Preparation, separation and physicochemical properties of α-glucosidase inhibitory peptides from Miichthys miiuy
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摘要: 为实现鮸 (Miichthys miiuy) 加工副产物的高值化利用，选用鮸加工副产物鱼碎肉作为原料，以α-葡萄糖苷酶 (α-glucosidase) 抑制率为评价指标，通过单因素实验和响应面法优化α-葡萄糖苷酶抑制肽的制备工艺条件，确定胰酶的最佳酶解条件为：时间4.8 h，加酶量0.21%，pH 8.5，料液比1∶2 (m∶V)，温度46 ℃。在此基础上，分析了酶解产物体外模拟胃肠液消化前后其抑制活性的变化，绘制酶抑制动力学曲线，并采用SephadexG-25对酶解物进行分离，测定酶解物的相对分子质量分布及氨基酸组成。结果表明：活性肽经体外模拟胃肠液消化后α-葡萄糖苷酶抑制率提高至61.97%；其对α-葡萄糖苷酶抑制作用为混合型抑制；酶解物的相对分子质量集中分布在3 kD以下 (占91.85%) ；经G-25分离，F4组分对α-葡萄糖苷酶抑制率为58.05%，其<1 kD的肽组分占78.28%；酶解物中天冬氨酸、谷氨酸、精氨酸、酪氨酸、缬氨酸、丙氨酸、亮氨酸和赖氨酸等相对含量较高。
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关键词:
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- α-葡萄糖苷酶抑制活性肽 /
- 酶解 /
- 响应面优化法
Abstract: In order to achieve the high-value utilization of processing by-products of Miichthys miiuy, taking α-glucosidase inhibition rate as an index, we carried out a single factor experiment and applied the response surface methodology to optimize the preparation process of α-glucosidase inhibitory peptides from minced fish muscle of M. miiuy processing by-products. The optimal enzymatic hydrolysis conditions with trypsin were as follows: hydrolysis time 4.8 h, enzyme dosage 0.21%, hydrolysis pH 8.5, material-liquid ratio 1∶2 (m/V), hydrolysis temperature 46 ℃. On this basis, we analyzed the inhibitory activity of the enzymatic hydrolysates before and after the simulated gastrointestinal digestion in vitro, and drew the kinetic curve of enzyme inhibition. Then we separated the enzymatic hydrolysate by SephadexG-25 so as to investigate the molecular weight distribution and amino acid composition of the enzymatic hydrolysates. The results show that the α-glucosidase inhibitory rate increased to 61.79% after that the α-glucosidase inhibitory peptide was simulated gastrointestinal digestion in vitro. Its inhibition type on α-glucosidase was mixed type inhibition. The molecular mass of the enzymatic hydrolysate was concentrated below 3 kD, accounting for 91.85%. After the separation by G-25, the α-glucosidase inhibition rate of F4 fraction was 58.05%. In Group F4, 78.28% of peptides were less than 1 kD. The amino acid analysis shows that the enzymatic hydrolysate was rich in Asp, Glu, Arg, Tyr, Val, Ala, Leu and Lys. -
图 1 最佳蛋白酶的选择
注：字母不同表示差异显著 (P<0.05)，后图同此。
Figure 1. Selection of best protease
Note: Different letters indicate significant differences (P<0.05). The same case in the following figures.
图 2 时间、加酶量、pH、料液比和温度对α-葡萄糖苷酶抑制活性的影响
Figure 2. Effects of time, enzyme dosage, pH, liquid-material ratio and temperature on α-glucosidase inhibition activity
图 4 鮸酶解物消化后的α-葡萄糖苷酶抑制活性
Figure 4. α-glucosidase inhibitory activity of M. miiuy enzymatic hydrolysates after digestion
图 5 鮸酶解物抑制α-葡萄糖苷酶的Lineweaver-Burk曲线
Figure 5. Lineweaver -Burk plots for inhibition of M. miiuy enzymatic hydrolysates on α-glucosidase
图 6 鮸酶解物HPLC色谱图及分子质量分布图
Figure 6. HPLC chromatogram of M. miiuy enzymatic hydrolysates and its relative molecular mass distribution
图 7 Sephadex G-25洗脱组分和α-葡萄糖苷酶活性
Figure 7. α-glucosidase activity of Sephadex G-25 eluents
图 8 Sephadex G-25 洗脱各组分的HPLC色谱图及相对分子质量分布图
Figure 8. HPLC chromatogram and molecular mass distribution of Sephadex G-25 eluents
表 1 蛋白酶的酶解条件
Table 1. Enzymolysis conditions of protease
酶种类
Enzyme type最适温度
Optimal
temperature/℃最适pH
Optimal
pH木瓜蛋白酶 Papain 50 6.5 中性蛋白酶 Neutral protease 50 7.0 碱性蛋白酶 Alkaline protease 50 8.0 复合蛋白酶 Compound protease 50 7.5 胰酶 Trypsin 37 8.0 
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表 2 响应面实验因素和水平
Table 2. Factors and levels in response surface design
水平
Level因素 Factor A：酶解时间
Enzymolysis
time/hB：加酶量
Enzyme
dosage/%C：酶解温度
Enzymolysis
temperature/℃−1 2 0.1 42 0 4 0.2 47 1 6 0.3 52
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表 3 Box-Behnken 实验设计及结果
Table 3. Design and results of Box-Behnken experiment
序号
No.A：
酶解时间
Enzymolysis
time/hB：
加酶量
Enzyme
dosage/%C：
酶解温度
Enzymolysis
temperature/℃Y：α-葡萄糖苷酶
抑制率
α-glucosidase
inhibition rate/%1 4 0.1 52 51.87 2 6 0.2 42 52.37 3 2 0.1 47 50.39 4 2 0.2 52 53.66 5 4 0.2 47 55.44 6 2 0.2 42 42.33 7 4 0.2 47 54.41 8 4 0.3 52 49.56 9 4 0.3 42 48.45 10 2 0.3 47 46.00 11 6 0.2 52 47.40 12 6 0.1 47 49.04 13 4 0.2 47 53.91 14 6 0.3 47 50.79 15 4 0.2 47 53.08 16 4 0.2 47 53.29 17 4 0.1 42 46.63
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表 4 Box-Behnken实验回归模型方差分析
Table 4. AVOVA of Box-Behnken experiment
来源
Source平方和
Sum of squares自由度
df均方
Mean squareF P Prob>F 模型 Model 195.83 9 21.76 36.36 < 0.0001 ** A 6.52 1 6.52 10.89 0.0131 * B 1.22 1 1.22 2.05 0.1957 C 20.19 1 20.19 33.74 0.0007 ** AB 9.42 1 9.42 15.75 0.0054 ** AC 66.42 1 66.42 110.99 < 0.0001 ** BC 4.26 1 4.26 7.13 0.0320 * A2 28.01 1 28.01 46.80 0.0002 ** B2 24.09 1 24.09 40.25 0.0004 ** C2 26.46 1 26.46 44.21 0.0003 ** 残差 Residual 4.19 7 0.60 失拟项 Lack of fit 0.59 3 0.20 0.22 0.8784 净误差 Pure error 3.60 4 0.90 总误差 Cor total 200.02 16 注：**. P<0.01；*. P<0.05。
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表 5 鮸鱼肉及酶解物的氨基酸组成
Table 5. Amino acid compositions of minced M. miiuy and its hydrolysates mg·g−1
氨基酸Amino acid 鱼碎肉Minced fish 酶解物Hydrolysate 洗脱组分F4Eluent component F4 天冬氨酸 Asp 106.48 116.74 88.47 谷氨酸 Glu 158.78 175.42 118.77 丝氨酸 Ser 38.10 41.17 36.34 甘氨酸 Gly 28.77 32.80 29.87 组氨酸 His 17.39 18.00 28.43 精氨酸 Arg 50.56 56.44 73.54 苏氨酸 Thr 35.80 37.80 30.41 丙氨酸 Ala 49.28 53.03 48.38 脯氨酸 Pro 22.72 25.25 19.57 酪氨酸 Tyr 25.49 24.83 40.42 缬氨酸 Val 38.79 38.20 36.32 蛋氨酸 Met 21.37 13.86 25.15 半胱氨酸 Cys 1.11 1.24 1.06 异亮氨酸 Ile 34.96 33.82 30.40 亮氨酸 Leu 56.67 57.35 54.74 苯丙氨酸 Phe 27.04 25.40 39.72 赖氨酸 Lys 66.91 73.13 118.76 色氨酸 Trp 4.92 3.04 7.56
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