高血壓，與多種代謝途徑有關，例如腎素-血管緊張素-醛固酮系統（RAAS）。在該系統中，血管緊張素I轉換酶（ACE）將血管緊張素I轉換為有效的血管收縮劑血管緊張素II，從而增加血壓。臨床上已經使用了幾種合成的ACE抑制藥物，但合成藥物長期使用可能對人體健康產生副作用。長期以來，源自食用食品蛋白質的ACE抑制（ACEI）肽被視為更溫和，更安全的替代品。牛奶是哺乳動物常見的可食用蛋白質來源之一，可提供功能性化合物，包括生物活性肽。因此，胃腸道蛋白酶用於釋放生物活性肽。這項研究旨在探索負責ACE抑制活性的商業牛奶的ACE抑制肽。
本研究使用改良式生物活性指引的分群，從“安佳”牛奶蛋白水解物中，有效地篩選ACEI肽。親水作用液相層析（HILIC）(可視作適用於水相的正相層析法)，以固相萃取（SPE）短柱的形式，用於第一次分群，具有最佳ACEI活性（IC50 61.75±5.74 µg / mL）的HILIC分群為95％ACN + 0.1％FA洗脫液。此分液再用逆相（RP）SPE短柱繼續進行第二次生物活性指引的分群。最佳的RP-SPE分液為20％ACN + 0.1％FA之洗脫液，其ACEI活性為IC50 = 36.22±1.18 µg / mL。接著，將最佳的RP-SPE分液進行LC-MS / MS鑑定，鑑定可提供ACEI活性的活性胜肽HL-7。HL-7為從β-酪蛋白衍生的短鏈胜肽，其IC50值為16.87±0.3 µM。
第三純化步驟後，HL-7之IC50值顯著降低了8.92倍。同時，根據多重反應監測（MRM）實驗，從奶粉的胃腸道蛋白酶水解產物(1mg / ml)和RP-SPE分液(1mg / ml)中，可定量出其分別含有19.86±1.14 pg和14545±572.9 pg ACEI肽HL-7。此外，對HL-7的動力學研究表明，HL-7同時為競爭性及受質類型之抑製劑，此特性與分子對接的模擬結果一致，發現HL-7藉由3個氫鍵與ACE相互作用在三個關鍵殘基Ala354（S1），Gln281（S2'）和Lys511（S2'），同時在活性位點與Lys511（S2'）發生1個電荷相互作用。因此，我們認為HL-7可以作為替代性的天然ACE抑制劑，可用於控制血壓以及開發功能性食品

Hypertension is also known as high blood pressure that has associated with multi-metabolic pathways such as the renin-angiotensin-aldosterone system (RAAS). In this system, angiotensin I-converting enzyme (ACE) converts angiotensin I into a potent vasoconstrictor angiotensin II that increases blood pressure. There have been several synthetic ACE inhibitory drugs used clinically. However, synthetic drugs show side effects on human health after long-term use. ACE inhibitory (ACEI) peptides derived from edible food proteins have long been regarded as milder and safer alternatives. Milk is one of the common edible protein sources from mammalian, which provides functional compounds, including bioactive peptides. Therefore, gastrointestinal proteases were used for releasing bioactive peptides. The study aims to explore ACE-inhibitory peptides of commercial milk that responsible for ACE-inhibitory activity.
In this study, a modified bioassay-guided fractionation was performed to efficiently screen ACEI peptides from milk protein hydrolysate. The aqueous normal phase chromatography namely hydrophilic interaction liquid chromatography (HILIC) was used as a format of solid-phase extraction (SPE) short column for the first fractionation, then the HILIC-SPE fraction with the best ACEI activity (IC50 61.75±5.74 µg/mL) was obtained when eluted by 95%ACN+0.1%FA. The best HILIC-SPE fraction was continued to the second fractionation using reversed-phase (RP) SPE short column. The best RP-SPE fraction was eluted by 20%ACN+0.1%FA with an ACEI activity of IC50 36.22±1.18 µg/mL Afterward, the best RP-SPE fraction was subjected to LC-MS/MS to identify bioactive peptides that responsible for ACEI activity. HL-7 was successfully screened from the milk protein hydrolysate that derived by β-casein with an IC50 value of 16.87±0.3 µM.
The IC50 value of HL-7 was significantly decreased by 8.92 folds after the third purification step. According to a multiple reaction monitoring (MRM) experiment, 19.86±1.14 pg and 14545±572.9 pg of ACEI peptide HL-7 were obtained in the gastrointestinal protease hydrolysate and RP-SPE fraction derived from 1mg/ml of milk protein. Furthermore, the kinetic study of HL-7 showed characteristics of a competitive inhibitor and substrate-type inhibitor, as well as HL-7, was consistent with the simulation results of molecular docking, which found that HL-7 interacts with ACE via 3 hydrogen bonds at three key residues such as Ala354 (S1), Gln281(S2’), and Lys511 (S2’), and 1 charge interaction with Lys511 (S2’) in the active site. Hence, we believe HL-7 could be an alternative natural ACE inhibitor for controlling blood pressure as well as for the development of functional food.

Acharya, K. R., Sturrock, E. D., Riordan, J. F., & Ehlers, M. R. (2003). Ace revisited: a new target for structure-based drug design. Nature Reviews Drug Discovery, 2(11), 891-902.
Ackermann, B. L., Berna, M. J., & Murphy, A. T. (2002). Recent advances in use of LC/MS/MS for quantitative high-throughput bioanalytical support of drug discovery. Current Topics In Medicinal Chemistry, 2(1), 53-66.
Alting, A. C., Meijer, R. J., & van Beresteijn, E. C. (1997). Incomplete elimination of the ABBOS epitope of bovine serum albumin under simulated gastrointestinal conditions of infants. Diabetes Care, 20(5), 875-880.
Amorim, F. G., Coitinho, L. B., Dias, A. T., Friques, A. G. F., Monteiro, B. L., de Rezende, L. C. D., Vasquez, E. C. (2019). Identification of new bioactive peptides from kefir milk through proteopeptidomics: bioprospection of antihypertensive molecules. Food Chemistry, 282, 109-119.
Atherton, E., Fox, H., Harkiss, D., Logan, C., Sheppard, R., & Williams, B. (1978). A mild procedure for solid phase peptide synthesis: use of fluorenylmethoxycarbonylamino-acids. Journal of the Chemical Society, Chemical Communications(13), 537-539.
Atkinson, A., & Robertson, J. (1979). Captopril in the treatment of clinical hypertension and cardiac failure. The Lancet, 314(8147), 836-839.
Bidlingmeyer, B. A., & Warren, F. V. (1984). An inexpensive experiment for the introduction of high performance liquid chromatography. Journal of Chemical Education, 61(8), 716.
Boersema, P. J., Mohammed, S., & Heck, A. J. (2008). Hydrophilic interaction liquid chromatography (HILIC) in proteomics. Analytical and Bioanalytical Chemistry, 391(1), 151-159.
Brenneman, C. A., & Ebeler, S. E. (1999). Chromatographic separations using solid-phase extraction cartridges: Separation of wine phenolics. Journal of Chemical Education, 76(12), 1710.
Buszewski, B., & Noga, S. (2012). Hydrophilic interaction liquid chromatography (HILIC) powerful separation technique. Analytical And Bioanalytical Chemistry, 402(1), 231-247.
Carpino, L. A. (1987). The 9-fluorenylmethyloxycarbonyl family of base-sensitive amino-protecting groups. Accounts of Chemical Research, 20(11), 401-407.
Chakraborty, A. B., & Berger, S. J. (2005). Optimization of reversed-phase peptide liquid chromatography ultraviolet mass spectrometry analyses using an automated blending methodology. Journal Of Biomolecular Techniques : JBT, 16(4), 327-335.
Chen, J., Wang, Y., Zhong, Q., Wu, Y., & Xia, W. (2012). Purification and characterization of a novel angiotensin-I converting enzyme (ACE) inhibitory peptide derived from enzymatic hydrolysate of grass carp protein. Peptides, 33(1), 52-58.
Cheung, H.-S., Wang, F.-L., Ondetti, M. A., Sabo, E. F., & Cushman, D. W. (1980). Binding of peptide substrates and inhibitors of angiotensin-converting enzyme. Importance of the COOH-terminal dipeptide sequence. Journal of Biological Chemistry, 255(2), 401-407.
Costa, F. F., Brito, M. A. V. P., Furtado, M. A. M., Martins, M. F., de Oliveira, M. A. L., de Castro Barra, P. M., dos Santos, A. S. d. O. (2014). Microfluidic chip electrophoresis investigation of major milk proteins: study of buffer effects and quantitative approaching. Analytical Methods, 6(6), 1666-1673.
Cushman, D., & Cheung, H. (1971). Spectrophotometric assay and properties of the angiotensin-converting enzyme of rabbit lung. Biochemical Pharmacology, 20(7), 1637-1648.
David, D. K., & Katie, W. (2003). Bioactive proteins and peptides from food sources. Applications of bioprocesses used in isolation and recovery. Current Pharmaceutical Design, 9(16), 1309-1323.
Du, L., Fang, M., Wu, H., Xie, J., Wu, Y., Li, P., Zhou, L. (2013). A novel angiotensin I-converting enzyme inhibitory peptide from Phascolosoma esculenta water-soluble protein hydrolysate. Journal of Functional Foods, 5(1), 475-483.
Dziuba, J., & Iwaniak, A. (2005). Database of protein and bioactive peptide sequences Nutraceutical Proteins And Peptides In Health and Disease (pp. 537-557): CRC Press.
Fujita, H., & Yoshikawa, M. (1999). LKPNM: a prodrug-type ACE-inhibitory peptide derived from fish protein. Immunopharmacology, 44(1-2), 123-127.
Fujita, H., Eiichiyo koyama, K., & Yoshikawa, M. (2000). Classification and antihypertensive activity of angiotensin I‐converting enzyme inhibitory peptides derived from food proteins. Journal of Food Science, 65(4), 564-569.
Geng, X., Tian, G., Zhang, W., Zhao, Y., Zhao, L., Wang, H., & Ng, T. B. (2016). A Tricholoma matsutake peptide with angiotensin converting enzyme inhibitory and antioxidative activities and antihypertensive effects in spontaneously hypertensive rats. Scientific reports, 6, 24130.
Gilar, M., Olivova, P., Daly, A. E., & Gebler, J. C. (2005). Orthogonality of separation in two-dimensional liquid chromatography. Analytical Chemistry, 77(19), 6426-6434.
Girgih, A. T., He, R., & Aluko, R. E. (2014). Kinetics and molecular docking studies of the inhibitions of angiotensin converting enzyme and renin activities by hemp seed (Cannabis sativa L.) peptides. Journal of Agricultural Food Chemistry, 62(18), 4135-4144.
Gobbetti, M., Corsetti, A., Smacchi, E., Zocchetti, A., & De Angelis, M. (1998). Production of crescenza cheese by incorporation of bifidobacteria. Journal of Dairy Science, 81(1), 37-47.
Gómez-Ruiz, J. Á., Ramos, M., & Recio, I. (2002). Angiotensin-converting enzyme-inhibitory peptides in Manchego cheeses manufactured with different starter cultures. International Dairy Journal, 12(8), 697-706.
Gómez‐Ruiz, J. Á., Ramos, M., & Recio, I. (2007). Identification of novel angiotensin‐converting enzyme‐inhibitory peptides from ovine milk proteins by CE‐MS and chromatographic techniques. Electrophoresis, 28(22), 4202-4211.
Hartmann, R., & Meisel, H. (2007). Food-derived peptides with biological activity: from research to food applications. Current opinion in biotechnology, 18(2), 163-169.
Hernández-Ledesma, B., Amigo, L., Ramos, M., & Recio, I. (2004). Release of angiotensin converting enzyme-inhibitory peptides by simulated gastrointestinal digestion of infant formulas. International Dairy Journal, 14(10), 889-898.
Hernández-Ledesma, B., del Mar Contreras, M., & Recio, I. (2011). Antihypertensive peptides: Production, bioavailability and incorporation into foods. Advances in Colloid and Interface Science, 165(1), 23-35.
Hsieh, Y., & A Korfmacher, W. (2006). Increasing speed and throughput when using hplc-ms/ms systems fordrug metabolism and pharmacokinetic screening. Current Drug Metabolism, 7(5), 479-489.
Iwaniak, A., & Dziuba, J. (2009). Animal and plant proteins as precursors of peptides with ACE inhibitory activity–An in silico strategy of protein evaluation. Food Technology and Biotechnology, 47(4), 441-449.
Iwaniak, A., Minkiewicz, P., & Darewicz, M. (2014). Food‐originating ACE inhibitors, including antihypertensive peptides, as preventive food components in blood pressure reduction. Comprehensive Reviews in Food Science and Food Safety, 13(2), 114-134.
Jandera, P. (2008). Stationary phases for hydrophilic interaction chromatography, their characterization and implementation into multidimensional chromatography concepts. Journal of Separation Science, 31(9), 1421-1437.
Jia, J., Wu, Q., Yan, H., & Gui, Z. (2015). Purification and molecular docking study of a novel angiotensin-I converting enzyme (ACE) inhibitory peptide from alcalase hydrolysate of ultrasonic-pretreated silkworm pupa (Bombyx mori) protein. Process Biochemistry, 50(5), 876-883.
Kang, S.-M., Heo, S.-J., Kim, K.-N., Lee, S.-H., Yang, H.-M., Kim, A.-D., & Jeon, Y.-J. (2012). Molecular docking studies of a phlorotannin, dieckol isolated from Ecklonia cava with tyrosinase inhibitory activity. Bioorganic & Medicinal Chemistry, 20(1), 311-316.
Khueychai, S., Jangpromma, N., Choowongkomon, K., Joompang, A., Daduang, S., Vesaratchavest, M., Klaynongsruang, S. (2018). A novel ACE inhibitory peptide derived from alkaline hydrolysis of ostrich (Struthio camelus) egg white ovalbumin. Process Biochemistry, 73, 235-245.
Kim, J., Conlon, J. M., Iwamuro, S., & Knoop, F. C. (2001). Antimicrobial peptides from the skin of the Japanese mountain brown frog, Rana ornativentris. The Journal of Peptide Research, 58(5), 349-356.
Kim, S.-K., Byun, H.-G., Park, P.-J., & Shahidi, F. (2001). Angiotensin I converting enzyme inhibitory peptides purified from bovine skin gelatin hydrolysate. Journal of Agricultural and Food Chemistry, 49(6), 2992-2997.
Ko, S.-C., Jang, J., Ye, B.-R., Kim, M.-S., Choi, I.-W., Park, W.-S., Jung, W.-K. (2017). Purification and molecular docking study of angiotensin I-converting enzyme (ACE) inhibitory peptides from hydrolysates of marine sponge Stylotella aurantium. Process Biochemistry, 54, 180-187.
Kohmura, M., Nio, N., Kubo, K., Minoshima, Y., Munekata, E., & Ariyoshi, Y. (1989). Inhibition of angiotensin-converting enzyme by synthetic peptides of human β-casein. Agricultural And Biological Chemistry, 53(8), 2107-2114.
Kong, X., Zhou, H., & Qian, H. (2007). Enzymatic preparation and functional properties of wheat gluten hydrolysates. Food Chemistry., 101(2), 615-620.
Korfmacher, W. A. (2005). Foundation review: principles and applications of LC-MS in new drug discovery. Drug Discovery Today, 10(20), 1357-1367.
Korhonen, H., & Pihlanto, A. (2006). Bioactive peptides: production and functionality. International Dairy Journal, 16(9), 945-960.
Kumar, R., Singh, V. P., & Baker, K. M. (2009). The intracellular renin-angiotensin system in the heart. Current hypertension reports, 11(2), 104.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227(5259), 680-685.
Lapointe, J.-F., Mollé, D., Gauthier, S. F., & Pouliot, Y. (2004). Effect of calcium on thermolysin hydrolysis of β-casein tryptic peptides. International Dairy Journal, 14(3), 185-193.
Li, P., Jia, J., Fang, M., Zhang, L., Guo, M., Xie, J., Wei, D. (2014). In vitro and in vivo ACE inhibitory of pistachio hydrolysates and in silico mechanism of identified peptide binding with ACE. Process Biochemistry, 49(5), 898-904.
Li, Y., Sadiq, F. A., Fu, L., Zhu, H., Zhong, M., & Sohail, M. (2016). Identification of angiotensin I-converting enzyme inhibitory peptides derived from enzymatic hydrolysates of razor clam Sinonovacula constricta. Marine drugs, 14(6), 110.
Lin, K., Zhang, L.-w., Han, X., Xin, L., Meng, Z.-x., Gong, P.-m., & Cheng, D.-y. (2018). Yak milk casein as potential precursor of angiotensin I-converting enzyme inhibitory peptides based on in silico proteolysis. Food Chemistry, 254, 340-347.
Linden, J. C., & Lawhead, C. L. (1975). Liquid chromatography of saccharides. Journal of Chromatography A, 105(1), 125-133.
Liu, F., Baggerman, G., Schoofs, L., & Wets, G. (2008). The construction of a bioactive peptide database in Metazoa. Journal of Proteome Research, 7(9), 4119-4131.
Liu, M., Du, M., Zhang, Y., Xu, W., Wang, C., Wang, K., & Zhang, L. (2013). Purification and identification of an ACE inhibitory peptide from walnut protein. Journal of Agricultural and Food Chemistry, 61(17), 4097-4100.
López-Fandiño, R., Otte, J., & Van Camp, J. (2006). Physiological, chemical and technological aspects of milk-protein-derived peptides with antihypertensive and ACE-inhibitory activity. International Dairy Journal, 16(11), 1277-1293.
Luis, C.-G., Mario, D.-M., Alma, M.-A., Gloria, D.-O., & David, B.-A. (2012). Lima bean (Phaseolus lunatus) protein hydrolysates with ACE-I inhibitory activity. Food and Nutrition Sciences, 2012.
Lunow, D., Kaiser, S., Rückriemen, J., Pohl, C., & Henle, T. (2015). Tryptophan-containing dipeptides are C-domain selective inhibitors of angiotensin converting enzyme. Food chemistry, 166, 596-602.
Ma, B., Zhang, K., Hendrie, C., Liang, C., Li, M., Doherty‐Kirby, A., & Lajoie, G. (2003). PEAKS: powerful software for peptide de novo sequencing by tandem mass spectrometry. Rapid Communications in Mass Spectrometry, 17(20), 2337-2342.
Mäde, V., Els-Heindl, S., & Beck-Sickinger, A. G. (2014). Automated solid-phase peptide synthesis to obtain therapeutic peptides. Beilstein Journal of Organic Chemistry, 10(1), 1197-1212.
Madureira, A. R., Pereira, C. I., Gomes, A. M. P., Pintado, M. E., & Xavier Malcata, F. (2007). Bovine whey proteins – Overview on their main biological properties. Food Research International, 40(10), 1197-1211.
Maeno, M., Yamamoto, N., & Takano, T. (1996). Identification of an antihypertensive peptide from casein hydrolysate produced by a proteinase from Lactobacillus helveticus CP790. Journal of Dairy Science, 79(8), 1316-1321.
Manadas, B., Mendes, V. M., English, J., & Dunn, M. J. (2010). Peptide fractionation in proteomics approaches. Expert Review of Proteomics, 7(5), 655-663.
Manso, M., & Lopez-Fandino, R. (2003). Angiotensin I converting enzyme–inhibitory activity of bovine, ovine, and caprine κ-casein macropeptides and their tryptic hydrolysates. Journal of Food Protection, 66(9), 1686-1692.
Matsui, T., & Tanaka, M. (2010). Antihypertensive peptides and their underlying mechanisms. Bioactive proteins and peptides as functional foods and nutraceuticals, 1.
McLuckey, S. A. (1992). Principles of collisional activation in analytical mass spectrometry. Journal of the American Society for Mass Spectrometry, 3(6), 599-614.
Meisel, H., & FitzGerald, R. J. (2003). Biofunctional peptides from milk proteins: mineral binding and cytomodulatory effects. Current pharmaceutical design, 9(16), 1289-1296.
Miguel, M., Gómez‐Ruiz, J. Á., Recio, I., & Aleixandre, A. (2010). Changes in arterial blood pressure after single oral administration of milk‐casein‐derived peptides in spontaneously hypertensive rats. Molecular Nutrition & Food Research, 54(10), 1422-1427.
Mullally, M. M., Meisel, H., & FitzGerald, R. J. (1997). Angiotensin-I-converting enzyme inhibitory activities of gastric and pancreatic proteinase digests of whey proteins. International Dairy Journal, 7(5), 299-303.
Muñoz-Durango, N., Fuentes, C. A., Castillo, A. E., González-Gómez, L. M., Vecchiola, A., Fardella, C. E., & Kalergis, A. M. (2016). Role of the renin-angiotensin-aldosterone system beyond blood pressure regulation: molecular and cellular mechanisms involved in end-organ damage during arterial hypertension. International Journal Of Molecular Sciences, 17(7), 797.
Murphy, A. T., Berna, M. J., Holsapple, J. L., & Ackermann, B. L. (2002). Effects of flow rate on high‐throughput quantitative analysis of protein‐precipitated plasma using liquid chromatography/tandem mass spectrometry. Rapid communications in mass spectrometry, 16(6), 537-543.
Murumkar, P., Shinde, A., Sharma, M., Yamaguchi, H., Miniyar, P., & Yadav, M. (2016). Development of a credible 3D-QSAR CoMSIA model and docking studies for a series of triazoles and tetrazoles containing 11β-HSD1 inhibitors. SAR and QSAR in Environmental Research, 27(4), 265-292.
Nakamura, Y., Kajimoto, O., Kaneko, K., Aihara, K., Mizutani, J., Ikeda, N., Kajimoto, Y. (2004). Effects of the liquid yogurts containing “lactotripeptide (VPP, IPP)” on high-normal blood pressure. Journal Nutrition Food, 7(1), 123-137.
Nakamura, Y., Yamamoto, N., Sakai, K., Okubo, A., Yamazaki, S., & Takano, T. (1995). Purification and characterization of angiotensin i-converting enzyme inhibitors from sour milk. Journal of Dairy Science, 78(4), 777-783.
Nielsen, S. D., Beverly, R. L., Qu, Y., & Dallas, D. C. (2017). Milk bioactive peptide database: A comprehensive database of milk protein-derived bioactive peptides and novel visualization. Food Chemistry, 232, 673-682.
Ondetti, M. A., Rubin, B., & Cushman, D. W. (1977). Design of specific inhibitors of angiotensin-converting enzyme: new class of orally active antihypertensive agents. Science, 196(4288), 441-444.
Ondetti, M., & Cushman, D. (1982). Enzymes of the renin-angiotensin system and their inhibitors. Annual review of biochemistry, 51(1), 283-308.
Ong, L., Henriksson, A., & Shah, N. P. (2007). Angiotensin converting enzyme-inhibitory activity in Cheddar cheeses made with the addition of probiotic Lactobacillus casei sp. Le Lait, 87(2), 149-165.
Perez, M., & Musini, V. (2008). Pharmacological interventions for hypertensive emergencies: a Cochrane systematic review. Journal of Human Hypertension, 22(9), 596-607.
Phillips, I. (1987). Functions of angiotensin in the central nervous system. Annual review of physiology, 49(1), 413-433.
Pihlanto, A. (2000). Bioactive peptides derived from bovine whey proteins: Opioid and ACE-inhibitory peptides. Trends in Food Science & Technology, 11, 347-356.
Pihlanto-Leppälä, A., Rokka, T., & Korhonen, H. (1998). Angiotensin I converting enzyme inhibitory peptides derived from bovine milk proteins. International Dairy Journal, 8(4), 325-331.
Pina, A., & Roque, A. (2009). Studies on the molecular recognition between bioactive peptides and angiotensin‐converting enzyme. Journal of Molecular Recognition: An Interdisciplinary Journal, 22(2), 162-168.
Poole, C. F., & Lenca, N. (2017). Chapter 4 - Reversed-phase liquid chromatography. In S. Fanali, P. R. Haddad, C. F. Poole, & M.-L. Riekkola (Eds.), Liquid Chromatography (Second Edition) (pp. 91-123): Elsevier.
Priyanto, A. D., Doerksen, R. J., Chang, C.-I., Sung, W.-C., Widjanarko, S. B., Kusnadi, J., Hsu, J.-L. (2015). Screening, discovery, and characterization of angiotensin-I converting enzyme inhibitory peptides derived from proteolytic hydrolysate of bitter melon seed proteins. Journal of Proteomics, 128, 424-435.
Pujiastuti, D. Y., Shih, Y.-H., Chen, W.-L., Sukoso, & Hsu, J.-L. (2017). Screening of angiotensin-I converting enzyme inhibitory peptides derived from soft-shelled turtle yolk using two orthogonal bioassay-guided fractionations. Journal of Functional Foods, 28, 36-47.
Rai, A. K., Sanjukta, S., & Jeyaram, K. (2015). Production of angiotensin I converting enzyme inhibitory (ACE-I) peptides during milk fermentation and their role in reducing hypertension. Critical reviews in food science and nutrition, 57(13), 2789-2800.
Rawendra, R. D., Chen, S.-H., Chang, C.-I., Shih, W.-L., Huang, T.-C., Liao, M.-H., & Hsu, J.-L. (2014). Isolation and characterization of a novel angiotensin-converting enzyme-inhibitory tripeptide from enzymatic hydrolysis of soft-shelled turtle (Pelodiscus sinensis) egg white: in vitro, in vivo, and in silico study. Journal of Agricultural and Food Chemistry, 62(50), 12178-12185.
Roufik, S., Gauthier, S. F., & Turgeon, S. L. (2006). In vitro digestibility of bioactive peptides derived from bovine β-lactoglobulin. International Dairy Journal, 16(4), 294-302.
Ruiz, J. Á. G., Ramos, M., & Recio, I. (2004). Angiotensin converting enzyme-inhibitory activity of peptides isolated from Manchego cheese. Stability under simulated gastrointestinal digestion. International Dairy Journal, 14(12), 1075-1080.
Saito, T., Nakamura, T., Kitazawa, H., Kawai, Y., & Itoh, T. (2000). Isolation and structural analysis of antihypertensive peptides that exist naturally in Gouda cheese. Journal of Dairy Science, 83(7), 1434-1440.
Song, R., Wei, R.b., Ruan, G.q., & Luo, H.y. (2015). Isolation and identification of antioxidative peptides from peptic hydrolysates of half-fin anchovy (Setipinna taty). LWT-Food Science and Technology, 60(1), 221-229.
Suetsuna, K., & Chen, J.-R. (2001). Identification of antihypertensive peptides from peptic digest of two microalgae, Chlorella vulgaris and Spirulina platensis. Marine Biotechnology, 3(4), 305-309.
Tanzadehpanah, H., Asoodeh, A., Saberi, M. R., & Chamani, J. (2013). Identification of a novel angiotensin-I converting enzyme inhibitory peptide from ostrich egg white and studying its interactions with the enzyme. Innovative food science & emerging technologies, 18, 212-219.
Tikhomirova, V. E., Kryukova, O. V., Serov, R. A., Bulaeva, N., Bokeria, L. A., Golukhova, E., Danilov, S. M. (2015). Phenotyping of angiotensin-converting enzyme in the human heart. Journal of the American College of Cardiology, 65(10 Supplement), A404.
Tiller, P. R., Romanyshyn, L. A., & Neue, U. D. (2003). Fast LC/MS in the analysis of small molecules. Analytical and bioanalytical chemistry, 377(5), 788-802.
Udenigwe, C. C. (2014). Bioinformatics approaches, prospects and challenges of food bioactive peptide research. Trends in Food Science & Technology, 36(2), 137-143.
Uhlig, T., Kyprianou, T., Martinelli, F. G., Oppici, C. A., Heiligers, D., Hills, D., Verhaert, P. (2014). The emergence of peptides in the pharmaceutical business: From exploration to exploitation. EuPA Open Proteomics, 4, 58-69.
Vermeirssen, V., Van Camp, J., Devos, L., & Verstraete, W. (2003). Release of angiotensin I converting enzyme (ACE) inhibitory activity during in vitro gastrointestinal digestion: from batch experiment to semicontinuous model. Journal of Agricultural and Food Chemistry, 51(19), 5680-5687.
Vermeirssen, V., van der Bent, A., Van Camp, J., van Amerongen, A., & Verstraete, W. (2004). A quantitative in silico analysis calculates the angiotensin I converting enzyme (ACE) inhibitory activity in pea and whey protein digests. Biochimie, 86(3), 231-239.
Vukic, V. R., Vukic, D. V., Milanovic, S. D., Ilicic, M. D., Kanuric, K. G., & Johnson, M. S. (2017). In silico identification of milk antihypertensive di-and tripeptides involved in angiotensin I–converting enzyme inhibitory activity. Nutrition Research, 46, 22-30.
Walsh, D. J., Bernard, H., Murray, B. A., MacDonald, J., Pentzien, A. K., Wright, G. A., FitzGerald, R. J. (2004). In vitro generation and stability of the Lactokinin β-Lactoglobulin fragment (142–148). Journal of Dairy Science, 87(11), 3845-3857.
Wang, W., & De Mejia, E. G. (2005). A new frontier in soy bioactive peptides that may prevent age-related chronic diseases. Comprehensive Reviews in Food Science and Food Safety, 4(4), 63-78.
Wells, J. M., & McLuckey, S. A. (2005). Collision‐induced dissociation (CID) of peptides and proteins. Methods in enzymology, 402, 148-185.
Whitfield, E. J., Pruess, M., & Apweiler, R. (2006). Bioinformatics database infrastructure for biotechnology research. Journal of Biotechnology, 124(4), 629-639.
Wielsch, N., Thomas, H., Surendranath, V., Waridel, P., Frank, A., Pevzner, P., & Shevchenko, A. (2006). Rapid validation of protein identifications with the borderline statistical confidence via de novo sequencing and MS BLAST searches. Journal of Proteome Research, 5(9), 2448-2456.
Wu, J., Aluko, R. E., & Nakai, S. (2006). Structural requirements of angiotensin i-converting enzyme inhibitory peptides:  quantitative structure−activity relationship study of di- and tripeptides. Journal of Agricultural and Food Chemistry, 54(3), 732-738.
Yamada, Y., Matoba, N., Usui, H., Onishi, K., & Yoshikawa, M. (2002). Design of a highly potent anti-hypertensive peptide based on Ovokinin(2-7). Bioscience, Biotechnology, and Biochemistry, 66(6), 1213-1217.
Zhang, B., Sun, Q., Liu, H.-J., Li, S.-Z., & Jiang, Z.-Q. (2017). Characterization of actinidin from Chinese kiwifruit cultivars and its applications in meat tenderization and production of angiotensin I-converting enzyme (ACE) inhibitory peptides. LWT, 78, 1-7.
Zinieris, N., Leondiadis, L., & Ferderigos, N. (2005). N α-Fmoc removal from resin-bound amino acids by 5% piperidine solution. Journal of Combinatorial Chemistry, 7(1), 4-6.