龙须菜蛋白质的提取及其酶解产物的抗氧化特性

doi:10.3969/j.issn.1004-1524.2022.05.21

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (5): 1061-1072.DOI: 10.3969/j.issn.1004-1524.2022.05.21

龙须菜蛋白质的提取及其酶解产物的抗氧化特性

刘晶¹^,²(), 胡晓², 杨贤庆²^,^*(), 陈胜军², 吴燕燕², 李来好², 戚勃², 邓建朝²

1.上海海洋大学食品学院,上海 201306
2.中国水产科学研究院南海水产研究所,国家水产品加工技术研发中心,农业农村部水产品加工重点实验室,广东广州 510300

收稿日期:2020-11-16 出版日期:2022-05-25 发布日期:2022-06-06
通讯作者: 杨贤庆
作者简介:* 杨贤庆,E-mail: yxqgd@163.com
刘晶(1996—),女,湖南常德人,硕士研究生,研究方向为水产功能性肽。E-mail: lj15115772810@163.com
基金资助:
现代农业产业技术体系建设专项(CARS-50);广东省现代农业产业技术体系创新团队建设专项资金(2019KJ151);农业农村部水产品加工重点实验室开放基金(NYJG201706);广东省海洋与渔业发展专项资金(技术推广)(2017A0001);国家水产品质量安全风险评估专项(GJFP201800904);广东省重点领域研发计划(2020B1111030004)

Extraction and antioxidant activity of enzymolysis products of Gracilaria lemaneiformis protein

LIU Jing¹^,²(), HU Xiao², YANG Xianqing²^,^*(), CHEN Shengjun², WU Yanyan², LI Laihao², QI Bo², DENG Jianchao²

1. College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306,China
2. Key Laboratory of Aquatic Product Processing, Ministry of Agriculture and Rural Affairs, National Research and Development Center for Aquatic Product Processing, South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510300,China

Received:2020-11-16 Online:2022-05-25 Published:2022-06-06
Contact: YANG Xianqing

摘要/Abstract

摘要：

以干燥后的龙须菜(Gracilaria lemaneiformis)粉为原料,采用超声辅助碱提酸沉法提取龙须菜蛋白质。首先通过单因素实验选择了影响龙须菜蛋白质提取率的因素及水平范围,然后以Box-Behnken中心组合设计原理建立二次响应面回归模型,确定了最佳提取条件为:碱浓度0.2 mol·L^-1、液固比24:1 (mL·g^-1)、超声时间70 min、超声功率482 W,在此条件下的龙须菜蛋白质提取率为73.78%。此外,对提取得到的龙须菜蛋白质进行了酶解,分别研究了木瓜蛋白酶、碱性蛋白酶、植物蛋白复合酶、胰蛋白酶和胃蛋白酶对酶解产物抗氧化活性和分子量的影响。结果表明,在酶解4 h后,碱性蛋白酶酶解产物的抗氧化活性显著高于其他4种酶酶解产物和龙须菜蛋白质,其铁离子还原能力(ferric reducing antioxidant power, FRAP)、1,1-二苯基-2-苦基肼(DPPH)自由基清除率和2,2'-联氮-双(3-乙基苯并噻唑啉-6-磺酸)二铵盐(ABTS)自由基清除率分别为81.88 μg·mL^-1、63.29%、64.25%,分子量主要集中在1 500 u以下。本研究可为龙须菜蛋白质的提取及其高值化利用提供一定参考。

关键词: 龙须菜, 蛋白提取, 酶解产物, 抗氧化活性, 基础特性

Abstract:

The method for extracting protein from dried Gracilaria lemaneiformis powder using ultrasonic assisted alkali dissolution and acid precipitation was optimized by single-factor experiment and Box-Behnken central composite design. The results indicated that the optimal extraction conditions were as follows: alkali concentration 0.2 mol·L^-1, liquid-solid ratio 24:1 (mL·g^-1), ultrasonic time 70 min and ultrasonic power 482 W. The extraction rate of protein was 73.78%. The effects of papain, alkaline protease, plant protein complex enzyme, trypsin and pepsin on antioxidant activity and molecular weight of hydrolysates from G. lemaneiformis were investigated. The results showed that the antioxidant activity of alkaline protease enzymolysis products was significantly higher than that of the other four hydrolysates and G. lemaneiformis protein. After 4 h of hydrolysis, the ferric reducing antioxidant power (FRAP), the DPPH free radical scavenging rate and the ABTS free radical scavenging rate of alkaline protease enzymolysis products were 81.88 μg·mL^-1, 63.29% and 64.25%, respectively. The molecular weight of the hydrolysates was mainly below 1 500 u. This study could provide a reference for protein extraction and high value utilization of G. lemaneiformis.

Key words: Gracilaria lemanensis, protein extraction, enzymolysis products, antioxidant activity, basic characteristic

中图分类号:

TS254.9

刘晶, 胡晓, 杨贤庆, 陈胜军, 吴燕燕, 李来好, 戚勃, 邓建朝. 龙须菜蛋白质的提取及其酶解产物的抗氧化特性[J]. 浙江农业学报, 2022, 34(5): 1061-1072.

LIU Jing, HU Xiao, YANG Xianqing, CHEN Shengjun, WU Yanyan, LI Laihao, QI Bo, DENG Jianchao. Extraction and antioxidant activity of enzymolysis products of Gracilaria lemaneiformis protein[J]. Acta Agriculturae Zhejiangensis, 2022, 34(5): 1061-1072.

图/表 11

参考文献 30

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因素 Factor	水平 Level
因素 Factor	-1	0	1
碱浓度Alkali concentration/(mol·L^-1)	0.15	0.20	0.25
液固比Liquid-solid ratio/(mL·g^-1)	20:1	25:1	30:1
超声时间Ultrasonic time/min	60	70	80
超声功率Ultrasonic power/W	400	500	600

因素 Factor	水平 Level
因素 Factor	-1	0	1
碱浓度Alkali concentration/(mol·L^-1)	0.15	0.20	0.25
液固比Liquid-solid ratio/(mL·g^-1)	20:1	25:1	30:1
超声时间Ultrasonic time/min	60	70	80
超声功率Ultrasonic power/W	400	500	600

种类 Type	加酶量 Enzyme amount/%	时间 Time/h	pH	温度 Temperature/℃	料液比 Solid-liquid ratio/(g·mL^-1)
木瓜蛋白酶Papain	3	4	6.5	50	1:50
碱性蛋白酶Alkaline protease	3	4	8	50	1:50
植物蛋白复合酶Plant protein complex enzyme	3	4	8	50	1:50
胰蛋白酶Trypsin	3	4	8	37	1:50
胃蛋白酶Pepsin	3	4	3	37	1:50

种类 Type	加酶量 Enzyme amount/%	时间 Time/h	pH	温度 Temperature/℃	料液比 Solid-liquid ratio/(g·mL^-1)
木瓜蛋白酶Papain	3	4	6.5	50	1:50
碱性蛋白酶Alkaline protease	3	4	8	50	1:50
植物蛋白复合酶Plant protein complex enzyme	3	4	8	50	1:50
胰蛋白酶Trypsin	3	4	8	37	1:50
胃蛋白酶Pepsin	3	4	3	37	1:50

方差来源 Source	平方和 Sum of squares	自由度 df	均方 Mean squares	F值 F value	P值 P value	显著性 Significant
模型Model	1 277.71	14	91.27	54.45	<0.000 1	***
A	26.14	1	26.14	15.59	0.001 5	**
B	57.73	1	57.73	34.44	<0.000 1	***
C	11.12	1	11.12	6.63	0.022 0	*
D	12.40	1	12.40	7.40	0.016 6	*
AB	8.12	1	8.12	4.85	0.045 0	*
AC	15.44	1	15.44	9.21	0.008 9	**
AD	0.12	1	0.12	0.07	0.793 8
BC	46.99	1	46.99	28.03	0.000 1	***
BD	145.32	1	145.32	86.70	<0.000 1	***
CD	102.21	1	102.21	60.98	<0.000 1	***
A²	77.91	1	77.91	46.48	<0.000 1	***
B²	260.06	1	260.06	155.15	<0.000 1	***
C²	555.38	1	555.38	331.34	<0.000 1	***
D²	337.61	1	337.61	201.42	<0.000 1	***
残差Residual	23.47	14	1.68
失拟项 Lack of fit	17.36	10	1.74	1.14	0.490 4	不显著 Not significant
纯误差Pure error	6.11	4	1.53
总变异Cor total	1 301.18	28

龙须菜蛋白质的提取及其酶解产物的抗氧化特性

Extraction and antioxidant activity of enzymolysis products of Gracilaria lemaneiformis protein

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必需氨基酸 Essential amino acids	含量 Content/ (g·kg^-1)	FAO评分标准模式 FAO scoring standard mode/(mg·g^-1)	全鸡蛋蛋白 Whole egg protein/(mg·g^-1)	氨基酸评分 AAS/%	化学评分 CS/%	非必需氨基酸 Nonessential amino acid	含量 Content/ (g·kg^-1)
缬氨酸Val	53.0	310	410	152.65	115.42	天冬氨酸Asp	77.6
蛋氨酸 Met	24.8	220	386	100.65	57.36	谷氨酸Glu	104.4
赖氨酸 Lys	26.6	340	441	69.85	53.85	丝氨酸Ser	51.9
亮氨酸 Leu	74.8	440	534	151.79	125.07	组氨酸His	2.3
异亮氨酸Ile	48.7	250	331	173.93	131.37	甘氨酸Gly	28.5
苏氨酸Thr	41.1	250	292	146.79	125.67	精氨酸Arg	62.3
苯丙氨酸 Phe	48.1					丙氨酸Ala	56.3
色氨酸Trp	4.1					酪氨酸Try	52.8
						脯氨酸Pro	26.9
苯丙氨酸+酪氨酸		380	565	237.07	159.45	半胱氨酸Cys	90.8
Phe+Tyr
E	321.2					N	553.8

分子量分布 Molecular weight distribution	酶种类The enzyme types
分子量分布 Molecular weight distribution	木瓜蛋白酶 Papain	碱性蛋白酶 Alkaline protease	复合蛋白酶 Complex enzyme	胰蛋白酶 Trypsin	胃蛋白酶 Pepsin
>10 000 u	18.13	0	0	0	0
5 000~<10 000 u	3.36	0.90	0.10	0.86	0
3 000~<5 000 u	3.65	2.05	0.84	2.16	0
1 500~<3 000 u	8.78	6.40	3.89	7.75	0.12
500~<1 500 u	22.99	28.42	25.91	27.86	14.79
<500 u	43.11	62.23	69.27	61.36	85.09

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