content(P<0.01).Compared to chicken meat,the high SOD,CAT,GSH-Px activities and muscle hydroxyl radical inhibition in yak meat indicated that low doses of sodium nitrite improved the endogenous antioxidant capacity of yak meat significantly more than chicken meat and helped to inhibit myoglobin oxidation in yak meat.3.Low dose of sodium nitrite promoted myoglobin nitrosylation and maintained the structural stability of myoglobin in yak meat,however,it was not conducive to maintaining myoglobin stability in chicken meat.The?-helix and irregular curl content gradually decreased,?-fold and?-turned content gradually increased in the yak meat sodium nitrite treated group,which were not significantly different from those in the non-sodium nitrite group;the?-helix,?-fold and irregular curl content first increased and then decreased,?-turned content first decreased and then increased in the chicken meat sodium nitrite treated group,which were significantly different from those in the non-sodium nitrite group(P<0.05);above results indicating that sodium nitrite maintained the stability of yak meat myoglobin diastasis and promoted the dynamic transformation of chicken meat myoglobin diastasis.Low dose of sodium nitrite reduced the size of yak and chicken meat myoglobin haemoglobin centres,resulting in a more compact structure.Yak and chicken meat showed an increase in nitroso-myoglobin content(P<0.05);34 nitrosated peptides and 19 unstable peptides were detected in yak meat;39 nitrosated peptides and 33 unstable peptides were detected in chicken meat,suggesting that exogenous sodium nitrite led to the interconversion of unstable nitrosylated peptides.This is related to the difference in amino acid sequence and sodium nitrite action site between yak meat and chicken myoglobin.4.Low dose of sodium nitrite improved the efficiency of electron transfer in both yak and chicken meat and maintained their myoglobin stability.Sodium nitrite reduced the swelling of yak meat mitochondria and membrane permeability,compared with the non-sodium nitrite group(P<0.05),transmission electron microscopy results showed a better mitochondrial integrity in sodium nitrite group,indicating that sodium nitrite slowed down the process of mitochondrial degradation and maintained mitochondrial integrity,and chicken meat was completely opposite to that of yak meat;sodium nitrite increased the activity of mitochondrial complexes I,II,III and IV in yak meat(P<0.05),which facilitated mitochondrial electron transfer;increased the activity of mitochondrial complexes I,III and IV in chicken meat(P<0.05)and decreased the activity of complex II,which was not favorable for mitochondria to gain electrons from FADH₂;the activity of mitochondrial complex V in yak and chicken meat increased while Na⁺/K⁺-ATPase activity decreased,contributing to the synthesis of ATP by mitochondria.The mitochondrial NADH-dependent MMb reductase activity was higher in yak meat than in chicken(P<0.01),indicating a more efficient electron transfer to methemoglobin in yak meat,which in turn maintains myoglobin stability.Mitochondrial integrity was better and more functional in yak meat compared to chicken meat(P<0.05).These results suggest that low dose of sodium nitrite contributed to the integrity of the mitochondrial structure of yak meat and thus better function,while chicken meat was in contrast with yak meat.5.Low dose of sodium nitrite inhibited the oxidative damage to yak and chicken meat myoglobin and mitochondria by hydroxyl radicals.In the hydroxyl radical system,the addition of sodium nitrite reduced the carbonyl content and turbidity of yak and chicken meat myoglobin(P<0.05).Sodium nitrite did not significantly differ in the secondary structure content of myoglobin in yak meat,whereas promoted the transformation of the secondary structure of chicken meat myoglobin.Both yak and chicken meat sodium nitrite groups had the strongest contraction of the myoglobin heme center and the porphyrin ring N atom,showing that sodium nitrite inhibited the oxidation of myoglobin,slowed down the expansion of the heme porphyrin ring of myoglobin caused by oxidation and maintained its structural stability.Sodium nitrite reduced mitochondrial swelling and membrane permeability(P<0.05)and increased Na⁺/K⁺-ATPase activity(P<0.05)in both yak and chicken meat;it enhanced the activity of mitochondrial NADH-dependent MMb reductase in yak meat and reduced the activity of this enzyme in chicken meat(P<0.05),indicating that sodium nitrite was beneficial in maintaining the integrity of yak and chicken meat mitochondria and promoting yak meat mitochondrial function,while having a dual effect on chicken meat mitochondrial function.In conclusion,during low-dose sodium nitrite curing process of yak meat,firstly,sodium nitrite was converted to NO with the catalytic effect of myoglobin and mitochondrial reduction and nitric oxide synthase,while MMb was produced;secondly,NO acted on mitochondrial genesis and damage repair,maintained the structural integrity of mitochondria,increased the activity of the electron respiratory chain complex,and promoted MMb reduction;meanwhile,it increased the activity of endogenous antioxidant enzymes and reduced the level of reactive oxygen species(ROS),which in turn hindered the oxidation of Mb by ROS.Furthermore,sodium nitrite can also act directly on Mb to form NOMb,resulting in a stable cherry red color of yak meat,thus maintaining stable meat color and prolonging freshness period.In terms of maintaining meat color stability,low-dose nitrite had similar effects in chicken and yak meat,while it increased the oxidation of chicken myoglobin,which may be related to the different myoglobin content,myoglobin amino acid sequence and sodium nitrite action sites in chicken and yak meat.