Abstract
The study aimed to investigate potent antioxidant activities (ABTS assay, Hydroxyl free radical scavenging assay, and Superoxide free radical assay), ACE inhibitory activity, and anti-inflammatory activity in the WPC (whey protein concentrate) hydrolysate using Alcalase. The hydrolysis conditions (addition rate and incubation times) for peptide synthesis were also optimized using proteolytic activity. The generation of proinflammatory cytokines by lipopolysaccharide-treated murine macrophages was reduced when the protein hydrolysate concentration was low. In comparison to unhydrolyzed WPC, SDS-PAGE examination revealed no protein bands in WPC hydrolysates. Two-Dimensional (2D) gel electrophoresis did not show any protein spots. Using the ‘In-solution trypsin digestion’ approach, the trypsin digested protein samples were put into RPLC/MS for amino acid sequencing. Peptides were also identified using RPLC/MS on fractions of 3 and 10 kDa permeates and retentates. The MASCOT database was used to look up the raw masses of LC/MS. By comparing hydrolyzed whey protein to the BLASTp (NCBI), PIR, BIOPEP, and AHTPDB databases, novel antioxidative and ACE inhibitory peptides were reported.
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References
Aluko RE (2015) Antihypertensive peptides from food proteins. Ann Rev Food Sci Technol 6:235–262. https://doi.org/10.1146/annurev-food-022814-015520
Athira S, Mann B, Saini P, Sharma R, Kumar R, Singh AK (2015) Production and characterization of whey protein hydrolysate having antioxidant activity from cheese whey. J Sci Food Agric 95(14):2908–2915. https://doi.org/10.1002/jsfa.7032
Boyacı D, Korel F, Yemenicioğlu A (2016) Development of activate-at-home-type edible antimicrobial films: an example pH-triggering mechanism formed for smoked salmon slices using lysozyme in whey protein films. Food Hydrocoll 60:170–178. https://doi.org/10.1016/j.foodhyd.2016.03.032
Cheison SC, Zhang SB, Wang Z, Xu SY (2009) Comparison of a modified spectrophotometric and the pH-stat methods for determination of the degree of hydrolysis of whey proteins hydrolysed in a tangential-flow filter membrane reactor. Food Res Int 42(1):91–97. https://doi.org/10.1016/j.foodres.2008.09.003
Colbert LB, Decker EA (1991) Antioxidant activity of an ultrafiltration permeate from acid whey. J Food Sci 56(5):1248–1250. https://doi.org/10.1111/j.1365-2621.1991.tb04744.x
del Mar Contreras M, Hernández-Ledesma B, Amigo L, Martín-Álvarez PJ, Recio I (2011) Production of antioxidant hydrolyzates from a whey protein concentrate with thermolysin: optimization by response surface methodology. LWT Food Sci Technol 44(1):9–15. https://doi.org/10.1016/j.lwt.2010.06.017
Dryáková A, Pihlanto A, Marnila P, Čurda L, Korhonen HJ (2010) Antioxidant properties of whey protein hydrolysates as measured by three methods. Eur Food Res Technol 230(6):865–874. https://doi.org/10.1007/s00217-010-1231-9
Ena JM, Van Beresteijn ECH, Robben AJPM, Schmidt DG (1995) Whey protein antigenicity reduction by fungal proteinases and a pepsin/pancreatin combination. J Food Sci 60(1):104–110. https://doi.org/10.1111/j.1365-2621.1995.tb05616.x
Farup J, Rahbek SK, Storm AC, Klitgaard S, Jørgensen H, Bibby BM, Vissing K (2016) Effect of degree of hydrolysis of whey protein on in vivo plasma amino acid appearance in humans. Springer plus 5(1):382. https://doi.org/10.1186/s40064-016-1995-x
Ferreira IMPLVO, Pinho O, Mota MV, Tavares P, Pereira A, Goncalves MP, Teixeira JA (2007) Preparation of ingredients containing an ACE-inhibitory peptide by tryptic hydrolysis of whey protein concentrates. Int Dairy J 17(5):481–487. https://doi.org/10.1016/j.idairyj.2006.06.023
Ghosh, S. (2015). Modulations of spleen and thymus function (s) in Indian Goat Capra hircus by melatonin and different hormones: an in vivo and in vitro study (Doctoral dissertation, BHU, Varanasi, UP, India).
Hati S, Sreeja V, Solanki J, Prajapati JB (2015) Influence of proteolytic lactobacilli on ACE inhibitory activity and release of bioactive peptides. Indian J Dairy Sci 68:1–8
Hernández-Ledesma B, Amigo L, Recio I, Bartolomé B (2007) ACE-inhibitory and radical-scavenging activity of peptides derived from β-lactoglobulin f (19–25). Interactions with ascorbic acid. J Agric Food Chem 55(9):3392–3397. https://doi.org/10.1021/jf063427j
Hogan S, Zhang L, Li J, Wang H, Zhou K (2009) Development of antioxidant rich peptides from milk protein by microbial proteases and analysis of their effects on lipid peroxidation in cooked beef. Food Chem 117:438–443. https://doi.org/10.1016/j.foodchem.2009.04.040
Hussein FA, Chay SY, Zarei M, Auwal SM, Hamid AA, Wan Ibadullah WZ, Saari N (2020) Whey protein concentrate as a novel source of bifunctional peptides with angiotensin-I converting enzyme inhibitory and antioxidant properties: RSM study. Foods 9(1):64. https://doi.org/10.3390/foods9010064
Jakubczyk A, Baraniak B (2014) Angiotensin I converting enzyme inhibitory peptides obtained after in vitro hydrolysis of Pea (PisumSativum var. Bajka) globulins. BioMed Res Int. https://doi.org/10.1155/2014/438459
Jauhiainen T, Korpela R (2007) Milk peptides and blood pressure. J Nutr 137:825S-829S. https://doi.org/10.1093/jn/137.3.825S
Jeewanthi RKC, Kim MH, Lee NK, Yoon YC, Paik HD (2017) Peptide analysis and the bioactivity of whey protein hydrolysates from cheese whey with several enzymes. Korean J Food Sci Anim Resour 37(1):62. https://doi.org/10.5851/kosfa.2017.37.1.62
Kinter M, Sherman NE (2005) Protein sequencing and identification using tandem mass spectrometry, Vol 9: John Wiley & Sons
Kopf-Bolanz KA, Schwander F, Gijs M, Vergères G, Portmann R, Egger L (2014) Impact of milk processing on the generation of peptides during digestion. Int Dairy J 35(2):130–138. https://doi.org/10.1016/j.idairyj.2013.10.012
Li Y, Jiang B, Zhang T, Mu W, Liu J (2008) Antioxidant and free radical-scavenging activities of chickpea protein hydrolysate (CPH). Food Chem 106(2):444–450. https://doi.org/10.1016/j.foodchem.2007.04.067
Li Z, Jiang A, Yue T, Wang J, Wang Y, Su J (2013) Purification and identification of five novel antioxidant peptides from goat milk casein hydrolysates. J Dairy Sci 96(7):4242–4251. https://doi.org/10.3168/jds.2012-6511
Lin S, Tian W, Li H, Cao J, Jiang W (2012) Improving antioxidant activities of whey protein hydrolysates obtained by thermal preheat treatment of pepsin, trypsin, alcalase and flavourzyme. Int J Food Sci Technol 47(10):2045–2051. https://doi.org/10.1111/j.1365-2621.2012.03068.x
Liu C, Hong J, Yang H, Wu J, Ma D, Li D, Lai R (2010) Frog skins keep redox homeostasis by antioxidant peptides with rapid radical scavenging ability. Free Radic Biol Med 48(9):1173–1181. https://doi.org/10.1016/j.freeradbiomed.2010.01.036
Ma Y, Liu J, Shi H, Yu L (2016) Isolation and characterization of anti-inflammatory peptides derived from whey protein. J Dairy Sci 99(9):6902–6912. https://doi.org/10.3168/jds.2016-11186
Madureira AR, Tavares T, Gomes AM, Pintado ME, Malcata FX (2010) Invited review: physiological properties of bioactive peptides obtained from whey proteins. J Dairy Sci 93(2):437–455. https://doi.org/10.3168/jds.2009-2566
Mann B, Kumari A, Kumar R, Sharma R, Prajapati K, Mahboob S, Athira S (2015) Antioxidant activity of whey protein hydrolysates in milk beverage system. J Food Sci Technol 52(6):3235–3241. https://doi.org/10.1007/s13197-014-1361-3
Morais HA, Silvestre MP, Silva MR, Silva VD, Batista MA, e Silva AC, Silveira JN (2015) Enzymatic hydrolysis of whey protein concentrate: effect of enzyme type and enzyme: substrate ratio on peptide profile. J Food Sci Technol 52(1):201–210. https://doi.org/10.1007/s13197-013-1005-z
Morifuji M, Ishizaka M, Baba S, Fukuda K, Matsumoto H, Koga J, Kanegae M, Higuchi M (2010) Comparison of different sources and degrees of hydrolysis of dietary protein: effect on plasma amino acids, dipeptides, and insulin responses in human subjects. J Agric Food Chem 58(15):8788–8797. https://doi.org/10.1021/jf101912n
Nakamura Y, Yamamoto N, Sakai K, Takano T (1995) Antihypertensive effect of sour milk and peptides isolated from it that are inhibitors to angiotensin I-converting enzyme. J Dairy Sci 78(6):1253–1267. https://doi.org/10.3168/jds.S0022-0302(95)76745-5
Nwachukwu ID, Aluko RE (2019) Structural and functional properties of food protein-derived antioxidant peptides. J Food Biochem 43(1):e12761. https://doi.org/10.1111/jfbc.12761
Ou K, Liu Y, Zhang L, Yang X, Huang Z, Nout MR, Liang J (2010) Effect of neutrase, alcalase, and papain hydrolysis of whey protein concentrates on iron uptake by Caco-2 cells. J Agric Food Chem 58(8):4894–4900. https://doi.org/10.1021/jf100055y
Pan D, Cao J, Guo H, Zhao B (2012) Studies on purification and the molecular mechanism of a novel ACE inhibitory peptide from whey protein hydrolysate. Food Chem 130(1):121–126. https://doi.org/10.1016/j.foodchem.2011.07.011
Panchal G, Hati S, Sakure A (2020) Characterization and production of novel antioxidative peptides derived from fermented goat milk by L. fermentum. LWT Food Sci Technol 119:108887. https://doi.org/10.1016/j.lwt.2019.108887
Patel R, Hati S (2018) Production of antihypertensive (angiotensin I-converting enzyme inhibitory) peptides derived from fermented milk supplemented with WPC70 and Calcium caseinate by Lactobacillus cultures. Rev Med Microbiol 29(1):30–40. https://doi.org/10.1097/MRM.0000000000000119
Pellegrini A, Dettling C, Thomas U, Hunziker P (2001) Isolation and characterization of four bactericidal domains in the bovine β-lactoglobulin. Biochim Biophys Acta 1526(2):131–140. https://doi.org/10.1016/S0304-4165(01)00116-7
Pena-Ramos EA, Xiong YL (2001) Antioxidative activity of whey protein hydrolysates in a liposomal system. J Dairy Sci 84(12):2577–2583. https://doi.org/10.3168/jds.S0022-0302(01)74711-X
Peng X, Kong B, Xia X, Liu Q (2010) Reducing and radical-scavenging activities of whey protein hydrolysates prepared with Alcalase. Int Dairy J 20(5):360–375. https://doi.org/10.1016/j.idairyj.2009.11.019
Piccolomini A, Iskandar M, Lands L, Kubow S (2012) High hydrostatic pressure pre-treatment of whey proteins enhances whey protein hydrolysate inhibition of oxidative stress and IL-8 secretion in intestinal epithelial cells. Food Nutr Res 56(1):17549. https://doi.org/10.3402/fnr.v56i0.17549
Pihlanto A (2006) Antioxidative peptides derived from milk proteins. Int Dairy J 16(11):1306–1314. https://doi.org/10.1016/j.idairyj.2006.06.005
Pouliot Y, Guy MM, Tremblay M, Gaonac’h AC, Chay Pak Ting BP, Gauthier SF, Voyer N (2009) Isolation and characterization of an aggregating peptide from a tryptic hydrolysate of whey proteins. J Agric Food Chem 57(9):3760–3774. https://doi.org/10.1021/jf803539f
Ricci-Cabello I, Olalla Herrera M, Artacho R (2012) Possible role of milk-derived bioactive peptides in the treatment and prevention of metabolic syndrome. Nutr Rev 70(4):241–255. https://doi.org/10.1111/j.1753-4887.2011.00448.x
Samaranayaka AG, Li-Chan EC (2011) Food-derived peptidic antioxidants: a review of their production, assessment, and potential applications. J Funct Foods 3(4):229–254. https://doi.org/10.1016/j.jff.2011.05.006
Silveira ST, Martínez-Maqueda D, Recio I, Hernández-Ledesma B (2013) Dipeptidyl peptidase-IV inhibitory peptides generated by tryptic hydrolysis of a whey protein concentrate rich in β-lactoglobulin. Food Chem 141(2):1072–1087. https://doi.org/10.1016/j.foodchem.2013.03.056
Silvestre MP, Silva MR, Silva VD, Souza MW, Lopes Junior CD, Afonso WD (2012) Analysis of whey protein hydrolysates: peptide profile and ACE inhibitory activity. Braz J Pharm Sci 48(4):747–757
Spellman D, Mcevoy E, O’Cuinn G, Fitzgerald RJ (2003) Proteinase and exopeptidase hydrolysis of whey protein: comparison of the TNBS, OPA and pH stat methods for quantification of degree of hydrolysis. Int Dairy J 13(6):447–453. https://doi.org/10.1016/S0958-6946(03)00053-0
Spellman D, O’cuinn G, FitzGerald RJ (2009) Bitterness in Bacillus proteinase hydrolysates of whey proteins. Food Chem 114(2):440–446. https://doi.org/10.1016/j.foodchem.2008.09.067
Steel RGD, Torrie JH (1980) Principles and procedure of statistics- a biometrical approach. Japan: Mcgraw Hill Kogakusha Ltd.
Suthar J, Jana A, Balakrishnan S (2017) High protein milk ingredients-A tool for value-addition to dairy and food products. J Dairy Vet Anim Res 6:00171
Tagliazucchi D, Martini S, Bellesia S, Conte A (2015) Identification of ACE-inhibitory peptides from Phaseolus vulgaris after in vitro gastrointestinal digestion. Int J Food Sci Nutr 66(7):774–782. https://doi.org/10.3109/09637486.2015.1088940
Tavares TG, Malcata FX (2013) Whey proteins as source of bioactive peptides against hypertension. In: Hernández-Ledesma B, Chien Hsieh C (eds) Bioactive food peptides in health and disease. Spain: IntechOpen, p 75. https://doi.org/10.5772/3318
Tian M, Fang B, Jiang L, Guo H, Cui J, Ren F (2015) Structure-activity relationship of a series of antioxidant tripeptides derived from β-Lactoglobulin using QSAR modeling. Dairy Sci Technol 95(4):451–463. https://doi.org/10.1007/s13594-015-0226-5
Toldra F, Gallego M, Reig M, Aristoy MC, Mora L (2020) Recent progress in enzymatic release of peptides in foods of animal origin and assessment of bioactivity. J Agric Food Chem 68(46):12842–12855. https://doi.org/10.1021/acs.jafc.9b08297
Van der Ven C, Gruppen H, de Bont DB, Voragen AG (2002) Optimisation of the angiotensin converting enzyme inhibition by whey protein hydrolysates using response surface methodology. Int Dairy J 12:813–820. https://doi.org/10.1016/S0958-6946(02)00077-8
Vasiljevic T, Jelen P (2002) Lactose hydrolysis in milk as affected by neutralizers used for the preparation of crude β-galactosidase extracts from Lactobacillus bulgaricus 11842. Innov Food Sci Emerg Technol 3(2):175–184. https://doi.org/10.1016/S1466-8564(02)00016-4
Whetstine MC, Croissant AE, Drake MA (2005) Characterization of dried whey protein concentrate and isolate flavor. J Dairy Sci 88(11):3826–3839. https://doi.org/10.3168/jds.S0022-0302(05)73068-X
Wróblewska B, Karamać M, Amarowicz R, Szymkiewicz A, Troszyńska A, Kubicka E (2004) Immunoreactive properties of peptide fractions of cow whey milk proteins after enzymatic hydrolysis. Int J Food Sci Technol 39(8):839–850. https://doi.org/10.1111/j.1365-2621.2004.00857.x
Wu Q, Zhang X, Jia J, Kuang C, Yang H (2018) Effect of ultrasonic pretreatment on whey protein hydrolysis by alcalase: thermodynamic parameters, physicochemical properties and bioactivities. Process Biochem 67:46–54. https://doi.org/10.1016/j.procbio.2018.02.007
Yamamoto N, Maeno M, Takano T (1999) Purification and characterization of an antihypertensive peptide from a yogurt-like product fermented by Lactobacillus helveticus CPN4. J Dairy Sci 82(7):1388–1393. https://doi.org/10.3168/jds.S0022-0302(99)75364-6
Yang Y, Zheng N, Yang J, Bu D, Wang J, Ma L, Sun P (2014) Animal species milk identification by comparison of two-dimensional gel map profile and mass spectrometry approach. Int Dairy J 35:15–20. https://doi.org/10.1016/j.idairyj.2013.09.008
Yu XX, Liu C, Lu MH, Liu YL, Yin JY, Zhang YH (2019) Impact of enzymatic hydrolysis followed by transglutaminase-induced cross-linking on decreasing antigenicity and reserving partial interfacial properties of whey protein isolate. Food Funct 10(3):1653–1660. https://doi.org/10.1039/C8FO01880D
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CHM: Methodology, Investigation, Formal analysis, Data curation, Writing- Original draft preparation. AS: Data curation; Formal analysis; Methodology; Project administration; Resources; Software; RM: Data curation; Formal analysis; Writing—review & editing; Methodology; MB: Data curation; Formal analysis; Writing—review & editing; Methodology; KKK: Data curation; Formal analysis; Writing—review & editing; Methodology; SD: Writing- Reviewing and Editing, Visualization. SH: Supervision, Conceptualization, Funding acquisition, Investigation, Project administration, Resources, Validation.
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Mansinhbhai, C.H., Sakure, A., Maurya, R. et al. Significance of whey protein hydrolysate on anti-oxidative, ACE-inhibitory and anti-inflammatory activities and release of peptides with biofunctionality: an in vitro and in silico approach. J Food Sci Technol 59, 2629–2642 (2022). https://doi.org/10.1007/s13197-021-05282-3
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DOI: https://doi.org/10.1007/s13197-021-05282-3