Preparation and properties of collagen ACE inhibitory peptides from bone of eel (Anguilla japonica)
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摘要: 为研究鳗鱼加工副产物的综合利用，以鳗鱼骨胶原蛋白为原料，采用酶解法制备血管紧张素转化酶 (Angiotensin-converting enzyme, ACE) 抑制肽。以ACE抑制活性和水解度为评价指标，通过单因素和响应面试验确定最佳的酶解制备条件，并分析酶解产物的分子质量分布和氨基酸组成。结果显示，碱性蛋白酶为最适水解酶，最佳酶解条件为：温度50 ℃，质量浓度15 g·L–1，酶解时间5.25 h，加酶量3.1% (质量分数)，pH 9.2。在此条件下，鳗鱼胶原蛋白肽的ACE抑制活性为70.33%，与预测值接近；酶解产物中分子质量小于1 kD的肽占57.02%，1~3 kD的肽占36.55%；氨基酸组成分析表明，酶解产物中与ACE抑制活性有关的疏水性氨基酸 (如脯氨酸、缬氨酸、异亮氨酸、亮氨酸、苯丙氨酸) 含量增加。Abstract: To study the comprehensive utilization of by-products from eel processing, the angiotensin-converting enzyme inhibitory peptide was prepared from eel (Anguilla japonica) bone collagen by enzymatic hydrolysis. The ACE inhibitory activity and degree of hydrolysis were used as evaluation indexes, we determined the optimal hydrolysis conditions by single factor and response surface experiments. Furthermore, the amino acid composition and molecular mass distribution of the hydrolysate prepared under the optimized conditions were determined. The results show that alkaline protease was the optimal enzyme, and the optimal hydrolysis conditions were as follows: temperature of 50 ℃, mass concentration of 15 g·L−1, hydrolysis time of 5.25 h, enzyme dosage of 3.1% (Mass fraction) and pH of 9.2. Under these conditions, the ACE inhibitory activity was 70.33%, which was close to the predicted value. The molecular weight of peptides below 1 000 D and 1 000–3 000 D in enzymatic hydrolysate accounted for 57.02% and 36.55%, respectively. Amino acid composition analysis shows that the content of hydrophobic amino acids related to ACE inhibition activities (Such as Pro、Val、Ile、Leu、Phe) increased.
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图 1 不同蛋白酶对ACE抑制活性的影响
Figure 1. Effect of different proteases on ACE inhibitory activity
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图 2 pH、加酶量、时间、胶原蛋白质量浓度对水解度和ACE抑制活性的影响
Figure 2. Effects of pH, enzyme dosage, enzymatic hydrolysis time and collagen mass concentration on degree of hydrolysis and ACE inhibitory activity
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图 3 各因素相互作用对ACE抑制活性的响应面及等高线图
Figure 3. Response surface and contour lines of various factors on ACE inhibitory activity
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图 4 鳗鱼骨胶原蛋白酶解产物的高效体积排阻色谱图 (a) 及分子质量分布图 (b)
Figure 4. HPSEC chromatogram (a) of hydrolysates of eel bone collagen and its molecular weight distribution (b)
表 1 5种蛋白酶的最适酶解条件
Table 1 Optimal enzymatic hydrolysis conditions of five proteases
蛋白酶
Protease温度
Temperature/℃pH 木瓜蛋白酶 Papain 55 7.6 碱性蛋白酶 Alcalase 50 9.0 中性蛋白酶 Neutrase 45 7.0 胰蛋白酶 Trypsin 50 7.5 胃蛋白酶 Pepsin 37 2.0 
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表 2 响应面试验因素水平表
Table 2 Response surface test factors and levels
水平
Level因素 Factor A：酶解时间
Enzymatic hydrolysis time/hB：加酶量
Enzyme dosage/%C：pH −1 4 2 8 0 5 3 9 1 6 4 10
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表 3 响应面试验设计和结果
Table 3 Design and results of response surface experiment
编号
No.A：酶解时间
Enzymatic
hydrolysis
time/hB：加酶量
Enzyme
dosage/%C：pH ACE抑制率
ACE
inhibitory
rate/%1 –1 1 0 40.83 2 0 1 –1 62.17 3 –1 0 –1 34.38 4 0 0 0 73.55 5 0 0 0 68.16 6 1 1 0 57.42 7 0 0 0 72.82 8 –1 0 1 51.8 9 –1 –1 0 39.67 10 0 –1 –1 59.4 11 0 0 0 70.05 12 1 –1 0 51.77 13 0 0 0 72.75 14 0 1 1 62.76 15 1 0 –1 65.43 16 1 0 1 62.73 17 0 –1 1 60.83
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表 4 回归模型方差分析
Table 4 Variance analysis of regression model
方差来源
Variance source自由度
df平方和
SSF P 模型 Model 9 2 236.3 27.19 0.000 1*** A：时间 Time 1 624.63 68.36 <0.000 1*** B：加酶量
Enzyme dosage1 16.56 1.81 0.220 2 C：pH 1 35.11 3.84 0.090 8 AB 1 5.04 0.551 6 0.481 9 AC 1 101.4 11.1 0.012 6* BC 1 0.176 4 0.019 3 0.893 4 A2 1 1 062.69 116.3 <0.000 1*** B2 1 281.51 30.81 0.000 9*** C2 1 17.17 1.88 0.212 8 残差 Residual 7 63.96 失拟项 Lack of fit 3 42.93 2.72 0.179 纯误差 Pure error 4 21.04 总和 Cor total 16 2 300.27 注：***. 差异极显著 (P<0.001)；**. 差异较显著 (P<0.01)；*. 差异显著 (P<0.05)。 Note: ***. Extremely significant difference (P<0.001); **. Very significant difference (P<0.01); *. Significant difference (P<0.05).
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表 5 鳗鱼胶原蛋白氨基酸组成
Table 5 Amino composition of collagen from eel bone
氨基酸
Amino acid氨基酸质量分数
Amino acid mass fraction/%胶原蛋白
Collagen酶解产物
Enzymatic hydrolysis product天门冬氨酸 Asp 7.23 6.87 苏氨酸 Thr 2.04 2.24 丝氨酸 Ser 4.8 4.37 谷氨酸 Glu 11.79 11.29 脯氨酸 Pro* 10.72 11.25 甘氨酸 Gly 22.79 20.11 丙氨酸 Ala* 12.21 11.52 半胱氨酸 Cys 0.11 — 缬氨酸 Val* 1.24 2.04 蛋氨酸 Met* 0.99 0.38 异亮氨酸 Ile* 0.55 1.42 亮氨酸 Leu* 2.31 3.21 酪氨酸 Tyr 0.64 0.94 苯丙氨酸 Phe* 1.89 2.49 赖氨酸 Lys 2.94 3.79 组氨酸 His 1.29 1.59 精氨酸 Arg 8.98 9.37 羟脯氨酸 Hyp 7.48 7.12 疏水性氨基酸总量
Hydrophobic amino acid29.91 32.31 注：*. 疏水性氨基酸。 Note: *. Hydrophobic amino acid.
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