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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  
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Faculty of Food Science, Chair of Food Biochemistry, University of Warmia and Mazury in Olsztyn, Poland
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
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.
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.
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).
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