纳豆中抗氧化肽的分离纯化与活性研究

doi:10.13560/j.cnki.biotech.bull.1985.2021-0379

摘要/Abstract

摘要：

从发酵的纳豆中提取具有抗氧化活性的多肽,通过分子筛层析和反相高效液相色谱对纳豆上清液进行分离纯化,并采用电喷雾串联质谱进行结构鉴定。结果表明：纳豆发酵后的蛋白（多肽）混合物经Sephadex G-50 凝胶色谱进行分离纯化后得到3个组分（F1、F2和F3）,其中组分F3的抗氧化活性最强,总还原力达到（8.4±0.6）mmol/g,显著高于其他组分;组分F3经半制备RP-HPLC液相色谱分离纯化得到4个组分（G1、G2、G3和G4）,选取含量最高的G1和G3进行活性测定,测得G1的DPPH 自由基清除率达到32.67%。通过 ESI-MS/MS对G1组分抗氧化肽进行一级序列鉴定,得到含量最高和活性最强的10种抗氧化肽序列,并用化学合成的方法对其活性进行验证,得到肽段SFEWVLEH的活性最强,其DPPH清除率为46.66%±0.96%。

关键词: 纳豆, 抗氧化肽, 分离纯化, 鉴定

Abstract:

High antioxidant polypeptides were extracted from fermented natto,the supernatant of the natto were isolated and purified by SephadexG-50 and RP HPLC,and their structures were identified by electrospray ionization tandem mass spectrometry. The results showed that 3 components（F1,F2 and F3）from the polypeptide mixture of fermented natto were obtained after the separation and purification of the antioxidant peptide via Sephadex G-50 gel. The antioxidant activity of the component F3 was the strongest,the total reduction power reached（8.4+0.6）mmol/g,higher than that of other components. F3 was separated and purified by semi preparative RP-HPLC,and 4 components（G1,G2,G3,and G4）were obtained. The G1 and G3 in the highest contents were selected for determining DPPH scavenging rate,and G1’s DPPH free radical scavenging rate reached 32.67% The antioxidant peptides in G1 were sequenced and identified by ESI-MS/MS. and the 10 antioxidant peptides with the highest content and the strongest activity were obtained. The activity of peptide SFEWVLEH was the strongest verified by chemical synthesis method. The DPPH scavenging rate was 46.66% ± 0.96%.

Key words: natto, antioxidant peptide, isolation and purification, identification

张丰文, 周丽亚, 董超, 史延茂. 纳豆中抗氧化肽的分离纯化与活性研究[J]. 生物技术通报, 2022, 38(2): 158-165.

ZHANG Feng-wen, ZHOU Li-ya, DONG Chao, SHI Yan-mao. Purification of Antioxidant Peptides from Natto Supernatant and Study on Its Activity[J]. Biotechnology Bulletin, 2022, 38(2): 158-165.

图/表 10

表1 半制备RP-HPLC的洗脱条件

Table 1 Elution conditions of semi preparative RP-HPLC

时间Time/min	流动相B Mobile phase B%
0-5	5-10
5-35	10-80
35-45	80-100
45-50	100-100
50-55	100-80
55-75	80-10
75-80	10-5

表2 洗脱时间表

Table 2 Elution schedule

时间Time/min	流动相Mobile phase B%
0	4
2	8
45	28
55	40
56	95
66	95

图1 Tricine-SDS-PAGE电泳图 1、2 大豆蛋白上清液;3 纳豆冻干粉;M标准蛋白混合物

Fig. 1 Tricine SDS PAGE electrophoretogram 1 and 2：Soybean protein supernatant. 3：Natto lyophilized powder. M：Standard protein mixture

图2 SephadexG-50凝胶层析图谱

Fig. 2 SephadexG-50 chromatogram

表3 SephadexG-50组分抗氧化活性

Table 3 Antioxidant activities of fractions from Sephade-xG-50 column

组分 Fraction	蛋白或多肽浓度 Protein or peptide concentration/（μg·mL^-1）	FRAP值 FRAP value/ （μmol·L^-1）	每克蛋白的还原力 Reducing power per gram protein/ （mmol·g^-1）
F1	237±7^A	419±10.97^A	3.9±0.5^C
F2	84.4±7.6^B	408±49.5^A	5±0.2^B
F3	50±2^B	422±12.6^A	8.4±0.6^A

图 3 F3 组分的 RP-HPLC图

Fig. 3 RP-HPLC analysis of fraction F3

图 4 G1和G3的DPPH清除率

Fig. 4 DPPH scavenging rates of G1 and G3

图5 G1的各肽段二级质谱图

Fig. 5 Secondary mass spectra of G1 peptides

表4 G1的各肽段信息

Table 4 Peptide information of G1

编号 Code	Peptide < ProteinMetrics Confidential >	Observed （M+H）	Calc. mass（M+H）
1	RAELSEDDVFVIPA	1560.785	1560.790
2	LSEDDVFVIPA	1204.607	1204.610
3	DVFRAIPSEVL	1245.681	1245.684
4	FEEINRVLL	1132.633	1132.636
5	SLLNALPEEVIQH	1462.787	1462.790
6	LAVLI	528.375	528.376
7	SFEWVLEH	1046.493	1046.494
8	GIFGM	540.248	540.249
9	QVFL	506.296	506.297
10	FPF	410.207	410.207

图6 各肽段抗氧化活性

Fig. 6 Antioxidant activity of each peptide segment

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