黄鳍金枪鱼酶解物免疫活性及其氨基酸分析

doi:10.11978/2020136

热带海洋学报 ›› 2021, Vol. 40 ›› Issue (6): 128-134.doi: 10.11978/2020136CSTR: 32234.14.2020136

黄鳍金枪鱼酶解物免疫活性及其氨基酸分析

巫楚君¹(), 潘剑宇², 蔡冰娜², 陶曙红¹()

1.广东药科大学中药学院, 广东广州 510006
2.中国科学院南海海洋研究所热带海洋生物资源与生态重点实验室, 广东广州 510301

收稿日期:2020-11-18 修回日期:2020-12-27 出版日期:2021-11-10 发布日期:2021-01-04
通讯作者: 陶曙红
作者简介:巫楚君(1995—), 女, 广东省清远市人, 硕士研究生, 主要从事中药提取分离技术与应用。email: 511786458@qq.com
基金资助:
广东省重点领域研发计划(2020B1111030004);国家重点研发计划项目(2018YFC0311202);广东省重点领域研发计划资助(2020B1111030004);广东省自然科学基金(2018A030313088);广东省自然科学基金(2018A0303130144);广东省自然科学基金(2018A030313626)

Immunoactivity and Amino Acid Analysis of Enzymatic Hydrolysates of Thunnus albacares

WU Chujun¹(), PAN Jianyu², CAI Bingna², TAO Shuhong¹()

1. School of Traditional Chinese Medicine, Guangdong Pharmaceutical University, Guangzhou 510006, China
2. Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China

Received:2020-11-18 Revised:2020-12-27 Online:2021-11-10 Published:2021-01-04
Contact: TAO Shuhong
Supported by:
Research and Development Plans in Key Areas of Guangdong Province(2020B1111030004);National Key Research and Development Program of China(2018YFC0311202);Research and Development Plans in Key Areas of Guangdong Province(2020B1111030004);Natural Science Foundation of Guangdong Province(2018A030313088);Natural Science Foundation of Guangdong Province(2018A0303130144);Natural Science Foundation of Guangdong Province(2018A030313626)

摘要/Abstract

摘要：

本文旨在研究黄鳍金枪鱼(Thunnus albacares)碎肉中4种不同蛋白酶酶解物的3k Da超滤组分对RAW264.7细胞的免疫活性。以RAW264.7细胞为实验模型, 采用噻唑蓝比色法检测细胞增殖活性, 中性红吞噬实验检测细胞吞噬能力, Griess试剂检测细胞培养上清液中NO含量, 并分析酶解物氨基酸组成和分子量分布。实验结果表明, 胰蛋白酶酶解物在12.5~200μg·mL^-1范围内无明显细胞毒性, 能增强RAW264.7细胞吞噬能力, 浓度为400μg·mL^-1时达到最大值132.50%(P<0.01), 浓度在50~200μg·mL^-1范围内能促进巨噬细胞NO释放(P<0.01), 且NO释放量与酶解液浓度存在一定的量效关系; 胰蛋白酶酶解物中必需氨基酸(essential amino acid, EAA)含量最高, 为51.09%, 其碱性氨基酸组氨酸、赖氨酸和精氨酸的含量分别为11.59%、11.00%和6.90%, 均高于其他蛋白酶酶解物, 并且分子量小于1k Da的小肽含量高达97.48%。研究结果表明, 4种蛋白酶酶解物中, 黄鳍金枪鱼碎肉胰蛋白酶酶解物的3k Da组分具有较明显的免疫活性, 这为其作为免疫活性肽的开发利用提供了理论依据。

关键词: 黄鳍金枪鱼, 多肽, 免疫活性, 氨基酸

Abstract:

In this study, we evaluated the immune activity of ultrafiltration components from yellowfin tuna (Thunnus albacares) meat hydrolyzed by four different proteases on RAW264.7 cells. The RAW264.7 cell line was used as the cell activity screening model in vitro. Methyl thiazolyl tetrazolium (MTT), neutral red and Griess reagent methods were used to detect cell activity, phagocytosis capability and nitric oxide levels in cell culture medium. The amino-acids composition and molecular weight distribution of the hydrolysate were also analyzed. The results show that trypsin hydrolysate had no obvious cytotoxicity in the range of 12.5-200 μg·mL^-1 for RAW264.7 cells. It could enhance the phagocytic capacity, and reached the maximum value of 132.50% (P<0.01) at the concentration of 400 μg·mL^-1. In the concentration range of 50-200 μg·mL ^-1, the trypsin hydrolysate could promote nitric oxide release of macrophages (P<0.01), and there was a dose-effect relationship between NO release and the concentration of enzymolysis solution. The content of essential amino acid (EAA, 51.09%) of trypsin hydrolysate was higher than those of the other enzymatic hydrolysates, and the contents of histidine (His, 11.59%), lysine (Lys, 11.00%) and arginine (Arg, 6.90%) were also higher than those of the other enzymatic hydrolysates. The content of small peptides with molecular weight less than 1k Da was as high as 97.48% in trypsin hydrolysate. In the four protease hydrolysates, the 3k Da peptide fraction of trypsin hydrolysate of tuna meat had obvious immune activity, which provides a theoretical basis for its development and utilization as a functional ingredient.

Key words: Thunnus albacares, polypeptide, immunomodulatory activity, amino acid

中图分类号:

TS254.1

巫楚君, 潘剑宇, 蔡冰娜, 陶曙红. 黄鳍金枪鱼酶解物免疫活性及其氨基酸分析[J]. 热带海洋学报, 2021, 40(6): 128-134.

WU Chujun, PAN Jianyu, CAI Bingna, TAO Shuhong. Immunoactivity and Amino Acid Analysis of Enzymatic Hydrolysates of Thunnus albacares[J]. Journal of Tropical Oceanography, 2021, 40(6): 128-134.

图/表 7

表1

图1

图2

图3

图4

表2

表3

参考文献 23

[1]	白生宾, 陈红香, 钟近洁, 等, 2011. MTT法检测RAW264.7细胞活力及可能因素分析[J]. 中国现代医学杂志, 21(23):2831-2833.
	BAI SHENGBIN, CHEN HONGXIANG, ZHONG JINJIE, et al, 2011. RAW264.7 cell viability via MTT assay and possible factor analysis[J]. China Journal of Modern Medicine, 21(23):2831-2833 (in Chinese with English abstract).
[2]	杜帅, 宋茹, 罗红宇, 2014. 水解金枪鱼碎肉制备高F值寡肽[J]. 中国食品学报, 14(10):124-133.
	DU SHUAI, SONG RU, LUO HONGYU, 2014. High fischer ratio oligo-peptide from hydrolysates of tuna meat[J]. Journal of Chinese Institute of Food Science and Technology, 14(10):124-133 (in Chinese with English abstract).
[3]	纪丽娜, 2012. 金枪鱼头酶解物免疫活性肽的分离及对小鼠腹腔巨噬细胞功能的影响[D]. 湛江: 广东海洋大学: 9-10.
	JI LINA, 2012. Studies on separation of immune activity from tuna head hydrolysates and its effect on mouse peritoneal macrophages[D]. Zhanjiang: Guangdong Ocean University: 9-10 (in Chinese with English abstract).
[4]	农业农村部渔业渔政管理局, 全国水产技术推广总站, 中国水产学会, 2019. 中国渔业统计年鉴[M]. 北京: 中国农业出版社: 38-40(in Chinese).
[5]	王晓芳, 陈同辛, 2013. 氨基酸与免疫功能的关系[J]. 国际儿科学杂志, 40(1):28-31.
	WANG XIAOFANG, CHEN TONGXIN, 2013. Amino acids and immune functions[J]. International Journal of Pediatrics, 40(1):28-31 (in Chinese with English abstract).
[6]	杨萍, 蔡本利, 洪鹏志, 等, 2009. 黄鳍金枪鱼头蛋白酶解液及其分级组分对小鼠免疫功能的影响[J]. 食品与生物技术学报, 28(1):131-135.
	YANG PING, CAI BENLI, HONG PENGZHI, et al, 2009. Effect of tuna head enzymatic hydrolysate and its portions on inmmune function in mice[J]. Journal of Food Science and Biotechnology, 28(1):131-135 (in Chinese with English abstract).
[7]	杨萍, 蔡本利, 洪鹏志, 2010. 黄鳍金枪鱼头蛋白酶解液及分级组分对小鼠脾淋巴细胞增殖的影响[J]. 天然产物研究与开发, 22(3):498-501.
	YANG PING, CAI BENLI, HONG PENGZHI, 2010. Effect of tuna head enzymatic hydrolysate and its portions on mouse spleen lymphocyte proliferation[J]. Natural Product Research and Development, 22(3):498-501 (in Chinese with English abstract).
[8]	叶盛旺, 杨最素, 李维, 等, 2019. 青蛤酶解多肽对RAW264.7巨噬细胞的免疫调节作用[J]. 食品科学, 40(7):185-191.
	YE SHENGWANG, YANG ZUISU, LI WEI, et al, 2019. Immunomodulatory effects of peptides from enzymatic hydrolysate of Cyclina sinensis on RAW264.7 macrophages[J]. Food Science, 40(7):185-191 (in Chinese with English abstract).
[9]	张爱琳, 段筱筠, 任斐, 等, 2016. 牛乳中主要过敏原的分离及其氨基酸成分分析[J]. 中国乳品工业, 44(12):4-6, 10.
	ZHANG AILIN, DUAN XIAOJUN, REN FEI, et al, 2016. Study the separation on the main allergens in milk and its amino acid composition analysis[J]. China Dairy Industry, 44(12):4-6, 10 (in Chinese with English abstract).
[10]	张彩梅, 张红梅, 于业军, 等, 2005. 扇贝多肽对小鼠免疫功能调节的研究[J]. 中国海洋药物, 24(3):18-21.
	ZHANG CAIMEI, ZHANG HONGMEI, YU YEJUN, et al, 2005. Immunomodulation of polypeptides from Chlamys farreri (PCF) in mice[J]. Chinese Journal of Marine Drugs, 2005, 24(3):18-21 (in Chinese with English abstract).
[11]	张朋, 贺卯苏, 迟长凤, 等, 2014. 金枪鱼(Katsuwonus pelamis)碎肉蛋白降压肽的酶解制备及活性研究[J]. 海洋与湖沼, 45(5):1092-1098.
	ZHANG PENG, HE MAOSU, CHI CHANGFENG, et al, 2014. Preparation and characterisation of an antihypertensive peptide from protein hydrolysate of askipjack tuna (Katsuwonus pelamis) ground meat[J]. Oceanologia et Limnologia Sinica, 45(5):1092-1098 (in Chinese with English abstract).
[12]	GHOSH S, PELLETT P L, AW-HASSAN A, et al, 2008. Impact of lysine-fortified wheat flour on morbidity and immunologic variables among members of rural families in northwest Syria[J]. Food and Nutrition Bulletin, 29(3):163-171. doi: 10.1177/156482650802900302
[13]	GUERARD F, GUIMAS L, BINET A, 2002. Production of tuna waste hydrolysates by a commercial neutral protease preparation[J]. Journal of Molecular Catalysis B: Enzymatic, 19- 20:489-498.
[14]	HAN YUNA, BYUN S H, PARK J H, et al, 2015. Bioactive properties of enzymatic hydrolysates from abdominal skin gelatin of yellowfin tuna (Thunnus albacares)[J]. International Journal of Food Science and Technology, 50(9):1996-2003. doi: 10.1111/ijfs.2015.50.issue-9
[15]	HE WEIWEI, SU GUOWAN, SUN-WATERHOUSE D, et al, 2019. In vivo anti-hyperuricemic and xanthine oxidase inhibitory properties of tuna protein hydrolysates and its isolated fractions[J]. Food Chemistry, 272:453-461. doi: 10.1016/j.foodchem.2018.08.057
[16]	HOU HU, FAN YAN, LI BAFANG, et al, 2012. Preparation of immunomodulatory hydrolysates from Alaska pollock frame[J]. Journal of the Science of Food and Agriculture, 92(15):3029-3038. doi: 10.1002/jsfa.5719
[17]	HOU HU, FAN YAN, WANG SHIKAI, et al, 2016. Immunomodulatory activity of Alaska pollock hydrolysates obtained by glutamic acid biosensor- Artificial neural network and the identification of its active central fragment[J]. Journal of Functional Foods, 24:37-47. doi: 10.1016/j.jff.2016.03.033
[18]	LIN HUIWEN, LIU CHENGWEI, YANG D J, et al, 2017. Dunaliella salina alga extract inhibits the production of interleukin-6, nitric oxide, and reactive oxygen species by regulating nuclear factor-κB/Janus kinase/signal transducer and activator of transcription in virus-infected RAW264.7 cells[J]. Journal of Food and Drug Analysis, 25(4):908-918. doi: 10.1016/j.jfda.2016.11.018
[19]	LIND M, HAYES A, CAPMDA M, et al, 2017. Inducible nitric oxide synthase: Good or bad?[J]. Biomedicine & Pharmacotherapy, 93:370-375. doi: 10.1016/j.biopha.2017.06.036
[20]	MIZOCK B A, 2010. Immunonutrition and critical illness: an update[J]. Nutrition, 26(7-8):701-707. doi: 10.1016/j.nut.2009.11.010
[21]	PEZESHK S, OJAGH S M, REZAEI M, et al, 2019. Fractionation of protein hydrolysates of fish waste using membrane ultrafiltration: Investigation of antibacterial and antioxidant activities[J]. Probiotics and Antimicrobial Proteins, 11(3):1015-1022. doi: 10.1007/s12602-018-9483-y
[22]	SUTANBAWA Y, AKNES A, 2006. Tuna process waste an unexploited resource[J]. Infofish International, 3:37-40.
[23]	YANG FAN, COMTOIS A S, FANG LIWEI, et al, 2002. Nitric oxide-derived nitrate anion contributes to endotoxic shock and multiple organ injury/dysfunction[J]. Critical Care Medicine, 30(3):650-657. doi: 10.1097/00003246-200203000-00026

样品组别	酶种类	料液比(V/V)	pH	水解温度/℃	酶量/( U·g^-1)	水解时长/h
TPP	胃蛋白酶	1:3	2.0	37	2500	6
TPT	胰蛋白酶		8.0	37	3000	6
TPA	碱性蛋白酶		8.0	50	3000	4
TPPT	胃蛋白酶、胰蛋白酶		2.0、8.0	37	2500、3000	2、4

样品	氨基酸组成/%
样品	Asp	Thr	Ser	Glu	Gly	Ala	Val	Met	Ile	Leu	Tyr	Phe	Lys	His	Arg	Pro	Trp	EAA
TPA	9.49	4.84	4.16	14.09	5.08	6.47	5.01	3.14	4.21	8.11	3.66	3.85	9.39	8.43	5.97	3.19	0.92	47.89
TPP	9.95	4.81	4.08	13.61	4.49	6.03	5.35	3.49	4.69	8.68	4.22	4.63	6.85	9.95	5.25	2.81	1.12	49.56
TPPT	9.59	4.94	4.24	13.23	4.99	6.45	5.48	3.09	4.69	8.49	1.90	4.52	9.58	7.90	6.23	3.40	1.27	49.95
TPT	8.57	4.58	3.27	12.03	4.70	6.41	4.73	2.91	3.74	7.47	3.88	4.05	11.00	11.59	6.90	3.14	1.02	51.09

样品组别	各组分占峰面积比值/%
样品组别	2~3k Da	1~2k Da	0.5~1k Da	<0.5k Da
TPP	2.66	22.77	28.50	46.08
TPT	0.00	2.53	24.24	73.24
TPA	0.00	4.23	36.39	59.39
TPPT	0.00	0.00	11.92	88.08

黄鳍金枪鱼酶解物免疫活性及其氨基酸分析

Immunoactivity and Amino Acid Analysis of Enzymatic Hydrolysates of Thunnus albacares

RichHTML

PDF (PC)

赞

可视化

摘要/Abstract

引用本文

使用本文

图/表 7

参考文献 23

相关文章 6

Metrics

本文评价

推荐阅读 0

[1]	吴雪萍, 叶红霞, 姚又菊, 刘浩翔, 李瑞华, 童潼. 3个地区可口革囊星虫营养成分及重金属含量的分析与评价[J]. 热带海洋学报, 2024, 43(2): 92-107.
[2]	江志坚, Chanaka Isuranga PREMARATHNE, 方扬, 林基桢, 吴云超, 刘松林, 黄小平. 铵态氮加富对贝克喜盐草光合作用、谷氨酰胺合成酶和氨基酸成分的影响[J]. 热带海洋学报, 2023, 42(3): 116-125.
[3]	李云凯, 陈子昂, 贡艺, 陈新军. 海洋动物营养生态位研究方法及其应用[J]. 热带海洋学报, 2021, 40(4): 143-156.
[4]	陈峰, 郭爱, 朱文斌, 周永东, 徐汉祥, 张龙. 南太平洋所罗门群岛海域黄鳍金枪鱼繁殖生物学研究^*[J]. 热带海洋学报, 2014, 33(2): 45-51.
[5]	张立娟,何毛贤. 生长激素与胰岛素样生长因子 - Ⅰ对马氏珠母贝壳生长形成相关基因表达的影响[J]. 热带海洋学报, 2012, 31(2): 96-101.
[6]	区又君,李加儿. 野生波纹唇鱼Cheilinus undulates营养成分分析与评价[J]. 热带海洋学报, 2010, 29(3): 97-102.