燕麦麸皮蛋白抗氧化肽制备工艺优化及分离纯化

doi:10.3969/j.issn.1004-1524.20240542

浙江农业学报 ›› 2025, Vol. 37 ›› Issue (9): 1958-1968.DOI: 10.3969/j.issn.1004-1524.20240542

燕麦麸皮蛋白抗氧化肽制备工艺优化及分离纯化

李华鑫¹^,²(), 杨炫康¹, 陈颖¹^,², 吴晓婷², 刘思恋², 杨忠³, 周成刚³, 高丹丹¹^,²^,^*()

¹ 西北民族大学生物医学研究中心, 中国-马来西亚国家联合实验室, 甘肃兰州 730030
² 西北民族大学生命科学与工程学院, 甘肃兰州 730124
³ 兰州市第二人民医院, 甘肃兰州 730046

收稿日期:2024-06-22 出版日期:2025-09-25 发布日期:2025-10-15
作者简介:*高丹丹,E-mail: gaodan0322@163.com
李华鑫(1998—),男,河南周口人,硕士研究生,研究方向为食源性生物活性肽。E-mail: 1224094494@qq.com
通讯作者: 高丹丹
基金资助:
甘肃省高校青年博士支持项目(2023QB-001);甘肃省高校青年博士支持项目(甘肃省2024年重点人才项目);兰州市青年科技人才创新项目(2023-QN-69);甘肃省高等教育教学成果培育项目(2022GSJXCGI09);西北民族大学校级创新创业教育示范专业项目(2022XJCXCYSFZY01);甘肃省科技厅技术创新引导计划科技专员专项(23CXGA0078);西北民族大学中央高校基本科研业务费项目(3192024125-01);甘肃省科技厅技术创新引导计划科技型中小企业技术创新基金(23CXGP0002)

Optimization of the preparation process and separation purification of antioxidant peptides from oat bran protein

LI Huaxin¹^,²(), YANG Xuankang¹, CHEN Ying¹^,², WU Xiaoting², LIU Silian², YANG Zhong³, ZHOU Chenggang³, GAO Dandan¹^,²^,^*()

¹ China-Malaysia National Joint Laboratory, Biomedical Research Center, Northwest Minzu University, Lanzhou 730030, China
² College of Life Science and Engineering, Northwest Minzu University, Lanzhou 730124, China
³ The No.2 People's Hospital of Lanzhou, Lanzhou 730046, China

Received:2024-06-22 Online:2025-09-25 Published:2025-10-15
Contact: GAO Dandan

摘要/Abstract

摘要：

为提高燕麦麸皮蛋白的综合利用价值,本研究通过中性蛋白酶水解燕麦麸皮蛋白制备抗氧化肽,以2,2-二苯基-1-苦基肼(DPPH)自由基清除率为指标,考察酶解时间、酶解温度、pH值和燕麦麸皮蛋白质量分数对水解效果的影响,在单因素试验的基础上结合响应面法优化工艺条件。结果显示,当燕麦麸皮蛋白质量分数为6.0%,pH值为7.5,酶解温度为45℃,酶解时间为4.5 h时,DPPH自由基清除率为(73.78±0.67)%。将酶解产物通过超滤分离出4个组分,其中F4组分清除活性最强。采用葡聚糖凝胶Sephadex G-25柱层析后分离出3个组分,并以DPPH自由基、羟基自由基(·OH)及超氧自由基(${O}_{2}^{·-}$)的清除率为指标,检测分离组分的抗氧化活性。研究发现,Sephadex G-25分离的E2组分抗氧化活性最高,其DPPH自由基、羟基自由基(·OH)和超氧自由基(${O}_{2}^{·-}$)的清除率分别达到(84.8±0.5)%、(81.6±0.7)%和(80.1±0.7)%。结果表明,中性蛋白酶能有效酶解燕麦麸皮蛋白制备抗氧化肽,且抗氧化肽经分离纯化后可显著提高抗氧化活性,为燕麦麸皮蛋白活性肽的制备和提升其附加值提供参考依据。

关键词: 中性蛋白酶, 燕麦麸皮蛋白, 抗氧化肽, DPPH自由基

Abstract:

To enhance the comprehensive application value of oat bran protein, this study prepared antioxidant peptides by enzymatic hydrolysis of oat bran protein with neutral protease. The DPPH free radical scavenging rate was used as an indicator to investigate the effects of enzymolysis time, enzymolysis temperature, pH value, and mass fraction of oat bran protein on the hydrolysis efficiency. Based on single-factor experiments, response surface methodology was employed to optimize the process conditions. The results showed that when the mass fraction of oat bran protein was 6.0%, pH value was 7.5, the enzymolysis temperature was 45 ℃, and the enzymolysis time was 4.5 h, the DPPH free radical scavenging rate was (73.78±0.67)%. The enzymatic hydrolysates were separated into four fractions by ultrafiltration, with the F4 fraction exhibiting the highest scavenging ability. Further purification using Sephadex G-25 gel filtration chromatography yielded three sub-fractions, which were assessed for their antioxidant activities based on their abilities to scavenge DPPH free radicals, hydroxyl radicals(·OH), and superoxide radicals (${O}_{2}^{·-}$). The study found that the E2 sub-fraction separated by Sephadex G-25 had the highest antioxidant activity, with the scavenging ability of DPPH radicals, hydroxyl radicals(·OH), and superoxide radicals (${O}_{2}^{·-}$) were (84.8±0.5)%, (81.6±0.7)%, and (80.1±0.7)%. These findings indicate that neutral protease can effectively hydrolyze oat bran protein to produce antioxidant peptides, and the antioxidant activity of these peptides can be significantly enhanced through separation and purification processes. This study provides a reference for the preparation of bioactive peptides from oat bran protein and offers a basis for enhancing their functional value.

Key words: neutral protease, oat bran protein, antioxidant peptides, DPPH free radical

中图分类号:

TS21

李华鑫, 杨炫康, 陈颖, 吴晓婷, 刘思恋, 杨忠, 周成刚, 高丹丹. 燕麦麸皮蛋白抗氧化肽制备工艺优化及分离纯化[J]. 浙江农业学报, 2025, 37(9): 1958-1968.

LI Huaxin, YANG Xuankang, CHEN Ying, WU Xiaoting, LIU Silian, YANG Zhong, ZHOU Chenggang, GAO Dandan. Optimization of the preparation process and separation purification of antioxidant peptides from oat bran protein[J]. Acta Agriculturae Zhejiangensis, 2025, 37(9): 1958-1968.

图/表 11

表1 响应面试验因素水平

Table 1 Response surface test factor levels

水平 Levels	各因素的设定值Setting value of factors
水平 Levels	(A)燕麦麸皮蛋白质量分数 Mass fraction of oat bran protein/%	(B)pH值 pH value	(C)酶解温度 Enzymolysis temperature/℃	(D)酶解时间 Enzymolysis time/h
-1	5.0	7.0	40	4.0
0	6.0	7.5	45	4.5
1	7.0	8.0	50	5.0

图1 酶解时间对水解度和DPPH自由基清除率的影响不同处理间没有相同字母表示差异显著(p<0.05)。下同。

Fig.1 Effect of enzymolysis time on degree of hydrolysis and DPPH free radical scavenging rate The values marked without the same lowercase letter indicate the significant(p<0.05) difference. The same as below.

图2 酶解温度对水解度和DPPH自由基清除率的影响

Fig.2 Effect of enzymolysis temperature on degree of hydrolysis and DPPH free radical scavenging rate

图3 pH值对水解度和DPPH自由基清除率的影响

Fig.3 Effect of pH value on degree of hydrolysis and DPPH free radical scavenging rate

图4 燕麦麸皮蛋白质量分数对水解度和DPPH自由基清除率的影响

Fig.4 Effect of mass fraction of oat bran protein on degree of hydrolysis and DPPH free radical scavenging rate

表2 响应面试验设计及结果

Table 2 Response surface test design and test results

序号 Number	各因素的水平Level of factors				DPPH自由基清除率 DPPH free radical scavenging rate/%
序号 Number	A	B	C	D	DPPH自由基清除率 DPPH free radical scavenging rate/%
1	-1	-1	0	0	66.52±0.63
2	1	-1	0	0	67.24±0.72
3	-1	1	0	0	65.13±0.89
4	1	1	0	0	64.31±0.74
5	0	0	-1	-1	60.13±0.66
6	0	0	1	-1	67.39±0.88
7	0	0	-1	1	65.82±0.81
8	0	0	1	1	63.09±0.79
9	-1	0	0	-1	62.89±0.81
10	1	0	0	-1	65.47±0.74
11	-1	0	0	1	66.89±0.77
12	1	0	0	1	63.24±0.69
13	0	-1	-1	0	68.35±0.89
14	0	1	-1	0	60.26±0.85
15	0	-1	1	0	63.09±0.87
16	0	1	1	0	69.81±0.68
17	-1	0	-1	0	64.47±0.73
18	1	0	-1	0	63.66±0.76
19	-1	0	1	0	65.05±0.65
20	1	0	1	0	66.21±0.87
21	0	-1	0	-1	66.45±0.81
22	0	1	0	-1	63.29±0.85
23	0	-1	0	1	66.03±0.64
24	0	1	0	1	64.28±0.63
25	0	0	0	0	73.24±0.74
26	0	0	0	0	73.89±0.79
27	0	0	0	0	74.31±0.84
28	0	0	0	0	73.26±0.82
29	0	0	0	0	72.80±0.75

表3 模型方差分析结果

Table 3 Results of variance analysis of quadratic multinomial simulation

变异源 Sources	平方和 Sum of squares	自由度 Degree of freedom	均方 Mean square	F值 F value	p值 p value	显著性 Significance
总模型Model	422.65	14	30.19	106.36	<0.000 1	**
A	0.056	1	0.056	0.20	0.663 6
B	9.36	1	9.36	32.99	<0.000 1	**
C	11.90	1	11.90	41.92	<0.000 1	**
D	1.16	1	1.16	4.08	0.062 8
AB	0.59	1	0.59	2.09	0.170 4
AC	0.97	1	2.21	3.42	0.085 7
AD	9.70	1	9.70	34.18	<0.000 1	**
BC	54.83	1	54.83	193.18	<0.000 1	**
BD	0.50	1	0.50	1.75	0.207 0
CD	24.95	1	24.95	87.90	<0.000 1	**
A²	107.78	1	107.78	379.71	<0.000 1	**
B²	84.83	1	84.83	298.84	<0.000 1	**
C²	133.99	1	133.99	472.06	<0.000 1	**
D²	151.95	1	151.95	535.33	<0.000 1	**
残差Residual	3.97	14	0.28
失拟项Lack of fit	2.55	10	0.26	0.72	0.696 1
误差项Pure error	1.42	4	0.36
总和Cor total	426.62	28
R²	0.990 7	${R}_{Adj}^{2}$	0.981 4

图5 燕麦麸皮蛋白质量分数(A)、pH值(B)、酶解温度(C)和酶解时间(D)对DPPH自由基清除率交互影响的曲面图 a为燕麦麸皮蛋白质量分数与pH值交互曲面图(AB);b为燕麦麸皮蛋白质量分数与酶解温度交互曲面图(AC);c为燕麦麸皮蛋白质量分数与酶解时间交互曲面图(AD);d为pH值与酶解温度交互曲面图(BC);e为pH值与酶解时间交互曲面图(BD);f为酶解温度与酶解时间交互曲面图(CD)。

Fig.5 Surface map of the interaction between mass fraction of oat bran protein(A), pH value(B), enzymolysis temperature(C) and enzymolysis time(D) on DPPH free radical scavenging rate a is the interaction curve graph between mass fraction of oat bran protein and pH value (AB); b is the interaction curve graph between mass fraction of oat bran protein and enzymolysis temperature (AC); c is the interaction curve graph between mass fraction of oat bran protein and enzymolysis time (AD); d is the interaction curve graph between pH value and enzymolysis temperature (BC); e is the interaction curve graph between pH value and enzymolysis time (BD); f is the interaction curve graph between enzymolysis temperature and enzymolysis time (CD).

表4 超滤组分与酶解液的抗氧化活性

Table 4 Antioxidant activity of ultrafiltration fractions and enzymatic digests

组 Group	自由基清除能力Free radical scavenging ability/%
组 Group	DPPH自由基 DPPH radical	羟基自由基 Hydroxyl radical	超氧自由基 Superoxide radical
F1	65.80±0.82 d	45.90±0.94 d	61.20±0.91 d
F2	72.60±0.62 c	62.80±1.41 b	68.90±1.24 b
F3	78.90±1.02 b	75.30±0.47 a	76.40±0.93 a
F4	81.50±1.08 a	76.10±0.92 a	76.40±0.47 a
F5	73.70±0.85 c	59.40±0.95 c	65.90±0.89 c

图6 F4组分Sephadex G-50分离图谱和S1组分抗氧化活性

Fig.6 Separation spectrum of F4 component Sephadex G-50 and antioxidant activity of S1 component

图7 S1组分Sephadex G-25分离图谱和3种组分抗氧化活性

Fig.7 Separation spectrum of S1 component Sephadex G-25 and antioxidant activity of the three components

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燕麦麸皮蛋白抗氧化肽制备工艺优化及分离纯化

Optimization of the preparation process and separation purification of antioxidant peptides from oat bran protein

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