{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,8]],"date-time":"2026-10-08T05:42:59Z","timestamp":1791438179177,"version":"4.3.3"},"reference-count":55,"publisher":"MDPI AG","issue":"10","license":[{"start":{"date-parts":[[2021,5,18]],"date-time":"2021-05-18T00:00:00Z","timestamp":1621296000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"the National Science Council, Taiwan","award":["MOST 107-2635-E-229-001"],"award-info":[{"award-number":["MOST 107-2635-E-229-001"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>This research focuses on the proteolytic capacity of sea bass byproduct (SB) and their hypocholesterolemic activity via the cholesterol micelle formation (CMF) inhibition. SB was fermented with seven mixed lactic acid bacteria for 5 h at 42 \u00b0C. The lactic fermented SB was hydrolyzed with Protease N for 6 h under HHP to obtain the SB hydrolysates (HHP-assisted Protease N hydrolysis after fermentation, F-HHP-PN6). The supernatant was separated from the SB hydrolysate and freeze-dried. As the hydrolysis time extended to 6 h, soluble protein content increased from 187.1 to 565.8 mg\/g, and peptide content increased from 112.8 to 421.9 mg\/g, while inhibition of CMF increased from 75.0% to 88.4%. Decreasing the CMF inhibitory activity from 88.4% to 42.1% by simulated gastrointestinal digestion (FHHP-PN6 was further hydrolyzed by gastrointestinal enzymes, F-HHP-PN6-PP) reduced the CMF inhibitory activity of F-HHP-PN6. Using gel filtration chromatography, the F-HHP-PN6-PP was fractioned into six fractions. The molecular weight of the fifth fraction from F-HHP-PN6-PP was between 340 and 290 Da, and the highest inhibitory efficiency ratio (IER) on CMF was 238.9%\/mg\/mL. Further purification and identification of new peptides with CMF inhibitory activity presented the peptide sequences in Ser-Ala-Gln, Pro-Trp, and Val-Gly-Gly-Thr; the IERs were 361.7, 3230.0, and 302.9%\/mg\/mL, respectively.<\/jats:p>","DOI":"10.3390\/ijms22105295","type":"journal-article","created":{"date-parts":[[2021,5,18]],"date-time":"2021-05-18T06:01:33Z","timestamp":1621317693000},"page":"5295","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":21,"title":["Purification and Identification of Cholesterol Micelle Formation Inhibitory Peptides of Hydrolysate from High Hydrostatic Pressure-Assisted Protease Hydrolysis of Fermented Seabass Byproduct"],"prefix":"10.3390","volume":"22","author":[{"given":"Guan-Wen","family":"Chen","sequence":"first","affiliation":[{"name":"Department of Food Science, National Taiwan Ocean University, No. 2 Pei-Ning Road, Keelung 202, Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8737-208X","authenticated-orcid":false,"given":"Hong-Ting Victor","family":"Lin","sequence":"additional","affiliation":[{"name":"Department of Food Science, National Taiwan Ocean University, No. 2 Pei-Ning Road, Keelung 202, Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Li-Wen","family":"Huang","sequence":"additional","affiliation":[{"name":"Department of Food Science, National Taiwan Ocean University, No. 2 Pei-Ning Road, Keelung 202, Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4103-9617","authenticated-orcid":false,"given":"Chia-Hua","family":"Lin","sequence":"additional","affiliation":[{"name":"Department of Biotechnology, National Formosa University, No. 64, Wunhua Rd, Yunlin 632, Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yu-Hsin","family":"Lin","sequence":"additional","affiliation":[{"name":"Department of Food Science and Technology, Taipei University of Marine Technology, No. 212, Section 9, Yan Ping North Road, Taipei 111, Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2021,5,18]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"512","DOI":"10.1056\/NEJM199502233320807","article-title":"Cholesterol reduction in cardiovascular disease; clinical benefits and possible mechanisms","volume":"32","author":"Levine","year":"1995","journal-title":"N. Engl. J. Med."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"580","DOI":"10.1016\/0002-8703(95)90369-0","article-title":"Preventing and arresting coronary atherosclerosis","volume":"130","author":"Roberts","year":"1995","journal-title":"Am. Heart J."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"208","DOI":"10.1007\/s11418-016-1050-9","article-title":"Hypocholesterolemic effect of sericin-derived oligopeptides in high-cholesterol fed rats","volume":"71","author":"Lapphanichayakool","year":"2017","journal-title":"J. Nat. Med."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"409","DOI":"10.1016\/j.dld.2010.03.015","article-title":"Bile salts and cholestasis","volume":"42","author":"Beuers","year":"2010","journal-title":"Dig. Liver Dis."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"337","DOI":"10.1016\/j.jhep.2006.06.001","article-title":"The emerging role of bile acids as integrators of intermediary metabolism","volume":"45","author":"Groen","year":"2006","journal-title":"J. Hepatol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1006\/bbrc.2001.4298","article-title":"Identification of novel hypocholesterolemic peptides derived from bovine milk \u03b2-lactoglobulin","volume":"281","author":"Nagaoka","year":"2001","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"373","DOI":"10.3177\/jnsv.47.373","article-title":"Hypocholesterolemic effect of indigestible fraction of Chlorella regularis in cholesterol-fed rats","volume":"47","author":"Shirata","year":"2001","journal-title":"J. Nutr. Sci. Vitaminol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1007\/s11883-011-0222-z","article-title":"A review of the rationale for additional therapeutic interventions to attain lower LDL-C when statin therapy is not enough","volume":"14","author":"Shanes","year":"2012","journal-title":"Curr. Atheroscler. Rep."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1994","DOI":"10.1002\/jsfa.5573","article-title":"Hypocholesterolaemic and antioxidant activities of chickpea (Cicer arietinum L.) protein hydrolysates","volume":"92","author":"Yust","year":"2012","journal-title":"J. Sci. Food Agric."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"756","DOI":"10.1016\/j.foodres.2007.01.005","article-title":"Fractionation and identification of a novel hypocholesterolemic peptide derived from soy protein Alcalase hydrolysates","volume":"40","author":"Zhong","year":"2007","journal-title":"Food Res. Int."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"661","DOI":"10.1016\/j.foodres.2006.11.011","article-title":"Preparation of hypocholesterol peptides from soy protein and their hypocholesterolemic effect in mice","volume":"40","author":"Zhong","year":"2007","journal-title":"Food Res. Int."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1261","DOI":"10.1039\/C4FO01171F","article-title":"Bile acid binding capacity of fish protein hydrolysates from discard species of the West Mediterranean Sea","volume":"6","author":"Guadix","year":"2015","journal-title":"Food Funct."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"395","DOI":"10.1016\/j.foodchem.2010.04.053","article-title":"Hypochlesterolemic effect of compounded freshwater clam protein hydrolysate and Gracilaria","volume":"123","author":"Lin","year":"2010","journal-title":"Food Chem."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"397","DOI":"10.1016\/j.foodchem.2010.09.016","article-title":"Plasma lipid regulatory of compounded freshwater clam protein hydrolysate and Gracilaria insoluble dietary fiber","volume":"125","author":"Lin","year":"2011","journal-title":"Food Chem."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"e12385","DOI":"10.1111\/jfbc.12385","article-title":"Purification and identification of hypocholesterolemic peptides from freshwater clam hydrolysate with in vitro gastrointestinal digestion","volume":"41","author":"Lin","year":"2017","journal-title":"J. Food Biochem."},{"key":"ref_16","first-page":"55","article-title":"Amino acids substitution of hypocholesterolemic peptide originated from glycinin hydrolysate","volume":"11","author":"Kwon","year":"2002","journal-title":"Food Sci. Biotechnol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"641","DOI":"10.1016\/j.idairyj.2006.07.004","article-title":"Purification of angiotensin I-converting enzyme inhibitory peptides and antihypertensive effect of milk produced by protease-facilitated lactic fermentation","volume":"17","author":"Chen","year":"2007","journal-title":"Int. Dairy J."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"639","DOI":"10.1111\/j.1745-4514.2007.00135.x","article-title":"Cardiovascular effects of whey from prozyme 6-facilitated lactic acid bacteria fermentation of milk","volume":"31","author":"Chen","year":"2007","journal-title":"J. Food Biochem."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"552","DOI":"10.1016\/j.foodchem.2007.06.039","article-title":"Antihypertensive effect of bioactive peptides produced by protease-facilitated lactic acid fermentation of milk","volume":"106","author":"Tsai","year":"2008","journal-title":"Food Chem."},{"key":"ref_20","unstructured":"Chen, G.W., and Liao, K.J. (2017). Method of Combining Pressure and Enzyme to Extract Hydrolysate of Animal-Derived Composition. (No. TWI600379B), Patent."},{"key":"ref_21","first-page":"85","article-title":"High-pressure biotechnology in medicine and pharmaceutical science","volume":"1","author":"Masson","year":"2001","journal-title":"J. Biomed. Biotechnol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1246","DOI":"10.1007\/s11947-013-1234-1","article-title":"Application of high hydrostatic pressure and enzymatic hydrolysis for the extraction of ginsenosides from fresh ginseng root (Panax ginseng CA Myer)","volume":"7","author":"Sunwoo","year":"2014","journal-title":"Food Bioprocess Technol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"187","DOI":"10.1016\/j.tifs.2018.08.013","article-title":"Enhancing enzymatic hydrolysis of food proteins and production of bioactive peptides using high hydrostatic pressure technology","volume":"80","author":"Marciniak","year":"2018","journal-title":"Trends Food Sci. Technol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"791","DOI":"10.1016\/S0958-6946(02)00078-X","article-title":"Effect of high hydrostatic pressure on the conformation of \u03b2-lactoglobulin A as assessed by proteolytic peptide profiling","volume":"12","author":"Knudsen","year":"2002","journal-title":"Int. Dairy J."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1734","DOI":"10.1016\/j.foodchem.2006.10.050","article-title":"The use of high hydrostatic pressure to promote the proteolysis and release of bioactive peptides from ovalbumin","volume":"104","author":"Recio","year":"2007","journal-title":"Food Chem."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"904","DOI":"10.1016\/j.foodchem.2012.05.097","article-title":"Combined effects of high-pressure and enzymatic treatments on the hydrolysis of chickpea protein isolates and antioxidant activity of the hydrolysates","volume":"135","author":"Zhang","year":"2012","journal-title":"Food Chem."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1730","DOI":"10.1021\/acs.jafc.5b06080","article-title":"High-pressure-assisted enzymatic release of peptides and phenolics increases angiotensin converting enzyme I inhibitory and antioxidant activities of Pinto bean hydrolysates","volume":"64","author":"Frias","year":"2016","journal-title":"J. Agric. Food Chem."},{"key":"ref_28","first-page":"17","article-title":"Application and development status of an ultra-high pressure processing technique in aquatic food","volume":"47","author":"Lin","year":"2017","journal-title":"Fish. Ext. Rep."},{"key":"ref_29","unstructured":"Chen, G.W., and Liao, K.J. (2016). Method of Combining Pressure and Enzyme to Extract Hydrolysate of Cubilose. (No. TWI516212B), Patent."},{"key":"ref_30","unstructured":"Fisheries Agency, Council of Agriculture Taiwan (2021, January 01). Fisheries Statistical Yearbook, Available online: https:\/\/www.fa.gov.tw\/cht\/PublicationsFishYear\/index.aspx."},{"key":"ref_31","first-page":"374","article-title":"Fish processing waste: A promising source of type-I collagen","volume":"10","author":"Silvipriya","year":"2016","journal-title":"Curr. Trends Biotechnol. Pharm."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"123","DOI":"10.1089\/109662003322233521","article-title":"Beneficial effects of fish liver preparations of sea bass (Lates calcarifer) versus gemfibrozil in high fat diet\u2013induced lipid-intolerant rats","volume":"6","author":"Rizvi","year":"2003","journal-title":"J. Med. Food."},{"key":"ref_33","first-page":"68","article-title":"Enzymatic production of protein hydrolysates for food use","volume":"41","author":"Lahl","year":"1994","journal-title":"J. Food Sci. Technol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"659","DOI":"10.1016\/j.biotechadv.2010.04.001","article-title":"Development of high hydrostatic pressure in biosciences: Pressure effect on biological structures and potential applications in biotechnologies","volume":"28","author":"Rivalain","year":"2010","journal-title":"Biotechnol. Adv."},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Bamdad, F., Bark, S., Kwon, C.H., Suh, J.W., and Sunwoo, H. (2017). Anti-inflammatory and antioxidant properties of peptides released from \u03b2-lactoglobulin by high hydrostatic pressure-assisted enzymatic hydrolysis. Molecules, 22.","DOI":"10.3390\/molecules22060949"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"304","DOI":"10.1016\/j.foodchem.2017.12.037","article-title":"Effect of high pressure on structural modifications and enzymatic activity of a purified X-prolyl dipeptidyl aminopeptidase from Streptococcus thermophiles","volume":"248","author":"Giannoglou","year":"2018","journal-title":"Food Chem."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1139","DOI":"10.3168\/jds.S0022-0302(03)73696-0","article-title":"High pressure effects on proteolytic and glycolytic enzymes involved in cheese manufacturing","volume":"86","author":"Malone","year":"2003","journal-title":"J. Dairy Sci."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"50","DOI":"10.1016\/j.idairyj.2019.04.012","article-title":"Effect of high hydrostatic pressure treatment on the viability and acidification ability of lactic acid bacteria","volume":"96","author":"Giannoglou","year":"2019","journal-title":"Int. Dairy J."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"288","DOI":"10.1016\/j.foodchem.2014.07.049","article-title":"Peptides from cowpea present antioxidant activity, inhibit cholesterol synthesis and its solubilisation into micelles","volume":"168","author":"Marques","year":"2015","journal-title":"Food Chem."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"5375","DOI":"10.1021\/bi015613i","article-title":"Molecular dynamics simulations of mixed micelles modeling human bile","volume":"41","author":"Marrink","year":"2002","journal-title":"Biochemistry"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"86","DOI":"10.1007\/s11130-009-0108-1","article-title":"Sunflower protein hydrolysates reduce cholesterol micellar solubility","volume":"64","author":"Pedroche","year":"2009","journal-title":"Plant Foods Hum. Nutr."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"77","DOI":"10.1016\/j.foodres.2018.05.009","article-title":"Isolation and identification by high resolution liquid chromatography tandem mass spectrometry of novel peptides with multifunctional lipid-lowering capacity","volume":"111","author":"Prados","year":"2018","journal-title":"Int. Food Res. J."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"136","DOI":"10.1016\/j.jff.2019.01.001","article-title":"Evaluation of hypolipidemic peptide (Val-Phe-Val-Arg-Asn) virtual screened from chickpea peptides by pharmacophore model in high-fat diet-induced obese rat","volume":"54","author":"Shi","year":"2019","journal-title":"J. Funct. Foods."},{"key":"ref_44","unstructured":"(2021, January 01). UniProt Database. Available online: http:\/\/www.uniprot.org."},{"key":"ref_45","first-page":"1217","article-title":"Involvement of post-digestion \u2018hydrophobia\u2019 peptides in plasma cholesterol-lowering effect of dietary plant proteins","volume":"50","author":"Iwami","year":"1986","journal-title":"Agric. Biol. Chem."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"173","DOI":"10.1016\/0003-2697(81)90175-5","article-title":"Modified colorimetric ninhydrin methods for peptidase assay","volume":"118","author":"Doi","year":"1981","journal-title":"Anal. Biochem."},{"key":"ref_47","doi-asserted-by":"crossref","unstructured":"Lin, Y.H., Chen, C.A., Tsai, J.S., and Chen, G.W. (2019). Preparation and identification of novel antihypertensive peptides from the in vitro gastrointestinal digestion of marine cobia skin hydrolysates. Nutrients, 11.","DOI":"10.3390\/nu11061351"},{"key":"ref_48","unstructured":"Cooper, T.G. (1977). Spectrophotometry. The Tools of Biochemistry, Wiley-Interscience. [1st ed.]."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"265","DOI":"10.1016\/S0021-9258(19)52451-6","article-title":"Protein measurement with the folin phenol reagent","volume":"193","author":"Lowry","year":"1951","journal-title":"J. Biol. Chem."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"1219","DOI":"10.3168\/jds.S0022-0302(83)81926-2","article-title":"Spectrophotometric assay using o-phthaldialdehyde for determination of proteolysis in milk and isolated milk proteins","volume":"66","author":"Church","year":"1983","journal-title":"J. Dairy Sci."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"367","DOI":"10.1016\/S0963-9969(01)00131-4","article-title":"Characterization of inhibition and stability of soy-protein-derived angiotensin I-converting enzyme inhibitory peptides","volume":"35","author":"Wu","year":"2002","journal-title":"Food Res. Int."},{"key":"ref_52","doi-asserted-by":"crossref","unstructured":"Lin, Y.H., Chen, G.W., Yeh, C.H., Song, H., and Tsai, J.S. (2018). Purification and identification of angiotensin I-converting enzyme inhibitory peptides and the antihypertensive effect of Chlorella sorokiniana protein hydrolysates. Nutrients, 10.","DOI":"10.3390\/nu10101397"},{"key":"ref_53","doi-asserted-by":"crossref","unstructured":"Huang, C.Y., Tsai, Y.H., Hong, Y.H., Hsieh, S.L., and Huang, R.H. (2018). Characterization and antioxidant and angiotensin I-converting enzyme (ACE)-inhibitory activities of gelatin hydrolysates prepared from extrusion-pretreated milkfish (Chanos chanos) scale. Mar. Drugs, 16.","DOI":"10.3390\/md16100346"},{"key":"ref_54","unstructured":"(2021, January 01). Pairwise Sequence Alignment Software. Available online: https:\/\/www.ebi.ac.uk\/Tools\/psa\/."},{"key":"ref_55","unstructured":"SAS Institute Inc (2019). SAS\u00ae9.4 TS1M5 User\u2019s Guide, SAS Institute Press."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/10\/5295\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T06:03:10Z","timestamp":1760162590000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/10\/5295"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,5,18]]},"references-count":55,"journal-issue":{"issue":"10","published-online":{"date-parts":[[2021,5]]}},"alternative-id":["ijms22105295"],"URL":"https:\/\/doi.org\/10.3390\/ijms22105295","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,5,18]]}}}