芋头球蛋白的提取纯化及其对<i>α</i>-淀粉酶和<i>α</i>-葡萄糖苷酶抑制活性研究

马二兰; 林莹; 涂连; 张帆; 王伟良

doi:10.13386/j.issn1002-0306.2020100266

芋头球蛋白的提取纯化及其对α-淀粉酶和α-葡萄糖苷酶抑制活性研究

doi: 10.13386/j.issn1002-0306.2020100266

广西大学轻工与食品工程学院，广西南宁 530004

基金项目: 广西重点研发计划（桂科AB1850012）

详细信息

作者简介:
马二兰（1994−），女，硕士研究生，研究方向：粮食与油脂加工，E-mail：1875479910@qq.com

通讯作者:
林莹（1971−），女，博士，教授，研究方向：粮食与油脂加工，E-mail：ly9a9@126.com

中图分类号: TS201.2
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出版历程
- 收稿日期: 2020-11-03
- 网络出版日期: 2021-06-08
- 刊出日期: 2021-07-07

Extraction, Purification of Taro Globulin and Its Inhibitory Activity on α-Amylase and α-Glucosidase

College of Light Industry and Food Engineering, Guangxi University, Nanning 530004, China

摘要

摘要: 目的：探究芋头球蛋白体外调节血糖活性。方法：以磷酸缓冲液提取芋头球蛋白，以蛋白质提取率为指标考察了料液比、温度、时间和次数对蛋白质提取的影响，在此基础上采用响应面优化提取工艺。采用DEAE-52离子纤维素柱层析法纯化粗蛋白，通过高效液相色谱法检测纯化所得球蛋白的纯度，并测定其等电点和分子量。研究了芋头球蛋白对α-淀粉酶和α-葡萄糖苷酶的抑制活性及抑制动力学，以阿卡波糖为阳性对照，评价其体外调节血糖活性。结果：最佳提取条件为：料液比1:16 g/mL、温度41 ℃、时间124 min，此时提取率为36.75%±0.31%，得率0.70%±0.04%，纯度85.72%±0.47%。纯化后的球蛋白经高效液相色谱检测得一主峰，纯度为93.27%，得率为0.20%±0.01%，等电点pI=5.6，分子量为22 kDa左右。芋头球蛋白对两种酶的抑制活性与蛋白浓度存在量效关系，对α-淀粉酶的IC₅₀为0.75±0.10 mg/mL，对α-葡萄糖苷酶的IC₅₀为（2.09±0.19） mg/mL，而阿卡波糖对α-淀粉酶的IC₅₀为（0.61±0.13） mg/mL，对α-葡萄糖苷酶的IC₅₀为（0.69±0.16） mg/mL，说明芋头球蛋白对α-淀粉酶略低于阿卡波糖，而对α-葡萄糖苷酶抑制活性远低于阿卡波糖，二者的抑制类型均为可逆性的非竞争性抑制，对α-淀粉酶抑制的K_i=（0.61±0.05） mg/mL，对α-葡萄糖苷酶抑制的K_i=（0.26±0.02） mmol/L。结论：本研究优化了芋头球蛋白的提取工艺，纯化得到芋头球蛋白，研究发现其有一定的体外调节血糖的活性，对功能食品的研发和芋头产品提高附加值具有一定的指导意义。
- 芋头 /
- 球蛋白 /
- 提取 /
- 纯化 /
- 血糖调节
Abstract: Objective: Exploring taro globulin regulating activity in vitro. Methods: Taro globulin was extracted by phosphate buffer. The effects of solid-liquid ratio, temperature, time and times on protein extraction were investigated with protein extraction rate as an index. Based on the test, response surface methodology was used to optimize the extraction process. The crude protein was purified by DEAE-52 ion cellulose column chromatography. The purity of purified globulin was detected by high performance liquid chromatography, and its isoelectric point and molecular weight were determined. The inhibitory activity and inhibition kinetics of taro globulin on α -amylase and α-glucosidase were studied, and acarbose was used as positive control to evaluate globulin activity of regulating blood glucose in vitro. Results: The optimum extraction conditions were as follows: The solid-to-liquid ratio was 1:16 g/mL, the temperature was 41 ℃, and the time was 124 min. Under these condition, the extraction rate was 36.75%±0.31%, the yield was 0.70%±0.04%, and the purity of the protein was 85.72%±0.47%. The purified globulin was detected by high performance liquid chromatography with a main peak with purity of 93.27%, yield of 0.20%±0.01%, isoelectric point of 5.6 and molecular weight of about 22 kDa. The taro globulin exhibited inhibition on the two enzymes with dose-effect relationship. The IC₅₀ was (0.75±0.10) mg/mL for α-amylase and was (2.09±0.19) mg/mL for α-glucosidase The IC₅₀ of acarbose were (0.61±0.13) mg/mL and (0.69±0.16) mg/mL. The results showed that the inhibitory activity of taro globulin on α-amylase was slightly lower than acarbose, while the inhibitory activity on α-glucosidase was much lower than acarbose. The inhibition type was reversible and uncompetitive inhibitor, with K_i=(0.61±0.05) mg/ml for α-amylase and K_i=(0.26±0.02) mmol/L/L for α-glucosidase. Conclusion: The extraction process of taro globulin was optimized, and taro globulin was purified. It was found that taro globulin has certain activity of regulating blood sugar in vitro, which would have certain guiding significance for the research and development of functional food and the increasing of added value of taro products.
- taro /
- globulin /
- extraction /
- purification /
- glucose regulating activity

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图 1 料液比对芋头球蛋白提取率的影响

Figure 1. Effect of solid-to-liquid ratio on the extraction yield of taro globulin

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图 2 温度对芋头球蛋白提取率的影响

Figure 2. Effect of extraction temperature on the taro globulin extraction rate

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图 3 时间对芋头球蛋白提取率的影响

Figure 3. Effect of extraction time on taro globulin extraction rate

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图 4 次数对芋头球蛋白提取率的影响

Figure 4. Effect of extraction times on taro globulin extraction rate

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图 5 各因素交互作用响应面图和等高线图

Figure 5. Response surface and contour plots showing the interaction of various factors

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图 6 DEAE-52纤维素层析柱纯化芋头球蛋白洗脱曲线

Figure 6. Elution curve of taro globulin purified by deae-52 ion exchange chromatography

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图 7 芋头球蛋白经纯化在HPLC流出曲线

Figure 7. Taro globulin HPLC outflow curve after purified

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图 8 上清液球蛋白浓度随pH变化曲线

Figure 8. Curve of globulin concentration in supernatant with pH

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图 9 芋头球蛋白电泳图

Figure 9. Electropherogram of taro globulin

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图 10 芋头球蛋白和阿卡波糖对α-淀粉酶的抑制作用

Figure 10. Inhibitory effects of taro globulin and acarbose on α-amylase

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图 11 芋头球蛋白和阿卡波糖对α-葡萄糖苷酶的抑制作用

Figure 11. Inhibitory effects of taro globulin and acarbose on α-glucosidase

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图 12 芋头球蛋白对α-淀粉酶的抑制动力学曲线

Figure 12. Inhibitory kinetic curve of taro globulin on α-amylase

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图 13 芋头球蛋白对α-葡萄糖苷酶的抑制动力学曲线

Figure 13. Inhibitory kinetic curve of taro globulin on α-glucosidase

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图 14 芋头球蛋白与α-淀粉酶反应的Lineweaver-Burk双倒数曲线

Figure 14. Lineweaver-Burk plots of the reactions of α-amylase with taro globulin

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图 15 芋头球蛋白与α-葡萄糖苷酶反应的Lineweaver-Burk双倒数曲线

Figure 15. Lineweaver-Burk plots of the reactions of α-glucosidase with taro globulin

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表 1 响应面试验因素和水平

Table 1. Independent variables and their coded levels used in response surface methodology

因素		编码水平（Xi）
因素	−1	0	1
A 料液比（g/mL）	10	15	20
B 提取温度（℃）	30	40	50
C 提取时间（min）	90	120	150

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表 2 响应面试验设计与结果

Table 2. Design and results of response urface methodology

实验号	因素			响应值
实验号	料液比（mL/g）	温度（℃）	时间（min）	蛋白质提取率（%）
1	10	30	120	31.81±0.83
2	20	30	120	34.53±1.16
3	10	50	120	32.94±0.95
4	20	50	120	34.96±0.66
5	10	40	90	30.25±0.79
6	20	40	90	33.39±0.98
7	10	40	150	32.97±0.58
8	20	40	150	34.67±0.85
9	15	30	90	33.45±1.07
10	15	50	90	35.11±0.84
11	15	30	150	35.62±0.72
12	15	50	150	36.48±0.86
13	15	40	120	36.56±0.59
14	15	40	120	36.51±0.62
15	15	40	120	36.59±0.74
16	15	40	120	36.68±0.84
17	15	40	120	36.74±0.69

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表 3 回归模型方差分析表

Table 3. Analysis of variance of regression model

来源	平方和	自由度	均方	F值	P值	显著性
模型	59.99229	9	6.66581	262.9955	< 0.0001	**
A-料液比	11.47205	1	11.47205	452.6229	< 0.0001	**
B-提取温度	2.0808	1	2.0808	82.09672	< 0.0001	**
C-提取时间	7.10645	1	7.10645	280.3807	< 0.0001	**
AB	0.1225	1	0.1225	4.833164	0.0639
AC	0.5184	1	0.5184	20.45316	0.0027	**
BC	0.16	1	0.16	6.312704	0.0402	*
A²	30.70611	1	30.70611	1211.491	< 0.0001	**
B²	0.532127	1	0.532127	20.99477	0.0025	**
C²	5.053138	1	5.053138	199.3685	< 0.0001	**
残差	0.17742	7	0.025346
失拟项	0.1429	3	0.047633	5.519506	0.0662
净误差	0.03452	4	0.00863
总离差	60.16971	16
注：：差异显著（P<0.05），*：差异极显著（P<0.01）。

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表 4 DEAE-52纤维素柱层析不同盐浓度洗脱组分的蛋白质含量和酶抑制活性

Table 4. Protein content and enzyme inhibitory activity of elution components with different salt concentrations on DEAE-52 cellulose column chromatography

NaCl浓度（mol/L）	蛋白质含量（mg）	1.0mg/mL蛋白质对α-淀粉酶的抑制率（%）	1.0mg/mL蛋白质对α-葡萄糖苷酶的抑制率（%）
0	13.84±0.15	7.62±0.16	5.32±0.38
0.1	65.58±0.41	58.62±0.45	35.92±0.87
0.2	53.24±0.47	12.32±0.23	9.05±0.59
0.3	15.24±0.28	8.43±0.26	5.86±0.27
0.4	3.49±0.52	4.72±0.45	2.12±0.33

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表 5 芋头蛋白提取率和纯度

Table 5. Extraction rate and purity of taro protein

样品	蛋白质提取率（%）	蛋白质得率（%）	蛋白质纯度（%）
粗蛋白	36.75±0.31^a	0.70±0.04^a	85.72±0.47^a
球蛋白	10.47±0.07^b	0.20±0.01^b	93.27±0.29^b
注：同列不同字母代表差异显著（P<0.05）。

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表 6 芋头球蛋白与α-淀粉酶和α-葡萄糖苷酶反应的Lineweaver-Burk双倒数曲线方程

Table 6. Lineweaver-Burk plots equation of the reactions of α-amylase and α-glucosidase with taro globulin

球蛋白浓度（mg/mL）	α-淀粉酶反应的双倒数曲线方程	α-葡萄糖苷酶反应的双倒数曲线方程
0	y=25.385x+11.77，R²=0.997	y=23.689x+12.706，R²=0.9934
1.0	y=59.583x+30.255，R²=0.9974	y=33.67x+18.526，R²=0.9948
2.0	y=119.7x+62.758，R²=0.996	y=39.143x+22.322，R²=0.9967

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