罗非鱼皮胶原肽的酪氨酸酶抑制活性与铜离子结合活性研究

宋玉琼; 胡晓; 相欢; 陈胜军; 李来好; 杨贤庆; 吴燕燕; 马海霞

doi:10.12131/20220097

罗非鱼皮胶原肽的酪氨酸酶抑制活性与铜离子结合活性研究

doi: 10.12131/20220097

宋玉琼^{1, 2,},
胡晓^{2, 3, ,},
相欢^{2, 3},
陈胜军^{2, 3},
李来好^{2, 3},
杨贤庆^{2, 3},
吴燕燕^{2, 3},
马海霞^{2, 3}

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/农业农村部水产品加工重点实验室，广东广州 510300
3.
大连工业大学/海洋食品精深加工关键技术省部共建协同创新中心，辽宁大连 116034

基金项目: 广东省重点领域研发计划资助项目(2020B020226005)；国家特色淡水鱼产业技术体系 (CARS-46)；广东省基础与应用基础研究基金 (2019A1515011588)；茂名市引进创新创业团队项目 (200201095834980); 广东省企业科技特派员专项 (GDKTP2021035300)；中国水产科学研究院中央级公益性科研院所基本科研业务费专项资金资助 (2020TD69)；中国水产科学研究院南海水产研究所中央级公益性科研院所基本科研业务费专项资金资助 (2021SD06)

详细信息

作者简介:
宋玉琼 (1997—)，女，硕士研究生，研究方向为水产品加工与质量安全、食品生物技术。E-mail: SYQ1010730529@163.com

通讯作者:
胡　晓 (1981—)，男，研究员，博士，研究方向为水产品加工与质量安全、食品生物技术。E-mail: hnhuxiao@163.com

中图分类号: TS 254.1
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- 文章访问数: 207
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- 被引次数: 0
出版历程
- 收稿日期: 2022-04-07
- 修回日期: 2022-06-06
- 录用日期: 2022-07-19
- 网络出版日期: 2022-07-27
- 刊出日期: 2023-02-03

Study on tyrosinase inhibitory activity and Cu²⁺ binding activity of tilapia skin peptides

SONG Yuqiong^{1, 2
,},
HU Xiao^{2, 3
, ,},
XIANG Huan^{2, 3},
CHEN Shengjun^{2, 3},
LI Laihao^{2, 3},
YANG Xianqing^{2, 3},
WU Yanyan^{2, 3},
MA Haixia^{2, 3}

1.
College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China
2.
South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences/Key Laboratory of Aquatic Product Processing, Ministry of Agriculture and Rural Affairs, Guangzhou 510300, China
3.
Dalian Polytechnic University/Collaborative Innovation Center of Provincial and Ministerial Co-Construction for Marine Food Deep Processing, Dalian 116034, China

摘要

摘要: 为探明罗非鱼 (Oreochromis) 鱼皮胶原蛋白肽能否有效抑制黑色素的生成，采用酶解法制备出罗非鱼皮酪氨酸酶抑制肽 (Tilapia skin tyrosinase inhibitory peptides, TSTIP)，并开展其酪氨酸酶 (Tyrosinase, TYR) 抑制活性与铜离子 (Cu²⁺) 结合活性的相关性研究。结果显示，罗非鱼皮经碱性蛋白酶酶解4 h后的产物具有最高的TYR抑制活性和Cu²⁺结合活性，且两者呈极显著正相关 (R=0.856)。荧光光谱分析显示，TSTIP与TYR或Cu²⁺结合后其内源荧光吸收强度的下降趋势一致，同时紫外吸收增强，且最大吸收波长发生红移。傅里叶变换红外光谱分析显示，TYR与Cu²⁺主要通过羰基及氨基与TSTIP结合。圆二色谱分析显示，两种结合物中β-转角及无规则卷曲含量相对减少，β-折叠含量相对增多，该现象在TSTIP-Cu²⁺结合物中更加明显。结果表明，TSTIP结合TYR产生的结构变化与结合Cu²⁺相似，TSTIP可通过螯合TYR的活性中心Cu²⁺来抑制其活性。
- 罗非鱼皮 /
- 胶原蛋白肽 /
- 酪氨酸酶 /
- 肽-Cu²⁺结合物 /
- 结构特征
Abstract: To find out whether the tilapia (Oreochromis) skin collagen peptides can effectively inhibit melanin production, we used enzymatic method to prepare the tilapia skin tyrosinase (TYR) inhibitory peptides (TSTIP) and studied the relevance between TYR inhibitory activity and Cu²⁺ binding activity. The results show that the product of tilapia skin hydrolyzed by alcalase for 4 h exhibited both the highest TYR inhibitory activity and Cu²⁺ binding activity, which were significantly positively correlated (R=0.856). When TSTIP was bound to Cu²⁺ or tyrosinase, the intrinsic fluorescence absorption had a consistent decreasing trend, but the UV absorption increased and the maximum absorption wavelength had a red shift. For FTIR results, TYR and Cu²⁺ bound to TSTIP mainly by carbonyl and amino groups. Circular dichroism shows that the β-turn and random curl contents of the two conjugates decreased relatively, while the β-fold content increased relatively, which was more obvious for TSTIP-Cu²⁺ conjugate. In conclusion, the structural change of TSTIP-TYR is similar with that of TSTIP-Cu²⁺ conjugate, which indicates that TSTIP can inhibit TYR's activity by binding to its Cu²⁺ active site.
- Tilapia skin /
- Collagen peptides /
- Tyrosinase /
- Peptide-Cu²⁺ conjugates /
- Structural characteristics

HTML全文

图 1 不同酶解物的TYR抑制率与Cu²⁺结合率和相关性分析

注：a. 酶解物的单酚酶抑制活性；b. 酶解物的二酚酶抑制活性；c. 酶解物的Cu²⁺结合率；d. TSTIP二酚酶抑制活性与Cu²⁺结合率和相关性分析；不同小写字母表示同种蛋白酶在不同酶解时间所得酶解物的抑制率差异显著 (P<0.05)；不同大写字母表示不同蛋白酶在同一酶解时间所得酶解物的抑制率差异显著 (P<0.05)。

Figure 1. Tyrosinase inhibition rate and Cu²⁺ binding rate of different hydrolysates and correlation analysis

Note: a. Monophenolase inhibitory activity of hydrolysates; b. Diphenolase inhibitory activity of hydrolysates; c. Cu²⁺ binding rate of hydrolysates; d. Correlation analysis between tyrosinase inhibitory activity and Cu²⁺ binding rate. Different lowercase letters indicate that the same protease at different hydrolysis time had significant difference (P<0.05), while different uppercase letters indicate that different proteases at the same hydrolysis time had significant difference (P<0.05).

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图 2 碱性蛋白酶酶解物分子量分布及其占比

注：a. 酪氨酸酶抑制肽的分子量分布；b. 碱性蛋白酶酶解物不同组分的占比。

Figure 2. Molecular weight distribution and proportion of alcalase hydrolysate

Note: a. Molecular weight distribution of TSTIP; b. Proportion of alcalase hydrolysate with different components.

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图 3 TSTIP-TYR及TSTIP-Cu²⁺的荧光光谱

注：a、b分别为TSTIP与不同浓度的TYR或Cu²⁺结合后的荧光强度变化；c、d分别为TSTIP与TYR或Cu²⁺结合后荧光强度随时间的变化。

Figure 3. Fluorescence intensity of TSTIP-TYR and TSTIP-Cu²⁺

Note: The a and b represent the changes in fluorescence intensity of TSTIP combining with TYR or Cu²⁺ at different concentrations, respectively; the c and d represent the changes in fluorescence intensity of TSTIP combining with TYR or Cu²⁺ at different time, respectively.

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图 4 TSTIP-TYR和TSTIP-Cu²⁺的紫外与红外光谱分析

注：a. TSTIP-TYR及TSTIP-Cu²⁺的紫外吸收光谱；b. TSTIP-TYR及TSTIP-Cu²⁺的红外吸收光谱。

Figure 4. UV and FTIR spectrum of TSTIP-TYR and TSTIP-Cu²⁺

Note: a. UV spectrums of TSTIP-TYR and TSTIP-Cu²⁺; b. FTIR spectrums of TSTIP-TYR and TSTIP-Cu²⁺.

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图 5 TSTIP-TYR及TSTIP-Cu²⁺的圆二色谱

注：a. TSTIP-TYR和TSTIP-Cu²⁺的圆二色谱图；b. TSTIP-TYR和TSTIP-Cu²⁺的二级结构；不同小写字母表示同一样品在不同二级结构的质量比例有显著性差异 (P<0.05)；不同大写字母表示不同样品在同种二级结构的质量比例有显著性差异 (P<0.05)。

Figure 5. CD analysis of TSTIP-TYR and TSTIP-Cu²⁺

Note: a. Circular dichroism spectrum of TSTIP-TYR and TSTIP-Cu²⁺; b. Proportions of secondary structures of TSTIP-TYR and TSTIP-Cu²⁺. Different lowercase letters indicate that the mass proportion of the same sample in different secondary structures had significant difference (P<0.05). Different uppercaseletters indicate that the mass proportion of different samples in the same secondary structure had significant difference (P<0.05).

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表 1 不同蛋白酶的酶解条件

Table 1. Enzymatic conditions for different proteases

蛋白酶 Protease	酶解条件 Hydrolysis condition
蛋白酶 Protease	酶活力 Enzymatic activity/ (U·mg⁻¹)	pH	温度 Temperature/ ℃	加酶量 Enzyme content/%	酶解时间 Hydrolysis time/h
碱性蛋白酶 Alcalase	200	9.0	50	2	1、2、4、6、8
木瓜蛋白酶 Papain	1 500	6.5	50	2	1、2、4、6、8
胰蛋白酶 Trypsin	250	8.0	37	2	1、2、4、6、8

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表 2 罗非鱼皮和酪氨酸酶抑制肽中氨基酸成分和质量分数

Table 2. Amino acids composition and mass fraction of 　　　　　　　tilapia skin and TSTIP　　　　　　%

氨基酸 Amino acids	罗非鱼皮 Tilapia skin	罗非鱼皮酪氨酸酶抑制肽 TSTIP
天冬氨酸 Asp	6.20±0.23^f	6.00±0.28^f
谷氨酸 Glu	10.78±0.14^d	10.65±0.21^d
赖氨酸 Lys	3.69±0.23^g	3.62±0.26^g
组氨酸 His	0.90±0.10^jk	0.87±0.25^kl
精氨酸 Arg	9.45±0.18^e	9.20±0.23^e
色氨酸 Trp	0.15±0.20^l	1.80±0.22ⁱ
酪氨酸 Tyr	0.65±0.23^k	0.61±0.18^l
苯丙氨酸 Phe	2.21±0.25ⁱ	2.11±0.15ⁱ
甘氨酸 Gly	26.28±0.21^a	27.70±0.21^a
丙氨酸 Ala	11.22±0.17^c	11.16±0.23^c
缬氨酸 Val	2.21±0.21ⁱ	2.19±0.26ⁱ
蛋氨酸 Met	1.18±0.17^k	1.47±0.34^ij
异亮氨酸 Ile	1.17±0.24^j	1.10±0.40^jk
脯氨酸 Pro	14.32±0.15^b	13.68±0.23^b
亮氨酸 Leu	2.95±0.09^h	2.92±0.22^h
苏氨酸 Thr	2.95±0.04^h	2.94±0.17^h
丝氨酸 Ser	3.84±0.14^g	3.72±0.32^g
胱氨酸 Cys	0.01±0.12^l	0.04±0.13^m
注：不同小写字母表示同种原料在不同氨基酸种类的相对质量分数差异显著 (P<0.05)。 Note: Different lowercase letters indicate that there are significant differences in the relative mass fraction of the same material in different amino acids (P<0.05).

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参考文献(36)

[1]	胡俊康. 罗非鱼养殖现状与健康管理——访中国水产科学研究院珠江水产研究所王广军研究员[J]. 广东饲料, 2021, 30(3): 8-10.
[2]	李敏雄. 罗非鱼皮胶原蛋白肽的制备及活性研究[D]. 广州: 华南农业大学, 2018: 1-2.
[3]	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. 2021中国渔业统计年鉴[M]. 北京: 中国农业出版社, 2021: 78.
[4]	陈胜军, 李来好, 杨贤庆, 等. 罗非鱼综合加工利用与质量安全控制技术研究进展[J]. 南方水产科学, 2011, 7(4): 85-90. doi: 10.3969/j.issn.2095-0780.2011.04.013
[5]	梁小明, 韦倩妮, 吴军, 等. 罗非鱼加工废弃物的综合利用探讨[J]. 现代食品, 2020(17): 29-31. doi: 10.16736/j.cnki.cn41-1434/ts.2020.17.010
[6]	ZHANG X G, NOISA P, YONGSAWATDIGUL J. Identification and characterization of tilapia antioxidant peptides that protect AAPH-induced HepG2 cell oxidative stress[J]. J Funct Foods, 2021, 86: 104662. doi: 10.1016/j.jff.2021.104662
[7]	CHEN J D, SUN S S, LI Y S, et al. Proteolysis of tilapia skin collagen: identification and release behavior of ACE-inhibitory peptides[J]. Food Sci Technol, 2021, 139: 110502.
[8]	JIN L, ZHENG D X, YANG G Y, et al. Tilapia skin peptides ameliorate diabetic nephropathy in stz-induced diabetic rats and HG-induced GMCs by improving mitochondrial dysfunction[J]. Mar Drugs, 2020, 18(7): 363-378. doi: 10.3390/md18070363
[9]	王堡煊. 灰星鲨鱼皮肽的提取及生物活性的研究[D]. 厦门: 厦门大学, 2019: 12-60.
[10]	李娜. 玉米源单体肽TPM对酪氨酸酶活性的影响[D]. 长春: 吉林大学, 2018: 1-25.
[11]	TENG H, FAN X Y, LV Q Y, et al. Folium nelumbinis (lotus leaf) volatile-rich fraction and its mechanisms of action against melanogenesis in B16 cells[J]. Food Chem, 2020, 330(15): 127030.
[12]	PENG Z Y, WANG G C, ZENG Q H, et al. Synthesis, antioxidant and anti-tyrosinase activity of 1, 2, 4-triazole hydrazones as antibrowning agents[J]. Food Chem, 2021, 341(2): 128265.
[13]	KUBGLOMSONG S, THEERAKULKAIT C, REED R L, et al. Isolation and identification of tyrosinase-inhibitory and copper-chelating peptides from hydrolyzed rice-bran-derived albumin[J]. J Agric Food Chem, 2018, 66(31): 8346-8354. doi: 10.1021/acs.jafc.8b01849
[14]	ISABELLA T, MARCO B, FRANCESCO V, et al. The reaction of alpha-synuclein with tyrosinase: possible implications for Parkinson disease[J]. J Biol chem, 2008, 283(24): 16808-16817. doi: 10.1074/jbc.M709014200
[15]	SAMANEH Z, ASIEH B, MAHMUD T H K, et al. A comprehensive review on tyrosinase inhibitors[J]. J Enzym Inhib Med Chem, 2019, 34(1): 279-309. doi: 10.1080/14756366.2018.1545767
[16]	JOOMPANG A, JANGPROMMA N, CHOOWONGKOMON K, et al. Evaluation of tyrosinase inhibitory activity and mechanism of Leucrocin I and its modified peptides[J]. J Biosci Bioeng, 2020, 130(3): 239-246. doi: 10.1016/j.jbiosc.2020.04.002
[17]	AGUILAR-TOAL'A J E, HERNΆNDEZ-MENDOZA A, GONZ'ALEZ-CΌRDOVA A F, et al. Potential role of natural bioactive peptides for development of cosmeceutical skin products[J]. Peptides, 2019, 122: 170170. doi: 10.1016/j.peptides.2019.170170
[18]	CARRASCO-CASTILLA J, HERNÁNDEZ-ÁLVAREZ A J, JIMĔNEZ-MARTĹNEZIMÉNEZ C, et al. Antioxidant and metal chelating activities of peptide fractions from phaseolin and bean protein hydrolysates[J]. Food Chem, 2012, 135(3): 1789-1795. doi: 10.1016/j.foodchem.2012.06.016
[19]	林善婷, 胡晓, 李来好, 等. 罗非鱼皮蛋白肽-Fe²⁺结合物的制备及性质分析[J]. 食品科学, 2021, 42(8): 157-164. doi: 10.7506/spkx1002-6630-20200307-117
[20]	GUO L D, HARNEDY P A, O'KEEFFE M B, et al. Fractionation and identification of Alaska pollock skin collagen-derived mineral chelating peptides[J]. Food Chem, 2015, 173(15): 536-542.
[21]	YU Q, LI J W, FAN L P. Effect of drying methods on the microstructure, bioactivity substances, and antityrosinase activity of asparagus stems[J]. J Agric Food Chem, 2019, 67(5): 1537-1545. doi: 10.1021/acs.jafc.8b05993
[22]	张金杨. 罗非鱼促钙吸收肽的制备分离及生物活性研究[D]. 上海: 上海海洋大学, 2018: 13-14.
[23]	WU W M, HE L C, LIANG Y H, et al. Preparation process optimization of pig bone collagen peptide-calcium chelate using response surface methodology and its structural characterization and stability analysis[J]. Food Chem, 2019, 284(30): 80-89.
[24]	SHEN Z W, WANG Y J, GUO Z, et al. Novel tyrosinase inhibitory peptide with free radical scavenging ability[J]. J Enzym Inhib Med Chem, 2019, 34(1): 1633-1640. doi: 10.1080/14756366.2019.1661401
[25]	CHEN M J, LIU J R, SHEU J F, et al. Study on skin care properties of milk kefir whey[J]. Asian-Australas J Anim Sci, 2006, 19(6): 905-908. doi: 10.5713/ajas.2006.905
[26]	DENG Y J, HUANG L X, ZHANG C H, et al. Skin-care functions of peptides prepared from Chinese quince seed protein: sequences analysis, tyrosinase inhibition and molecular docking study[J]. Ind Crops Prod, 2020, 148: 112331. doi: 10.1016/j.indcrop.2020.112331
[27]	XIE N, HUANG J, LI B, et al. Affinity purification and characterisation of zinc chelating peptides from rapeseed protein hydrolysates: possible contribution of characteristic amino acid residues[J]. Food Chem, 2015, 173(15): 210-217.
[28]	冯兰. 黑色素生成抑制肽的设计、筛选及作用机制探究[D]. 大连: 大连理工大学, 2018: 15-22.
[29]	NIE H L, LIU L, YANG H Q, et al. A novel heptapeptide with tyrosinase inhibitory activity identified from a phage display library[J]. Appl Biochem Biotechnol, 2017, 181(1): 219-232. doi: 10.1007/s12010-016-2208-3
[30]	TORRES-FUENTES C, ALAIZ M, VIOQUE J. Chickpea chelating peptides inhibit copper-mediated lipid peroxidation[J]. J Sci Food Agric, 2014, 94(15): 3181-3188. doi: 10.1002/jsfa.6668
[31]	MEGĺAS C, PEDROCHE J, YUST M, et al. Affinity purification of copper chelating peptides from chickpea protein hydrolysates[J]. J Agric Food Chem, 2007, 55(10): 3949-3954. doi: 10.1021/jf063401s
[32]	DAS D, MITRA S, KUMAR R, et al. Copper-induced spectroscopic and structural changes in short peptides derived from azurin[J]. Arch Biochem Biophys, 2020, 687(15): 108388.
[33]	SCHURINK M, van BERKEL W J H, WICHERS H J, et al. Novel peptides with tyrosinase inhibitory activity[J]. Peptides, 2007, 28(3): 485-495. doi: 10.1016/j.peptides.2006.11.023
[34]	ARICHAYA M, PORNLERT A, SUWIMON K. Chicken foot broth byproduct: a new source for highly effective peptide-calcium chelate[J]. Food Chem, 2021, 345(30): 128713.
[35]	MA Q F, HU J, WU W H, et al. Characterization of copper binding to the peptide amyloid-β (1-16) associated with Alzheimer's disease[J]. Biopolymers, 2006, 83(1): 20-31. doi: 10.1002/bip.20523
[36]	OOKUBO N, MICHIUE H, KITAMATSU M, et al. The transdermal inhibition of melanogenesis by a cell-membrane-permeable peptide delivery system based on poly-arginine[J]. Biomaterials, 2014, 35(15): 4508-4516. doi: 10.1016/j.biomaterials.2014.01.052