碱性氨基酸，L-组氨酸（L-histidine, L-His）、L-赖氨酸（L-lysine, L-Lys）和L-精氨酸（L-arginine, L-Arg）因可改善低盐肌肉蛋白功能特性，在肉品减盐保质中受到广泛的关注。近期的研究发现碱性氨基酸在低盐条件下具有调控肌肉蛋白磷酸化的潜力。无氧糖酵解是肌肉宰后的重要活动，多种糖酵解酶能够发生磷酸化反应后改变自身活性，进而影响糖酵解的速率和宰后肌肉的成熟。因此，本研究从蛋白质磷酸化角度出发，以宰后不同时间的黄羽肉鸡胸大肌为研究对象，探究低NaCl下碱性氨基酸预处理诱导的糖酵解酶磷酸化水平与活性的变化规律，建立二者之间的内在联系；明确差异磷酸化修饰位点，在不同磷酸化水平下，深入研究低盐下碱性氨基酸对关键糖酵解酶自身性质的影响规律，为肉品减盐保质及宰后品质改善提供理论基础。
主要研究结果如下：
（1）探究低盐条件下碱性氨基酸对糖酵解酶磷酸化水平及其活性的影响。对宰后1 h的胸大肌肉糜进行0% NaCl、1% NaCl、3% NaCl、1% NaCl+0.06% L-His/L-Lys/L-Arg处理，于宰后0、0.5、1、1.5、2、6、15和24 h测定丙酮酸激酶（pyruvate kinase, PK）、β-烯醇化酶（β-enolase, β-ENO）、甘油醛-3-磷酸脱氢酶（glyceraldehyde-3-phosphate dehydrogenase, GAPDH）、6-磷酸果糖激酶（6-phosphate fructokinase, PFK）的磷酸化水平和活性。结果表明：低盐条件下，碱性氨基酸对糖酵解酶的磷酸化水平整体上无显著影响，但对活性的影响显著。宰后1.5、2和6 h时，1%NaCl的基础上添加碱性氨基酸处理显著改变酶的磷酸化水平，正向增强酶活。相关性分析的结果显示，PK和GAPDH的磷酸化水平与活性之间均呈负相关，β-ENO和PFK的磷酸化水平与活性之间均呈正相关。
（2）体外验证PK的磷酸化水平与活性的关系，解析低盐下碱性氨基酸对丙酮酸激酶磷酸化的作用规律。PK与三磷酸腺苷（adenosine triphosphate, ATP）溶液、蛋白激酶A（protein kinases A, PKA）、4 ℃预冷的孵育缓冲液按比例混合，1 h后分六组添加0% NaCl、1% NaCl、3% NaCl、1% NaCl+0.06% L-His/L-Lys/L-Arg处理溶液，测定PK的磷酸化水平、活性、二级结构、磷酸化位点和降解。结果表明：体外体系中PK的磷酸化水平与活性呈负相关；碱性氨基酸处理组降低PK的磷酸化水平，增强PK的活性，同时PK的二级结构由有序转向无序，增大PK的降解程度；鉴定出不同处理组能够使不同位点的磷酸化水平发生上调或下调，集中在丝氨酸（serine, Ser）、苏氨酸（threonine, Thr）或酪氨酸（tyrosine, Tyr）的14个差异磷酸化位点，包括Ser22、Ser36、Ser66、Ser76、Ser96、Ser99、Ser126、Ser436、Thr79、Thr94、Thr128、Tyr82、Tyr369、Tyr520。
（3）基于宰后胸大肌主要糖酵解酶的磷酸化，探究低盐条件下碱性氨基酸对宰后胸大肌糖酵解潜力（glycolysis Potential, GP）、剪切力和肌原纤维小片化指数（myofibril fragmentation index, MFI）的影响。对宰后1 h的胸大肌肉糜进行0% NaCl、1% NaCl、3% NaCl、1% NaCl+0.06% L-His/L-Lys/L-Arg处理，于0、0.5、1、1.5、2、6、15和24 h分别测定与能量代谢相关的糖原、葡萄糖、葡萄糖-6-磷酸和乳酸含量，得到GP值，测定MFI指标；相同时间点对整块胸大肌进行相同分组处理，测定剪切力。结果显示：碱性氨基酸处理可以通过正向调节糖酵解酶的磷酸化水平，提高酶的活性，加速糖原代谢，显著降低GP值；宰后成熟期间剪切力先增大后减小，MFI值不断增大，与第二章糖酵解酶的磷酸化水平、活性数据结合分析，表明低盐条件下添加碱性氨基酸处理能够通过调节磷酸化水平增强酶的活性，显著降低宰后胸大肌的剪切力值、增大MFI值，进而改善肉的嫩度。

The basic amino acids, L-histidine (L-His), L-lysine (L-Lys) and L-arginine (L-Arg), have received much attention for their ability to improve the functional properties of low-salt muscle proteins in meat salt reduction and preservation. Recently, basic amino acids have been found to have the potential to regulate muscle protein phosphorylation under low salt conditions. Anaerobic glycolysis is an important activity in post-mortem muscle, and a variety of glycolytic enzymes are capable of altering their activity following phosphorylation reactions, which in turn affects the rate of glycolysis and post-mortem muscle maturation. Therefore, from the perspective of protein phosphorylation, this study took yellow-feathered broilers pectoralis major muscles at different times after slaughter as the research object, explored the change law of glycolytic enzyme phosphorylation level and activity induced by alkaline amino acid pretreatment under low NaCl, and established the intrinsic relationship between the two. The characteristics of differential phosphorylation modification were clarified, and the influence of basic amino acids on the properties of key glycolytic enzymes under different phosphorylation levels was deeply studied , which provided a theoretical basis for salt reduction and quality improvement of meat products.

The main findings are as follows:

(1) The effect of basic amino acids on the phosphorylation level and activity of glycolytic enzymes under low salt conditions was investigated. The minced pectoralis major meat was treated with 0% NaCl, 1% NaCl, 3% NaCl, 1% NaCl+0.06% L-His/L-Lys/L-Arg grouping for 1 h after slaughter. The phosphorylation levels and activities of pyruvate kinase (PK), β-enolase (β-ENO), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and 6-phosphate fructokinase (PFK) were measured at 0, 0.5, 1, 1.5, 2, 6, 15 and 24 h after slaughter. The results showed that basic amino acids had no significant effect on the overall phosphorylation level of glycolytic enzymes and a significant effect on the activity. At 1.5, 2 and 6 h after slaughter, basic amino acid treatment significantly altered the phosphorylation level of the enzyme and positively enhanced the activity under low salt conditions. Correlation analysis showed that there was negative correlations between phosphorylation levels and activities of PK and GAPDH, and positive correlations between phosphorylation levels and activities of β-ENO and PFK.

(2) The relationship between phosphorylation level and activity of PK was verified in vitro, and the effect of basic amino acids under low salt on pyruvate kinase phosphorylation was analyzed. PK was mixed with adenosine triphosphate (ATP) solution, protein kinases A (PKA), and incubation buffer precooled at 4 °C, and 0% NaCl, 1% NaCl, 3% NaCl, 1% NaCl+0.06% L-His/L-Lys/L-Arg treatment solution were added in six groups after 1 h, and the phosphorylation level, activity, secondary structure, Phosphorylation sites and degradation.The results showed that the phosphorylation level of PK in the in vitro system was negatively correlated with the activity; the basic amino acid treatment group decreased the phosphorylation level of PK and enhanced the activity of PK, while the secondary structure of PK shifted from order to disorder and the degradation of PK increased; different treatment groups were identified to stimulate different phosphorylation sites, resulting in up- or down-regulation of phosphorylation level at different sites, concentrating on serine (Ser) , threonine (Thr) and tyrosine (Tyr) in total Fourteen differential phosphorylation sites were identified, mainly Ser22, Ser36, Ser66, Ser76, Ser96, Ser99, Ser126, Ser436, Thr79, Thr94, Thr128, Tyr82, Tyr369 and Tyr520.

(3) Based on the phosphorylation of the main glycolytic enzymes of the postmortem pectoralis major muscle, the effects of alkaline amino acids on the glycolysis potential (GP), shear force and myofibril fragmentation index (MFI) of the postmortem pectoralis major muscle under low salt conditions were investigated. The round pectoralis major meat was treated with 0% NaCl, 1% NaCl, 3% NaCl, 1% NaCl+0.06% L-His/L-Lys/L-Arg at 1 h after slaughter. The contents of glycogen, glucose, glucose-6-phosphate and lactic acid related to energy metabolism were determined at 0, 0.5, 1, 1.5, 2, 6, 15 and 24 h respectively, and the GP value was obtained and the MFI index was measured. The whole pectoralis major muscle was grouped in the same time point to measure the shear force.The results showed that the basic amino acid treatment could increasing the activity of glycolysis enzymes by positively regulating phosphorylation levels , accelerated glycogen metabolism and significantly reduced the GP; the shear force increased and then decreased during post-slaughter maturation, and the MFI values increased continuously. The shear force and MFI values of pectoralis majors were increased, which in turn improved the tenderness of the meat.

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