Preparation and Characteristics of Low-Bitter Peptides from the Enzymatic Hydrolysate of Cod Bone
-
摘要: 本研究以鳕鱼骨为原料，通过控制水解度降低鳕鱼骨酶解液的苦味，并分析了水解物中的多肽与游离氨基酸对苦味的产生机制。结果表明通过木瓜蛋白酶适度水解2 h，鳕鱼骨酶解液水解度达到7.48%，苦味值为5.80，并且多肽主要分布在1500~2000 Da；另外，第一次酶解后将沉淀进行高压蒸煮热处理（121 ℃，30 min），使鱼骨表面变得松散，再次添加混合酶对沉淀进行酶解，以实现鳕鱼骨的深度水解，水解度达到49.24%，水解物中多肽分子量较小，主要分布在500~1000 Da，苦味值为6.03，游离氨基酸显著高于仅一次水解后的含量。通过控制水解度从而限制了苦味的产生，这种低苦味酶解肽的制备为其在食品中的应用奠定了基础。Abstract: In this study, cod bones were used as raw material. The bitterness of cod bone hydrolysate was reduced by controlling the degree of hydrolysis, and the mechanism of peptide and free amino acids in the hydrolysate on bitterness was analyzed. The results showed that the degree of hydrolysis reached 7.48% and the bitterness value was 5.80 through mild hydrolysis for 2 h. The peptides were mainly distributed between 1500 and 2000 Da. In addition, the precipitate was subjected to high-pressure cooking and heat treatment (121 °C, 30 min) to make the surface of the fish bone loose after the first enzymolysis. The degree of hydrolysis reached 49.24% and the bitterness value was 6.03 through extensive hydrolysis for 2 h. The peptides were mainly distributed between 500 and 1000 Da. Free amino acids were significantly higher than the content after only the first hydrolysis. By controlling the degree of hydrolysis to limit the production of bitterness, the preparation of this low-bitterness enzymatic peptide laid the foundation for its application in food.
-
Key words:
- cod bone /
- proteolysis /
- low bitterness /
- peptide
-
图 3 适度水解不同时间下苦味强度和涩味强度的变化
Figure 3. The change of bitterness intensity and astringency intensity under different time of mild hydrolysis
图 4 深度水解不同时间下水解度的变化
Figure 4. The change of degree of hydrolysis under different time of extensive hydrolysis
图 5 一段酶解后（A）与二段酶解后（B）鳕鱼骨表面的扫描电镜图（150000×）
Figure 5. The scanning electron micrograph of cod bone surface after the first (A) and second (B) enzymolysis(150000×)
图 6 适度水解（A）与深度水解（B）后鳕鱼骨酶解液中多肽分子量的分布
Figure 6. The molecular weight distribution of cod bone hydrolysates after mild (A) and extensive (B) hydrolysis
表 1 两段式酶解后鳕鱼骨酶解上清液电子舌味道值
Table 1. The taste value of enzymatic supernatant electronic tongue after the second enzymolysis
苦味 涩味 鲜味 咸味 一段酶解 7.33±0.26a 0.95±0.13a 15.98±0.05a 0.39±0.33a 二段酶解 6.03±0.45b 0.55±0.25b 16.64±0.18b 1.31±0.25b 注：同列不同小写字母表示差异显著（P<0.05）。 
下载: 导出CSV
表 2 两段式酶解后鳕鱼骨酶解上清液游离氨基酸的含量
Table 2. The free amino acid content of cod bone hydrolysate supernatant after the second enzymolysis
氨基酸 一段水解（nmol/L） 二段水解（nmol/L） 上升幅度（%） 天冬氨酸（Asp） 17.97±0.22 30.85±1.13 71.68 苏氨酸（Thr） 9.21±0.28 12.13±0.65 31.70 丝氨酸（Ser） 10.73±0.09 14.40±0.86 34.20 谷氨酸（Glu） 6.23±0.56 9.33±0.12 49.76 脯氨酸（Pro） 15.96±6.75 21.24±3.37 33.08 甘氨酸（Gly） 1.80±1.20 3.92±0.35 117.78 丙氨酸*（Ala） 36.78±2.88 51.31±1.89 39.51 半胱氨酸（Cys） 8.17±1.75 12.70±1.47 55.45 缬氨酸*（Val） 11.61±2.63 22.32±0.99 92.25 蛋氨酸*（Met） 3.41±0.30 5.71±0.57 67.45 异亮氨酸*（Ile） 17.16±0.23 25.53±0.84 48.78 亮氨酸*（Leu） 23.99±2.87 33.90±7.36 41.31 酪氨酸（Tyr） 51.58±2.32 57.15±0.37 10.80 苯丙氨酸*（Phe） 75.87±2.96 92.13±1.18 21.43 赖氨酸（Lys） 28.63±2.56 40.57±2.87 41.70 组氨酸（His） 2.06±0.51 2.55±0.49 23.79 精氨酸（Arg） 146.67±1.50 186.72±2.68 27.31 注：*为疏水性氨基酸。
下载: 导出CSV
表 3 适度水解后鳕鱼骨酶解物的多肽序列
Table 3. The polypeptide sequence of cod bone hydrolysates after mild hydrolysis
肽段 分子量（Da） 蛋白来源 LVVDGVK 728.4433 肌酸激酶M型 EAPLNPK 767.4161 肌动蛋白 IIAPPER 794.4624 肌动蛋白，α骨骼肌 KLEGDLK 801.4564 肌球蛋白重链，骨骼肌 FAGDDAPR 847.3817 肌动蛋白，α骨骼肌 LQDLVDKL 942.5384 肌球蛋白重链，骨骼肌 IHLDDALR 951.5119 肌球蛋白重链，骨骼肌 AGFAGDDAPR 975.4393 肌动蛋白，α骨骼肌 STHPHFVR 979.4984 肌球蛋白重链，骨骼肌 LTEAPLNPK 981.5467 肌动蛋白，α骨骼肌 EQIDNLQR 1014.5072 肌球蛋白重链，骨骼肌 FAGDDAPRAV 1017.4872 肌动蛋白，α骨骼肌 RDLTDYLM 1041.4806 肌动蛋白，α骨骼肌 LGEQIDNLQR 1184.6104 肌球蛋白重链，骨骼肌 GSLEQEKKLR 1186.6640 肌球蛋白重链，骨骼肌 HHTFYNELR 1215.5793 肌动蛋白，α骨骼肌 DLTDYLMKIL 1223.6437 肌动蛋白，α骨骼肌 NWDDMEKIW 1235.5254 肌动蛋白，α骨骼肌 ELEEISERLE 1245.6106 肌球蛋白重链，骨骼肌 RDLTDYLMKI 1266.6615 肌动蛋白，α骨骼肌 TNWDDMEKIW 1336.5770 肌动蛋白，α骨骼肌 WDDMEKIWHHTFY 1806.7776 肌动蛋白，α骨骼肌 GIITNWDDMEKIWHH 1893.8829 肌动蛋白 RDLTDYLMKIL 1395.7397 肌动蛋白，α骨骼肌 LGEQIDNLQRVK 1411.7757 肌球蛋白重链，骨骼肌 NWDDMEKIWHH 1509.6456 肌动蛋白，α骨骼肌 NWDDMEKIWHHT 1610.6924 肌动蛋白，α骨骼肌 WDDMEKIWHHTF 1643.7162 肌动蛋白，α骨骼肌 DLQHRLDEAEQLAL 1650.8277 肌球蛋白 NWDDMEKIWHHTF 1757.7578 肌动蛋白，α骨骼肌
下载: 导出CSV
表 4 深度水解后鳕鱼骨酶解物的多肽序列
Table 4. The polypeptide sequence of cod bone hydrolysates after extensive hydrolysis
肽段 分子量（Da） 蛋白来源 GAPGPAG 525.2548 胶原α-1（I）链 AGAPGPA 539.2696 胶原α-1（I）链 GAEGAPG 557.2469 胶原α-1（I）链 ANGLAGA 573.2981 胶原α-1（I）链 GAPGPAGAR 752.3880 胶原α-1（I）链 APPHIFS 767.3970 肌球蛋白 GPPGPTG 581.2809 胶原α-1（I）链 GHRGFTG 730.3507 胶原α-1（I）链 GAPGPQG 582.2762 胶原α-1（I）链 GPSGNIG 600.2865 胶原α-1（I）链 RGPPGPM 710.3534 胶原α-1（I）链 ASGPAGPR 711.3659 胶原α-1（I）链 TEAPLNPK 868.4612 肌动蛋白，α骨骼肌 GQKDSYVG 852.3982 肌动蛋白，α骨骼肌 AEREIVR 871.4828 肌动蛋白，α骨骼肌 LQDLVDKLQ 1070.5965 肌球蛋白重链，骨骼肌 GTTMYPGIADR 1180.5560 肌动蛋白，α骨骼肌 VAPEEHPVL 989.5160 肌动蛋白 IWHHTFY 1002.4736 肌动蛋白，α骨骼肌 PGPGPMG 611.2739 胶原α-2（I）链 SGPAGPR 640.3296 胶原α-1（I）链 GPMGPRG 670.3218 胶原α-1（I）链 GFSGLDGAKG 907.4393 胶原α-1（I）链 ALPHAIM 907.502 肌动蛋白，α骨骼肌 MYPGIADR 921.4372 肌动蛋白，α骨骼肌 SGPMGPR 700.3332 胶原α-1（I）链 VFPSIVG 717.4045 肌动蛋白，α骨骼肌 APGPVGPAG 721.3754 胶原α-1（I）链 NWDDMEKIWH 1372.5861 肌动蛋白，α骨骼肌 RDLTDYLMKIL 1379.7446 肌动蛋白，α骨骼肌
下载: 导出CSV
-
[1] 王鸿泽, 赵聪颖, 兰海铭, 等. 鳕鱼排加工工艺的研究[J]. 食品安全导刊,2018,203(12):149−150. [2] Toppe J, Albrektsen S, Hope B, et al. Chemical composition, mineral content and amino acid and lipid profiles in bones from various fish species[J]. Comparative Biochemistry Physiology B Biochemistry and Molecular Biology,2007,146(3):395−401. doi: 10.1016/j.cbpb.2006.11.020 [3] 李宏凯. 酶法水解植物蛋白概况[J]. 中国食品添加剂,2010(4):206−210. doi: 10.3969/j.issn.1006-2513.2010.04.033 [4] 刘伯业. 小麦蛋白低苦味肽的制备及其脱苦机理研究[D]. 无锡: 江南大学, 2017: 5−10. [5] Kukman I L, Zelenik-blatnik M, Abram V. Isolation of low-molecular-mass hydrophobic bitter peptides in soybean protein hydrolysates by reversed-phase high-performance liquid chromatography[J]. Journal of Chromatography A,1995,704(1):113−120. doi: 10.1016/0021-9673(95)00014-E [6] Shinoda I, Tada M, Okai H, et al. Bitter taste of h-pro-phe-pro-gly-pro-ile-pro-oh corresponding to the partial sequence (positions 61-67) of bovine b-casein, and related peptides[J]. Journal of Agricultural Chemical Society of Japan,1986,50(5):1247−1254. [7] Liu H, Li Y, Diao X, et al. Effect of porcine bone protein hydrolysates on the emulsifying and oxidative stability of oil-in-water emulsions[J]. Colloids and Surfaces A: Physicochemical and Engineering Aspects,2018,538:757−764. doi: 10.1016/j.colsurfa.2017.11.061 [8] Fan, W W, Tan X Y, Xu X B, et al. Relationship between enzyme, peptides, amino acids, ion composition, and bitterness of the hydrolysates of Alaska pollock frame[J]. Journal of Food Biochemistry 2019, 43(4): e12801. [9] 周慧江, 朱振宝, 易建华. 核桃蛋白水解物水解度测定方法比较[J]. 粮食与油脂,2012,25 (2):28−30. doi: 10.3969/j.issn.1008-9578.2012.02.008 [10] 中华人民共和国卫生部. GB 5009.5-2010 食品中蛋白质的测定[S]. 北京: 中国标准出版社, 2010: 2−6. [11] Burton L J, Rivera M, Hawsawi O, et al. Muscadine grape skin extract induces an unfolded protein response-mediated autophagy in prostate cancer cells: A TMT-based quantitative proteomic analysis[J]. PLoS One,2016,11(10):e0164115. doi: 10.1371/journal.pone.0164115 [12] 白艳红, 张相生, 赵电波, 等. 分步酶解猪骨粉提取猪骨素的工艺研究[J]. 现代食品科技,2012,28(12):1696−1699. [13] 周雪松. 蛋白质酶解物苦味形成机理及控制研究[J]. 粮食与油脂,2004,18(8):20−24. doi: 10.3969/j.issn.1008-9578.2004.08.007 [14] Dauksas E, Slizyte R, Rustad T, et al. Bitterness in fish protein hydrolysates and methods for removal[J]. Journal of Aquatic Food Product Technology,2004,13(2):101−114. doi: 10.1300/J030v13n02_09 [15] 曾晓房. 鸡骨架酶解及其产物制备鸡肉香精研究[D]. 广州: 华南理工大学, 2007: 5−10. [16] 安灿, 王欣, 陈美龄. 蛋白酶水解龙头鱼产生咸味的研究[J]. 中国食品添加剂,2017(1):135−140. doi: 10.3969/j.issn.1006-2513.2017.01.014 [17] 吴莉莉, 陈俊香. 双酶法降解核酸生产核苷酸食品增鲜剂: 酵母深加工高附加值产品[J]. 甘蔗糖业,2000(3):32−36, 41. [18] Chobert J M, Unklesbay N, Hsieh F H, et al. Hydrophobicity of bitter peptides from soy protein hydrolysates[J]. Journal of Agricultural & Food Chemistry,2004,52(19):5895−5901. [19] Matoba T, Hata T. Relationship between bitterness of peptides and their chemical structures[J]. Agricultural & Biological Chemistry,2004,36(8):1423−1431. [20] Ishibashi N, Ono I, Kato K, et al. Role of the hydrophobia amino acid residue in the bitterness of peptides[J]. Agricultural and Biological Chemistry,1988,52(1):91−94. [21] Zhao J, Xiong Y L. Interfacial peptide partitioning and undiminished antioxidative and emulsifying activity of oxidatively stressed soy protein hydrolysate in an O/W emulsion[J]. LWT-Food Science and Technology,2015,61(2):322−329. doi: 10.1016/j.lwt.2014.12.022 [22] Agboola S O, Singh H, Munro P A, et al. Destabilization of oil-in-water emulsions formed using highly hydrolyzed whey proteins[J]. Journal of Agricultural and Food Chemistry,1998,46(1):84−90. doi: 10.1021/jf970365b [23] Slattery H, Fitzgerald R J. Functional properties and bitterness of sodium caseinate hydrolysates prepared with a bacillus proteinase[J]. Journal of Food Science,2008,63(3):418−422. [24] Fitz Gerald R J, O’Cuinn G. Enzymatic debittering of food protein hydrolysates[J]. Biotechnology Advances,2006,24(2):234−237. doi: 10.1016/j.biotechadv.2005.11.002 [25] Kim H O, Li-Chan E C Y. Quantitative structure-activity relationship study of bitter peptides[J]. Journal of Agricultural and Food Chemistry,2006,54(26):10102−10111. doi: 10.1021/jf062422j -
点击查看大图
计量
- 文章访问数: 147
- HTML全文浏览量: 54
- PDF下载量: 16
- 被引次数: 0
