氧化对肌原纤维蛋白热诱导凝胶质构特性及保水性的影响

doi:10.3864/j.issn.0578-1752.2018.18.013

摘要/Abstract

摘要：

【目的】研究氧化对肌原纤维蛋白（myofibrillar proteins,MP）凝胶质构和保水性的影响,探讨凝胶特性随蛋白质氧化程度变化的根本原因,为MP凝胶特性控制和鸡肉制品的质量控制提供理论依据。【方法】活鸡屠宰,取鸡胸肉提取MP。利用质构仪研究在脂肪氧化酶-亚油酸体系中蛋白质氧化对MP凝胶质构的影响;用高速离心机测定凝胶保水性;用拉曼光谱法测定I760和I850/I830表示MP凝胶的疏水作用力和氢键,Zeta电位法测定电位值代表静电斥力;通过总巯基含量的变化反应二硫键的变化;通过扫描电镜观察凝胶的超微结构;通过氨基酸分析仪研究氧化对MP氨基酸含量的影响。【结果】在脂肪氧化酶-亚油酸-MP体系中,随着亚油酸浓度增加,MP中羰基含量逐步增加,氧化程度逐渐增高。亚油酸含量从0增加到2 mmol·L^-1时,凝胶硬度和保水性均逐渐增加到最大值,而后随亚油酸浓度增加均逐渐下降;凝胶弹性在低氧化程度下略有增加,但随着氧化程度继续增加而逐渐降低;亚油酸浓度为2 mmol·L^-1时,MP凝胶结构致密,多孔且孔径均一。高度氧化的MP凝胶孔径变大,空隙增多,胶束不均匀。随着氧化程度升高,拉曼光谱的I760在2 mmol·L^-1处达到最大值,表明疏水相互作用力在此处达到最大。Ser, Glu和Cys 3种氨基酸残基能够形成MP分子内氢键,这3种氨基酸含量随着氧化程度的升高而降低,同时拉曼光谱的I850/I830随氧化程度的升高而增加,最终大于1.25,表明MP分子间的氢键随着氧化程度的升高而减少。解离后带负电荷的Glu含量随氧化程度升高而降低,导致Zeta电位绝对值下降,表明静电相互作用随氧化程度增加而减弱。Cys的巯基在凝胶形成过程中能够形成二硫键,其含量随氧化程度的升高而降低,导致总巯基含量同向变化,表明氧化过程中二硫键生成。疏水性氨基酸（Ala,Met,Val,Leu,Ile和Phe）的总量随氧化程度升高而变化,在亚油酸为2 mmol·L^-1处达到最大值,这为疏水作用力在2 mmol·L^-1处达到最大值提供了证据。主成分分析表明疏水相互作用对脂质酶氧化体系下MP凝胶特性起决定性作用。【结论】适度氧化有助于改善MP凝胶的特性,在脂肪氧化酶-亚油酸体系中,亚油酸浓度为2 mmol·L^-1时,MP凝胶的硬度和保水性都获得最大值。其原因为氧化作用改变MP的组成和疏水作用力,在亚油酸2 mmol·L^-1时,MP分子中疏水性氨基酸总量最高,疏水作用力最大,形成的凝胶微观结构均匀致密,因此MP凝胶的质构和保水性均获得最大值。

关键词: 肌原纤维蛋白, 凝胶特性, 氧化, 氨基酸, 蛋白质分子作用力

Abstract:

【Objective】This study was designed to investigate the influence of protein oxidation on the textural properties and water holding capacity of myofibrillar protein (MP) gel, and to reveal the root cause of gel properties changes with the degree of protein oxidation, in order to provide the theoretical basis for controlling the gel properties and the quality of chicken products.【Method】Live chickens were slaughtered and the chicken breast muscle was used to extract MP. Effects of protein oxidation on the textural properties of MP gel were studied in the lipoxygenase-linoleic acid-MP system using a texture analyzer. Water holding capacity (WHC) of MP gel was measured by high-speed centrifuge. I760 and I850/I830 measured by Raman spectroscopy were used to represent the hydrophobic interaction and hydrogen bonds of MP gel, and the potential value was determined by Zeta potential to reflect electrostatic repulsion. The change of disulfide bond was determined by the change of total sulfhydryl group (SH). The ultrastructures of the gel were observed by scanning electron microscopy. The amino acid composition and content were investigated by an amino acid analyzer. 【Result】 The carbonyl content and the degree of oxidation of MP increased with increasing linoleic acid concentration in the lipoxygenase-linoleic acid-MP system. When the content of linoleic acid increased from 0 to 2 mmol·L^-1, the gel hardness and WHC increased to the maximum, and then gradually decreased as the concentration of linoleic acid increased. Springiness slightly increased at low oxidation degree, and then decreased with more linoleic acid added. When the concentration of linoleic acid was 2 mmol·L^-1, the network of the MP gel was dense, porous and uniform in pore size. The gel pore size became larger and uneven at higher linoleic acid concentration. I760 reached the maximum at 2 mmol·L^-1 with the increase of the degree of oxidation, which indicated that hydrophobic interaction force reached maximum. The intramolecular hydrogen bonds could be formed by the three amino acid residues Ser, Glu and Cys, and the content of these three amino acids decreased with the increase of the degree of oxidation. Meanwhile, the I850/I830 of the Raman spectrum increased with the increase of the degree of oxidation and finally >1.25, indicating that the hydrogen bonds between MP molecules decreased with the increase of the degree of oxidation. After dissociation, the content of negatively charged Glu decreased with the increase of the degree of oxidation, which led to the decrease of the absolute value of Zeta potential with the increase of the degree of oxidation, indicating that the electrostatic interaction decreased with the increase of the degree of oxidation. The sulfhydryl group of Cys could form disulfide bond in the gel formation process and the content of Cys decreased with the increase of the degree of oxidation, resulting in the change of the total sulfhydryl content in the same direction, which indicated the formation of disulfide bonds in the oxidation process. The total amount of hydrophobic amino acids (Ala, Met, Val, Leu, Ile and Phe) changed with increasing degree of oxidation and reached maximum at 2 mmol·L^-1 linoleic acid, which provided evidence that hydrophobic forces reached their maximum at 2 mmol·L^-1. The principal component analysis suggested that hydrophobic interaction was the key force controlling the gel properties in the lipoxygenase- linoleic acid-MP system. 【Conclusion】 Moderate oxidation of MP helped to improve the properties of MP gels, and the gel hardness of MP reached the maximum at 2 mmol·L^-1 in the lipoxygenase-linoleic acid-MP system. The reason was that oxidation changed the composition and hydrophobic forces of MP. When the linoleic acid was 2 mmol·L^-1, the total amount of hydrophobic amino acid in MP molecule was the highest and the hydrophobic force was the largest, and the microstructure of the gel was uniform and dense, so that the texture and WHC of MP gel were the highest.

Key words: myofibrillar protein, gel properties, oxidation, amino acid, protein molecule forces

杨玉玲, 周磊, 游远, 汤晓智, 魏苏萌. 氧化对肌原纤维蛋白热诱导凝胶质构特性及保水性的影响[J]. 中国农业科学, 2018, 51(18): 3570-3581.

YuLing YANG, Lei ZHOU, Yuan YOU, XiaoZhi TANG, SuMeng WEI. The Effects of Oxidation on Textural Properties and Water Holding Capacity of Heat-Induced Myofibrillar Protein Gel[J]. Scientia Agricultura Sinica, 2018, 51(18): 3570-3581.

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氨基酸 Amino acid	亚油酸浓度Linoleic acid concentration
氨基酸 Amino acid	0	0.2 mmol·L^-1	1 mmol·L^-1	2 mmol·L^-1	4 mmol·L^-1	10 mmol·L^-1
Ala	43.85±0.58a	41.47±0.34b	41.08±0.17b	41.64±0.94b	39.39±0.52b	39.83±0.61b
Arg	51.97±0.54a	49.81±0.84a	48.14±0.65a	51.23±0.28a	48.35±0.68a	49.05±0.94a
Asp	66.03±0.57a	68.67±0.34a	65.96±0.68a	67.76±0.84a	65.99±0.21a	66.63±0.52a
Cys	7.23±0.15a	6.97±0.11b	6.82±0.17b	6.68±0.21b	6.52±0.14b	6.27±0.35c
Glu	142.85±0.51a	140.38±0.27a	136.95±0.34b	132.62±0.84c	132.99±0.35c	133.78±0.68c
Gly	27.19±0.57a	23.12±0.36b	22.33±0.57b	23.58±0.95b	22.35±0.28b	18.64±0.58c
His	13.53±0.35a	13.21±0.68a	12.87±0.47a	13.71±0.33a	12.92±0.95a	13.25±0.62a
Ile	35.94±0.35a	35.27±0.94a	35.31±0.65a	36.21±0.34a	34.42±0.38a	34.92±0.52a
Leu	61.75±0.28a	62.64±0.97a	62.68±1.21a	64.44±0.57b	60.82±0.68a	61.89±0.21a
Lys	9.19±0.65a	8.69±0.52a	8.57±0.68a	8.96±0.62a	8.54±0.47a	8.74±0.95a
Met	25.16±0.84a	23.01±1.32b	22.28±0.56b	23.32±0.75b	22.53±0.35b	22.41±0.59b
Phe	28.68±0.54a	29.17±0.68a	28.12±0.24a	29.72±0.33a	28.13±0.37a	28.38±0.59a
Ser	29.13±0.28a	26.48±0.49b	23.73±0.36c	24.96±0.25c	23.67±0.98c	23.72±0.54c
Thr	33.75±0.58a	33.51±0.36a	31.36±0.27a	32.96±0.95a	31.37±0.84a	31.53±0.68a
Tyr	22.85±0.63a	20.47±0.21a	19.77±0.37a	20.96±0.57a	20.43±0.64a	20.25±0.84a
Val	34.23±0.97a	35.19±0.27a	35.81±0.86a	35.12±0.54a	33.44±0.67a	33.62±0.35a

主成分PC	特征值Eigenvalue	解释方差Total variance (%)	累积特征值Cumulative eigenvalue	累积解释方差Cumulative (%)
1	6.491	72.124	6.491	72.124
2	2.068	22.972	8.559	95.096