响应面法优化草菇抗氧化肽的酶法制备工艺

王耀冉; 陈明杰; 查磊; 魏晨颖; 李治平; 赵妍

doi:10.13386/j.issn1002-0306.2021110073

响应面法优化草菇抗氧化肽的酶法制备工艺

doi: 10.13386/j.issn1002-0306.2021110073

王耀冉^{1, 2,},
陈明杰²,
查磊²,
魏晨颖^{1, 2},
李治平^{1, 2},
赵妍^2, ,

1.
上海海洋大学食品学院，上海 201306
2.
上海市农业科学院食用菌研究所，上海市农业遗传育种重点开放实验室，上海 201403

基金项目: 上海市科技兴农重点攻关项目（2020-02-08-00-12-F01479）。

详细信息

作者简介:
王耀冉（1997−），女，硕士研究生，研究方向：食品生化，E-mail：1784835766@qq.com

通讯作者:
赵妍（1981−），女，博士，研究员，研究方向：食用菌生理生化，E-mail：jiandan289@126.com

中图分类号: TS201.2
计量
- 文章访问数: 75
- HTML全文浏览量: 15
- PDF下载量: 42
- 被引次数: 0
出版历程
- 收稿日期: 2021-11-08
- 网络出版日期: 2022-06-30
- 刊出日期: 2022-08-03

Optimization of Enzymatic Preparation Technology of Antioxidant Peptide from Volvariella volvacea by Response Surface Methodology

WANG Yaoran^{1, 2
,},
CHEN Mingjie²,
ZHA Lei²,
WEI Chenying^{1, 2},
LI Zhiping^{1, 2},
ZHAO Yan^{2
, ,}

1.
College of Food Sciences, Shanghai Ocean University, Shanghai 201306, China
2.
Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, Shanghai Key Laboratory of Agricultural Genetics and Breeding, Shanghai 201403, China

摘要

摘要: 以草菇为原料提取蛋白质，蛋白酶酶解蛋白制备抗氧化肽。以DPPH自由基清除率为指标，在单因素实验基础上，结合响应面法优化草菇抗氧化肽的提取工艺。通过超滤分离纯化获得不同分子量的肽段，采用DPPH自由基清除率、Fe²⁺螯合率和还原力法测定超滤组分的抗氧化活性。结果表明：中性蛋白酶为最优酶解蛋白酶，最佳酶解工艺条件为酶解时间3.70 h，加酶量3.81%，底物质量浓度3.11 g/100 mL，在此条件下，酶解产物的DPPH自由基清除率为69.85%±2.52%。通过超滤分级制备所得分子量最小的肽段F1（<3 kDa）具有最高的抗氧化活性，其DPPH自由基清除率、Fe²⁺螯合率和还原力分别为78.81%±1.56%、91.05%±1.65%、0.47±0.02。草菇抗氧化肽可作为潜在的天然抗氧化剂来源得到开发利用。
- 草菇 /
- 蛋白酶水解 /
- 抗氧化肽 /
- 响应面法 /
- 超滤
Abstract: The antioxidant peptides were prepared from Volvariella volvacea protein by protease hydrolysis. Based on the single-factor test, the extraction conditions of antioxidant peptides from V. volvacea protein were optimized using response surface methodology. At the same time, peptides with different retention molecular weights were isolated by ultrafitration, and their antioxidant activity were detected through 2,2-dipheny1-1-picrylhydrazyl (DPPH) radical scavenging activity, Fe²⁺ chelating ability and reducing power. The results showed that the optimum enzyme for enzymatic hydrolysis was neutral protease, and the optimum process for antioxidant peptide was as follows: The enzymatic hydrolysis time was 3.70 h, the enzyme dosage was 3.81%, and the substrate mass concentration was 3.11 g/100 mL. Under the optimal enzymalysis conditions, DPPH free radical scavenging rate of the optimal hydrolysis products was 69.85%±2.52%. The F1 fraction with the lowest molecular weight (<3 kDa) exhibited the highest antioxidant activity. DPPH free radical scavenging rate, Fe²⁺ chelating ability and reducing power of F1 were 78.81%±1.56%, 91.05%±1.65%, 0.47±0.02. The antioxidant peptides from V. volvacea protein can be explored as a potential natural antioxidant.
- V. volvacea /
- protease hydrolysis /
- antioxidant peptides /
- response surface methodology (RSM) /
- ultrafitration

HTML全文

图 1 酶种类对草菇蛋白酶解产物抗氧化活性的影响

Figure 1. Effect of enzyme types on antioxidant activity of V. volvacea protein hydrolysates

注：VPI：草菇蛋白；AH：碱性蛋白酶水解产物；TH：胰蛋白酶水解产物；NH：中性蛋白酶水解产物；PH：木瓜蛋白酶水解产物；ATH：碱性-胰蛋白酶水解产物；ANH：碱性-中性蛋白酶水解产物；APH：碱性-木瓜蛋白酶水解产物；不同小写字母表示差异显著P<0.05，图2~图4同。

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图 2 酶解时间对DPPH自由基清除率的影响

Figure 2. Effect of hydrolysis time on DPPH free radical scavenging rate

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图 3 加酶量对DPPH自由基清除率的影响

Figure 3. Effect of enzyme dosage on DPPH free radical scavenging rate

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图 4 底物质量浓度对DPPH自由基清除率的影响

Figure 4. Effect of substrate concentration on DPPH free radical scavenging rate

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图 5 各因素交互作用的曲面图和等高线图

Figure 5. Surface maps and contour maps for each factor interaction

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表 1 不同酶酶解条件

Table 1. Hydrolysis conditions for the various enzymatic hydrolyses

蛋白酶	温度（℃）	pH	酶解时间（h）	加酶量（%）	底物质量浓度（g/100 mL）
碱性蛋白酶	50	10.0	4	4	3
中性蛋白酶	45	7.5	4	4	3
胰蛋白酶	37	8.0	4	4	3
木瓜蛋白酶	37	7.0	4	4	3
碱性-中性蛋白酶	50~45	10.0~7.5	4	4	3
碱性-胰蛋白酶	50~37	10.0~8.0	4	4	3
碱性-木瓜蛋白酶	50~37	10.0~7.0	4	4	3

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表 2 响应面试验因素水平

Table 2. Factors and levels of response surface experiment

因素	水平
因素	−1	0	1
A 酶解时间（h）	3	4	5
B 加酶量（%）	3	4	5
C 底物质量浓度（g/100 mL）	2	3	4

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表 3 响应面试验设计方案与结果

Table 3. Experimental scheme and results of response surface

试验号	因素			响应值
试验号	A 酶解时间	B 加酶量	C 底物质量浓度	DPPH自由基清除率（%）
1	0	−1	−1	52.67
2	0	0	0	68.85
3	0	1	−1	46.42
4	0	1	1	49.03
5	−1	1	0	56.72
6	0	0	0	68.79
7	1	−1	0	48.46
8	−1	0	1	57.82
9	0	0	0	69.54
10	0	0	0	70.26
11	0	−1	1	53.15
12	−1	0	−1	43.50
13	0	0	0	68.81
14	−1	−1	0	60.77
15	1	0	1	37.65
16	1	0	−1	43.50
17	1	1	0	46.14

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表 4 回归模型方差分析及回归方程系数显著性检验

Table 4. Significance test for variance analysis and coefficient constants of regression models

来源	平方和	自由度	均方	F值	P值	显著性
模型	1854.93	9	206.10	175.68	<0.0001	**
A	231.77	1	231.77	197.56	<0.0001	**
B	35.03	1	35.03	29.86	0.0009	**
C	16.70	1	16.70	14.24	0.0070	**
AB	0.7482	1	0.7482	0.6378	0.4508
AC	101.71	1	101.71	86.69	<0.0001	**
BC	1.13	1	1.13	0.9668	0.3582
A²	461.01	1	461.01	392.96	<0.0001	**
B²	139.88	1	139.88	119.23	<0.0001	**
C²	730.17	1	730.17	622.39	<0.0001	**
残差	8.21	7	1.17
失拟	6.54	3	2.18	5.23	0.0720	不显著
净误差	1.67	4	0.4174
总和	1863.14	16
决定系数R²	0.9956
校正决定系数R_Adj²	0.9899
注：表示显著，P<0.05；*表示极显著，P<0.01。

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表 5 中性蛋白酶酶解产物及其超滤组分的抗氧化活性

Table 5. Antioxidant activities of neutral protease hydrolysates and their ultrafiltration components

	DPPH自由基清除率（%）	Fe²⁺螯合率（%）	还原力
酶解液	69.85±2.52^c	83.71±1.00^b	0.35±0.03^c
F1（<3 kDa）	78.81±1.56^a	91.05±1.65^a	0.47±0.02^a
F2（3~10 kDa）	73.32±0.49^b	83.18±1.16^b	0.42±0.01^b
F3（>10 kDa）	58.51±1.07^d	74.06±1.95^c	0.30±0.07^d
注：同列不同的字母表示差异显著（P<0.05）。

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