ORIGINAL ARTICLE
Hybrid Approach in the Analysis of Bovine Milk Protein Hydrolysates as a Source of Peptides Containing Di- and Tripeptide Bitterness Indicators
Anna Iwaniak 1  
,   Piotr Minkiewicz 1  
,   Monika Hrynkiewicz 1  
,   Justyna Bucholska 1  
,   Małgorzata Darewicz 1  
 
More details
Hide details
1
Faculty of Food Science, Chair of Food Biochemistry, University of Warmia and Mazury in Olsztyn, Poland
CORRESPONDING AUTHOR
Anna Iwaniak   

Faculty of Food Science, Chair of Food Biochemistry, University of Warmia and Mazury in Olsztyn, Pl. Cieszyński 1, 10-726, Olsztyn-Kortowo, Poland
Submission date: 2019-09-06
Final revision date: 2019-10-24
Acceptance date: 2019-10-29
Online publication date: 2020-01-02
Publication date: 2020-03-03
 
Pol. J. Food Nutr. Sci. 2020;70(2):139–150
 
KEYWORDS
TOPICS
ABSTRACT
The aim of this study was to employ a hybrid approach combined with a fragmentomic idea of research used to analyze bovine milk protein hydrolysates as a source of peptides with a potential bitter taste. Firstly, selected sequences of bovine milk proteins were in silico hydrolyzed using bromelain, ficin, papain, and proteinase K. Hydrolysis was simulated using the BIOPEP-UWM “Enzyme(s) action” tool. Potentially released peptides (called parent peptides) were analyzed for the presence of shorter peptide regions with bitter taste. Some of them were defined as peptide bitterness indicators. Then, in silico results were verified in the in vitro experiments with the use of a bovine milk protein concentrate (MPC) as a substrate. The verification included the MPC hydrolysis and identification of peptides in MPC hydrolysates using RP-HPLC and RP-HPLC-MS/MS, respectively. The hybrid analysis of bovine milk protein hydrolysates showed that all released peptides contained fragments with bitter taste and some of them were bitterness indicators, which could potentially determine the taste of a whole sequence. However, the results of in silico and in vitro hydrolysis were divergent. It was also reflected by the ranking of enzymes acting in silico and in vitro. Despite above discrepancies, our predictions concerning the release of peptides that may affect the bitter taste of a hydrolysate, contribute to bringing more insights into the taste of foods, especially if unwanted. However, before introducing a food product to the market, sensory studies are required to confirm (or not) its taste. Hybrid analysis of bovine milk protein hydrolysates showed that all released peptides contained fragments with bitter taste and some of them were bitterness indicators, which could potentially decide about the taste of a whole sequence. However, the results of in silico and in vitro hydrolysis were divergent. It was also reflected by the ranking of enzymes acting in silico and in vitro. Despite above discrepancies, our predictions concerning the release of peptides that may have influence on bitter taste of a hydrolysate, contribute to giving more insights on the taste of foods, especially if unwanted. However, before the introducing of food to the market, sensory studies confirming (or not) taste of the product are required.
ABBREVIATIONS
BSA, bovine serum albumin; B, bromelain; B-MPC, bromelain hydrolysate of milk protein concentrate; F, ficin; F-MPC, ficin hydrolysate of milk protein concentrate; MLR, multivariate linear regression; MPC, milk protein concentrate; O-MPC, non-hydrolyzed milk protein concentrate; P, papain; P-MPC, papain hydrolysate of milk protein concentrate; PK, proteinase K; PK-MPC, proteinase K hydrolysate of milk protein concentrate; RP-HPLC, reversed-phase high performance liquid chromatography; RP-HPLC-MS/MS, reversed-phase high performance liquid chromatography and mass spectrometry; Rcaf., the ratio of caffeine (the threshold concentration for 1 mM caffeine solution as a standard (Rcaf. = 1.0); tR predicted, theoretical retention time; tR experimental, experimental retention time; αs1, casein; αs2-CN, αs2- casein; α-La, α– lactalbumin; β-Lg, β– lactoglobulin; β-CN, β– casein; κ-CN, κ-casein; and TFA, trifluoracetic acid.
FUNDING
Project financially supported by Minister of Science and Higher Education in the range of the program entitled "Regional Initiative of Excellence" for the years 2019-2022, Project No. 010/RID/2018/19, amount of funding 12.000.000 PLN as well as the funds of the University of Warmia and Mazury in Olsztyn (Project No. 17.610.014-300).
 
REFERENCES (55)
1.
Al-Shamsi, K.A., Mudgil, P., Hassan, H.M., Maqsood, S. (2018). Camel milk protein hydrolysates with improved technofunctional properties and enhanced antioxidant potential in in vitro and in food model systems. Journal of Dairy Science, 101(1), 47-60.
 
2.
Arihara, K. (2006). Strategies for designing novel functional meat products. Meat Science, 74(1), 219-229.
 
3.
Bucholska, J., Minkiewicz, P. (2016). The use of peptide markers of carp and herring allergens as an example of detection of sequenced and non-sequenced proteins. Food Technology and Biotechnology, 54(3), 266–274.
 
4.
Bucholska, J., Minkiewicz, P., Darewicz, M., Iwaniak, A. (2018). Databases and associated bioinformatic tools in studies of food allergens, epitopes and haptens - a review. Polish Journal of Food and Nutrition Sciences, 68(2), 103-113.
 
5.
Bumberger, E., Belitz, H.D. (1993). Bitter taste of enzymic hydrolysates of casein. I. Isolation, structural and sensorial analysis of peptides from tryptic hydrolysates of β-casein. Zeitschrift für Lebensmittel-Untersuchung und Forschung, 197(1), 14-19.
 
6.
Cheung, L.K.Y., Aluko, R.E., Cliff, M.A., Li-Chan, E.C.Y. (2015). Effects of exopeptidase treatment on antihypertensive activity and taste attributes of enzymatic whey protein hydrolysates. Journal of Functional Foods, 13, 262-275.
 
7.
Choopinham, S., Jaturasitha, S., Rakariyatham, N., Suree, N., Hataichanoke, N. (2015). Antioxidant and antihypertensive activity of gelatin hydrolysate from Nile tilapia skin. Journal of Food Science and Technology, 52(5), 3134-3139.
 
8.
Ciosek, P., Wróblewski, R. (2011). Potentiometric electronic tongues for foodstuff and biosample recognition - an overview. Sensors, 11(5), 4688-4701.
 
9.
Daskaya-Dikmen, C., Yucetepe, A., Karbancioglu-Guler, F., Daskaya, H., Ozcelik, B. (2017). Angiotensin-I-converting enzyme (ACE)-inhibitory peptides from plants. Nutrients, 9(4), art. no. 316.
 
10.
Darewicz, M., Borawska, J., Vegarud, G.E., Minkiewicz, P., Iwaniak, A. (2014). Angiotensin I-converting enzyme (ACE) inhibitory activity and ACE inhibitory peptides of salmon (Salmo salar) protein hydrolysates obtained by human and porcine gastrointestinal enzymes. International Journal of Molecular Sciences, 15(8), 14077-14101.
 
11.
Deng, S., Lutema, P.C., Gwekwe, B., Li, Y., Akida, J.S, Pang, Z., Huang, Y., Dang, Y., Wang, S., Chen, M., Miao, W., Lin, H., Wang, L., Cheng, L. (2019). Bitter peptides increase engulf of phagocytes in vitro and inhibit oxidation of myofibrillar protein in peeled shrimp (Litopenaeus vannamei) during chilled storage. Aquaculture Reports, 15, art. no. e100234.
 
12.
Ding, Y., Li, X., Kan, J. (2017). Isolation and identification of flavor peptides identified in douchi (traditional Chinese soybean food). International Journal of Food Properties, 20(suppl. 2), 1982-1994.
 
13.
Dziuba, J., Minkiewicz, P., Mogut, D. (2011). Determination of theoretical retention times for peptides analyzed by reversed-phase high-performance liquid chromatography. Acta Scientiarium Polonorum Technologia Alimentaria, 10(2), 209-221.
 
14.
Esmaeilpour, M., Ehsani, M.R., Aminlari, M., Shekarforoush, S., Hoseini, E. (2016). Antimicrobial activity of peptides derived from enzymatic hydrolysis of goat milk caseins. Comparative Clinical Pathology, 25(3), 599-605.
 
15.
Gallego, M., Mora, L., Toldrá, F. (2019). The relevance of dipeptides and tripeptides in the bioactivity of dry-cured ham. Food Production, Processing, and Nutrition, 1, art. no. 2.
 
16.
Guetouache, M., Bettache, G., Medjekal, S. (2014). Composition and nutritional value of raw milk. Issues in Biological Sciences and Pharmaceutical Research, 2(10), 115-122.
 
17.
Iwaniak, A., Dziuba, J. (2009). Animal and plant origin proteins as the precursors of peptides with ACE inhibitory activity. Proteins evaluation by means of in silico methods. Food Technology and Biotechnology, 47(4), 441-449.
 
18.
Iwaniak, A., Dziuba, J. (2011). BIOPEP-PBIL tool for the analysis of the structure of biologically active motifs derived from food proteins. Food Technology and Biotechnology, 49(1), 118-127.
 
19.
Iwaniak, A., Minkiewicz, P., Darewicz, M., Protasiewicz, M., Mogut, D. (2015). Chemometrics and cheminformatics in the analysis of biologically active peptides from food sources. Journal of Functional Foods, 16, 334-351.
 
20.
Iwaniak, A., Minkiewicz, P., Darewicz, M., Hrynkiewicz, M. (2016a). Food protein-originating peptides as tastants - Physiological, technological, sensory, and bioinformatic approaches. Food Research International, 89(1), 27-38.
 
21.
Iwaniak, A., Minkiewicz, P., Darewicz, M., Sieniawski, K., Starowicz, P. (2016b). BIOPEP database of sensory peptides and amino acids. Food Research International, 85, 155-161.
 
22.
Iwaniak, A., Hrynkiewicz, M., Bucholska, J., Darewicz, M., Minkiewicz, P. (2018). Structural characteristics of food protein-originating di- and tripeptides using principal component analysis. European Food Research and Technology, 244(10), 1751-1758.
 
23.
Iwaniak, A., Minkiewicz, P., Mogut, D., Darewicz, M. (2019a). Elucidation of the role of in silico methodologies in approaches to studying bioactive peptides derived from foods. Journal of Functional Foods, 61, art. no. 103486.
 
24.
Iwaniak, A., Hrynkiewicz, M., Bucholska, J., Minkiewicz, P., Darewicz, M. (2019b). Understanding the nature of bitter-taste di- and tripeptides derived from food proteins based on chemometric analysis. Journal of Food Biochemistry, 43, art. no. e12500.
 
25.
Khaldi, N. (2012). Bioinformatic approaches for identifying new therapeutic bioactive peptides in food. Functional Foods in Health and Disease, 2(10), 325-338.
 
26.
Kilara, A., Panyam, D. (2003). Peptides from milk proteins and their properties. Critical Reviews in Food Science and Nutrition, 43(6), 607-633.
 
27.
Kim, H., Li-Chan, E.C.Y. (2006). Application of Fourier transform Raman spectroscopy for prediction of bitterness of peptides. Applied Spectroscopy, 60(11), 1297-1306.
 
28.
Krokhin, O.V. (2006). Sequence Specific Retention Calculator - a novel algorithm for peptide retention prediction in ion-pair RP-HPLC: application to 300 Å and 100 Å pore size C18 sorbents. Analytical Chemistry, 78(22), 7785-7795.
 
29.
Krokhin, O.V., Craig, R., Spicer, V., Ens, W., Standing, K.G., Beavis, R.C., Wilkins, J.A. (2004). An improved model for prediction of retention times of tryptic peptides in ion pair reversed-phase HPLC. Molecular & Cellular Proteomics, 3(9), 908-919.
 
30.
Lacroix, I.M.E., Meng, G., Cheung, I.W.Y., Li-Chan, E.C.Y. (2016). Do whey protein-derived peptides have dual dipeptidyl-peptidase IV and angiotensin I-converting enzyme inhibitory activities? Journal of Functional Foods, 21, 87-96.
 
31.
Lafarga, T., Hayes, M. (2017). Bioactive protein hydrolysates in the functional food ingredient industry: overcoming current challenges. Food Reviews International, 33(3), 217-246.
 
32.
Li, S.S., Bu, T.T., Zheng, J.X., Liu, L., He, G.Q., Wu, J.P. (2019). Preparation, bioavailability, and mechanism of emerging activities of Ile-Pro-Pro and Val-Pro-Pro. Comprehensive Reviews in Food Science and Food Safety, 18(4), 10-97-1110.
 
33.
Li-Chan, E.C.Y. (2015). Bioactive peptides and protein hydrolysates: research and challenges for application as nutraceuticals and functional food ingredients. Current Opinion in Food Science, 1, 28-37.
 
34.
Lim, J. (2011). Hedonic scaling: A review of methods and theory. Food Quality and Preference, 22(8), 733-747.
 
35.
Mallick, P., Schirle, M., Chen, S.S., Flory M.R., Lee H., Martin D., Ranisz J., Raught B., Schmitt R., Werner P., Kuster B., Aebersold R. (2007). Computational prediction of proteotypic peptides for quantitative proteomics. Nature Biotechnology, 25(1), 125-131.
 
36.
Minkiewicz, P., Dziuba, J., Iwaniak, A., Dziuba, M., Darewicz, M. (2008). BIOPEP and other programs for processing bioactive peptide sequences. Journal AOAC International, 91(4), 965-980.
 
37.
Minkiewicz, P., Miciński, J., Darewicz, M., Bucholska, J. (2013). Biological and chemical databases for research into the composition of animal source foods. Food Reviews International, 29(4), 321-351.
 
38.
Mooney, C., Haslam, N.J., Pollastri, G., Shields, D.C. (2012). Towards the improved discovery and design of functional peptides: common features of diverse classes permit generalized prediction of bioactivity. PLoS ONE, 7(10), art. no. e45012.
 
39.
Otagiri, K., Miyake, I., Ishibashi, N., Fukui, H., Kanehisa, H., Okai, H. (1983). Studies of bitter peptides from casein hydrolyzate. II. Syntheses of bitter peptide fragments and analogs of BPIa (Arg-Gly-Pro-Pro-Phe-Ile-Val) from casein hydrolysate. Bulletin of the Chemical Society of Japan, 56, 1116–1119.
 
40.
Paizs, B., Suhai, S. (2005). Fragmentation pathways of protonated peptides. Mass Spectrometry Reviews, 24(4), 508-548.
 
41.
Panjaitan, F.C.A., Gomez, H.L.R., Chang, Y.-W. (2018). In silico analysis of bioactive peptides released from giant grouper (Epinephelus lanceolatus) roe proteins identified by proteomics approach. Molecules, 23(11), art. no. e2910.
 
42.
Pripp, A.H., Ardö, S. (2007). Modelling relationship between angiotensin-(I)-converting enzyme inhibition and the bitter taste of peptides. Food Chemistry, 102(3), 880-888.
 
43.
Rawlings, N.D. (2009). A large and accurate collection of peptidase cleavages in the MEROPS database. Database (Oxf.), bap015.
 
44.
Roepstorff, P., Fohlman, J. (1984). Proposal for a common nomenclature for sequence ions in mass spectra of peptides. Biomedical Mass Spectrometry, 11(11), 601.
 
45.
Ryan, J.T., Ross, P.R., Bolton, D., FitzGerald, G.F., Stanton, C. (2011). Bioactive peptides from muscle sources: meat and fish. Nutrients, 3(9), 765-791.
 
46.
Sánchez, A., Vázquez, A. (2017). Bioactive peptides: a review. Food Quality and Safety, 1(1), 29-46.
 
47.
Savitzky, A., Golay, M.J.E. (1964). Smoothing and differentiation of data by simplified least squares procedures. Analytical Chemistry, 36(8), 1627-1638.
 
48.
Spicer, V., Yamchuk, A., Cortens, J., Sousa, S., Ens, W., Standing, K.G., Wilkins, J. A., Krokhin, O.V. (2007). Sequence-specific retention calculator. A family of peptide retention time prediction algorithms in reversed-phase HPLC: applicability to various chromatographic conditions and columns. Analytical Chemistry, 79(22), 8762-8768.
 
49.
Temussi, P.A. (2012). The good taste of peptides. Journal of Peptide Science, 18(2), 73-82.
 
50.
The UniProt Consortium, 2019. UniProt: a worldwide hub of protein knowledge. Nucleic Acids Research, 47(D1), D506–D515.
 
51.
Udenigwe, C.C. (2014). Bioinformatic approaches, prospects and challenges of food bioactive peptide research. Trends in Food Science and Technology, 36(2), 137-143.
 
52.
Vermeirssen, V., van der Bent, A., Van Camp, J., van Amerongen, A., Verstraete, W. (2004). A quantitative in silico analysis calculates angiotensin I converting enzyme (ACE) inhibitory activity in pea and whey protein digests. Biochimie, 86(3), 231-239.
 
53.
Visser, S., Slangen, C.J., Rollema, H.S. (1991). Phenotyping of bovine milk proteins by reversed-phase high-performance liquid chromatography. Journal of Chromatography A, 548(12), 361-370.
 
54.
Zambrowicz, A., Timmer, M., Polanowski, A., Lubec, G., Trziszka, T. (2013). Manufacturing of peptides exhibiting biological activity. Amino Acids, 44(2), 315-320.
 
55.
Zamyatnin, A.A. (2009). Fragmentomics of natural peptide structures. Biochemistry (Moscow), 74(13), 1575-1585.
 
 
CITATIONS (2):
1.
Characteristics of Biopeptides Released In Silico from Collagens Using Quantitative Parameters
Anna Iwaniak, Piotr Minkiewicz, Monika Pliszka, Damir Mogut, Małgorzata Darewicz
Foods
 
2.
Soybean (Glycine max) Protein Hydrolysates as Sources of Peptide Bitter-Tasting Indicators: An Analysis Based on Hybrid and Fragmentomic Approaches
Anna Iwaniak, Monika Hrynkiewicz, Piotr Minkiewicz, Justyna Bucholska, Małgorzata Darewicz
Applied Sciences
 
eISSN:2083-6007
ISSN:1230-0322