Identification and Relative Quantification of Bioactive Peptides

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Identification and Relative Quantification of Bioactive Peptides Sequentially Released during Simulated Gastrointestinal Digestion of Commercial Kefir Yufang Liu, and Monika Pischetsrieder J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05385 • Publication Date (Web): 14 Feb 2017 Downloaded from http://pubs.acs.org on February 20, 2017

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Journal of Agricultural and Food Chemistry

Identification and Relative Quantification of Bioactive Peptides Sequentially Released during Simulated Gastrointestinal Digestion of Commercial Kefir

Yufang Liu, Monika Pischetsrieder*

Department of Chemistry and Pharmacy, Food Chemistry, University of Erlangen-Nuremberg, Schuhstraße 19, 91052 Erlangen, Germany

* Corresponding author: Prof. Dr. Monika Pischetsrieder, Chair of Food Chemistry, Emil Fischer Center, Schuhstr. 19, 91052 Erlangen, Germany, telephone: +49-9131-8524102, telefax: +499131-8522587, Email: [email protected]

1 ACS Paragon Plus Environment


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Abstract

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Health-promoting effects of kefir may be partially caused by bioactive peptides. To evaluate

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their formation or degradation during gastrointestinal digestion, we monitored changes of the

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peptide profile in a model of oral (1), gastric (2) and small intestinal (3) digestion of kefir.

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MALDI-TOF-MS analyses revealed clearly different profiles between digests 2/3 and

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kefir/digest 1. Subsequent UPLC˗ESI-MS/MS identified 92 peptides in total (25, 25, 43, and 30,

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partly overlapping in kefir and digests 1, 2, and 3, respectively), including 16 peptides with

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ascribed bioactivity. Relative quantification in scheduled multiple reaction monitoring mode

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showed that many bioactive peptides were released by simulated digestion. Most prominently,

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the concentration of ACE inhibitor β-casein203-209 increased approximately 10,000-fold after

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combined oral, gastric, and intestinal digestion. Thus, physiological digestive processes may

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promote bioactive peptide formation from proteins and oligopeptides in kefir. Furthermore,

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bioactive peptides present in certain compartments of the gastrointestinal tract may exert local

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physiological effects.

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Keywords

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Kefir, peptide profile, bioactive peptides, gastrointestinal digestion


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Introduction

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Kefir, an acidic and mildly alcoholic fermented milk beverage with a refreshing and yeasty taste,

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has a long history of being beneficial to human health.1 Traditionally, it is obtained by culturing

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milk with kefir grains, which is a complex mixture of bacteria and yeast cohering in a

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polysaccharide matrix.2 Kefir grains are important to kefir production, but it is difficult to

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maintain the stability of their microbiological composition during industrial production. Thus,

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mostly well-defined starter cultures are applied for commercial products.3 Several in vitro and

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animal studies, but also a few human trials provide evidence that kefir and its constituents exert

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health-promoting effects, like anticarcinogenic, antimicrobial, and immunomodulatory activity.4

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These physiological effects are mainly attributed to the presence of a probiotic microflora.

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However, also metabolites of the microorganisms seem to contribute considerably to the

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observed biological effects of kefir.4 Peptides that are released from milk proteins exert, for

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example, antihypertensive, immunomodulating, or antimicrobial activity.5-7 Recently, it was

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shown that the conversion of milk to kefir increases the total yield of peptides and also

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substantially changes the composition of the peptide fraction compared to raw milk.8, 9 Sixteen of

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the newly identified kefir peptides were reported previously as bioactive compounds showing,

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for example, angiotensin-converting enzyme (ACE)-inhibitory, immunomodulating,

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antimicrobial, mineral-binding, antioxidant, and antithrombotic activities.8 Quiros et al.

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additionally identified two potent ACE inhibitors in the peptide fractions of caprine kefir.10

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Kefir-derived peptides may exert local physiological activity, for example in the oral cavity or in

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the upper gastrointestinal tract.11 However, the ability of bioactive peptides to show systemic

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effects or effects in the lower gastrointestinal tract depends on their stability during digestion

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(bioaccessibility), because digestion can lead to the complete or full degradation of peptides. 3 ACS Paragon Plus Environment


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Systemic effects of peptides additionally rely on their absorption rate into the blood stream

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(bioavailability).12 On the other hand, bioactive peptides may also be released during

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gastrointestinal digestion from protein precursors or larger peptides.13-15

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The present study monitored the peptide profile of kefir at different steps of an in vitro model of

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the human digestion system with the aim to better understand and predict the health benefits of

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kefir related to bioactive peptides. For this purpose, the digestive enzymes amylase, pepsin, and

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pancreatic proteases were sequentially used to simulate oral, gastric, and small intestinal

48

environment. The peptide profiles of the samples were recorded by matrix-assisted laser

49

desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF-MS). Sequences of the

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most abundant peptides were subsequently identified by ultra-performance liquid

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chromatography electrospray ionization tandem mass spectrometry (UPLC−ESI-MS/MS) and

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relative quantification of bioactive peptides was achieved by UPLC−ESI-MS/MS in scheduled

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multiple reaction monitoring mode (sMRM).

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Material and Methods

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In Vitro Digestion Procedure

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Three different batches of kefir from the same brand were purchased from a local supermarket.

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The kefir was industrially produced from pasteurized and homogenized low-fat (1.5 % fat)

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organic cow milk using a complex culture of yeast and lactic acid bacteria. In vitro digestive

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juices (salivary juice, gastric juice, duodenal juice, and bile juice) were prepared and digestion

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processes were carried out as reported previously by Versantvoort et al. with some


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modifications.16 Briefly, the saliva model contained α-amylase (catalogue number 10080, Sigma-

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Aldrich, Taufkirchen, Germany) and mucin (Roth, Karlsruhe, Germany), while the main

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ingredients of gastric, duonenal, and bile juices were pepsin (catalogue number P 7000),

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pancreatin (catalogue number P3292), and bile extract (all Sigma-Aldrich, Taufkirchen,

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Germany), respectively. Prior to use, all digestive juices were heated to 37±2 ℃. Five milliliters

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of the kefir sample or MilliQ water as blank control was mixed in a ratio of 1:1 (v/v) in an

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Erlenmeyer flask with in vitro salivary juice and incubated for 5 min at 37 ℃ (digest 1). In vitro

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gastric juice (7.5 mL) was added to the mixture and the pH was adjusted to 2−3 with 0.1 M HCl.

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The mixture was incubated for 2 h at 37 ℃ (digest 2). Finally, 15 mL of in vitro duodenal juice

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and 5 mL of bile juice were added to the mixture and the pH was adjusted to 7±0.2 with 0.1 M

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NaOH. The final mixture was incubated for another 2 h at 37 ℃ (digest 3). After the incubation,

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kefir sample and the digests 1, 2, and 3 were immediately cooled on ice and centrifuged for 30

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min at 1100×g at 4 ℃. Experiments were conducted in three independent triplicates using three

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different kefir batches. Prior to MALDI-TOF-MS and UPLC˗ESI-MS/MS, the kefir digests were

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filtered through an ultrafiltration membrane with a cutoff of 10 kDa (Merck, Darmstadt,

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Germany).

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Peptide Extraction

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For MALDI-TOF-MS and UPLC˗ESI-MS/MS analysis, peptides were extracted from the

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filtrates by stage-tip extraction according to Baum et al., with some modifications.17 For the

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preparation of a stage tip, three small layers with a diameter of 1 mm each were punched from a

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C18 Empore Disk (3 M, Neuss, Germany) with a biopsy punch (KAI Medical, Solingen,



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Germany) and placed into a 1−10 µL pipet tip (Eppendorf, Hamburg, Germany). The tip was

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inserted into a perforated cap of a 1.5 mL test tube. For the peptide extraction, 50 µL of

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acetonitrile was pipetted into a stage tip and centrifuged at 1845×g at 25 ℃ for 1 min followed

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by a washing step with 50 µL of formic acid. Then, 50 µL of the ultrafiltrate of the kefir sample,

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digests, or blank control was loaded into the stage tip and centrifuged for 5 min. Subsequently,

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the tip was washed with 0.1 formic acid and centrifuged for 3 min. Prior to elution, the tip was

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removed to a new 1.5 mL test tube. Finally, 10 µL of 60% acetonitrile in 0.1% formic acid was

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pipetted into the tip and centrifuged for 3 min. The eluents were collected and stored at −20℃ for

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further use.

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MALDI-TOF-MS

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MALDI matrix consisted of 5 mg of 4-chloro-α-cyanocinnamic acid (Sigma-Aldrich,

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Taufkirchen, Germany) in 1 mL of a mixture of acetonitrile and 0.1% trifluoroacetic acid (60:40,

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v/v). The eluates from stage-tip extraction were mixed 1:1 with matrix, spotted onto a ground

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steel target plate (Bruker Daltonics, Bremen, Germany) and air-dried. MALDI-TOF-MS (Bruker

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Daltonics, Bremen, Germany) was performed in the positive reflector mode with a nitrogen laser.

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For each sample, 200 single spectra were automatically generated from different spot positions in

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a mass range of 600−5000 Da and summed up. External calibration was performed using Bruker

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peptide calibration standard II.

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Analysis by UPLC˗ESI-MS/MS


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Peptide identification was performed on a Dionex Ultimate 3000 RS system (Dionex, Idstein,

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Germany) coupled to an API 4000 QTrap mass spectrometer (AB Sciex, Forster City, CA, USA),

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equipped with an ESI source (Applied Biosystems, Forster City, CA, USA). The peptide extracts

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were diluted 1:5 with eluent A (0.1% formic acid in MilliQ water) and injected into a C18

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column (Waters Acquity UPLC BEH 300; 2.1 mm×100 mm, 1.7 µm) with a flow rate of 0.3

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mL/min and a column temperature of 30 ℃. The peptides were separated using the following

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gradient: −6 min 5% B (acetonitrile, LC-MS grade, VWR International, Darmstadt, Germany), 0

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min 5% B, 5 min 5% B, 55 min 50% B, 56 min 95% B, and 60 min 95% B. The injection

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volume was 2 µL. After 1 min, the LC flow was directly led into the mass spectrometer for 59

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min. The ion source was operated at the voltage of 5000 V and declustering potential of 50 V. To

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determine the retention time of the peptides previously detected by MALDI-TOF-MS and their

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charge state, enhanced MS (EMS) scans were performed. The identity of the peptides was then

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confirmed by enhanced product ion (EPI) scans. For the untargeted analysis, EMS scans of

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peptide extracts were performed to determine the m/z values, retention time and charge state of

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the most abundant peptides. EPI scans were subsequently used for the identification of the

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peptides. Collision energy was set to 30 V and collision energy spread was 10 V. All MS

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experiments were carried out in the positive mode.

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Analyst version 1.5 (AB Sciex, Forster City, CA, USA) was used for data acquisition and

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processing. Identification of peptide sequences was aimed for by searching the spectra from

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UPLC˗ESI-MS/MS in the SwissProt database by Mascot (Matrix Science, London, U.K.).

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Because the proteases leading to peptide release were not known, the Mascot search provided the

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sequence information only in some cases. To determine the other sequences, a bovine milk

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peptide database was established by an ion fragment calculator in mMass (version 5.5.0) 7 ACS Paragon Plus Environment


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including precursor, position, and mass of all possible peptides derived from αs1-, αs2-, β-, and κ-

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casein, β-lactoglobulin, and α-lactalbumin. The peptides were then manually identified by

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comparing the experimentally determined m/z of the product ions in the EPI spectra with the

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theoretical m/z of product ions of all possible sequences in the database that have the same m/z

132

value as the analyzed peptide.

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Scheduled Multiple Reaction Monitoring

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Relative quantification of bioactive peptides was performed by the sMRM method employing the

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same LC and MS parameters as described above. Angiotensin I was used as internal standard for

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relative quantification at the concentration of 4 µM. Angiotensin I was mixed with diluted

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peptide extracts and was analyzed by UPLC˗ESI-MS/MS. An enhanced MS spectrum (EMS)

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was acquired to determine the retention time of the bioactive peptide and EPI spectra were

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recorded to determine b- and y-ions suitable for sMRM analysis. During the method

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development, the three most intensive transitions per target peptide were selected as one

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quantifier and two qualifiers (see Supporting Information, Table S1). Target scan time was set to

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1.2 s.

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The relative intensity for each target peptide was calculated from the area of extracted ion

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chromatograms of sMRM runs compared with the internal standard, which was performed by

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Analyst software version 1.5.

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Literature Search and Statistical Analysis


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A literature search for bioactivity was performed for all peptides, which had been detected in

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kefir sample and the three digests, using the databases Google Scholar, PubMed, and ISI Web of

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Knowledge with the respective peptide sequence in single letter code as the search term.

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Statistical data analysis was performed in Microsoft Office Excel 2010. Level of significance

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was calculated using unpaired Student’s t-test.

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Results and Discussion

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Analysis of the Peptide Profiles of Kefir and Kefir Digests by MALDI-TOF-MS

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The aim of this study was to investigate the effects of simulated gastrointestinal digestion on the

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profile of bioactive peptides in kefir. Gastrointestinal digestion can influence the peptide profile

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of dairy products, on the one hand by the release of new peptides from precursors and, on the

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other hand, by degradation processes. Thus, the digestive enzymes amylase, pepsin, and

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pancreatic proteases were sequentially applied to simulate the oral, gastric, and small intestinal

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environment. In vitro methods for simulating the human gastrointestinal tract provide a rapid and

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inexpensive alternative to in vivo studies on animals or humans.18 These methods have been used,

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for example, to test the ACE-inhibitory activity of cheese and infant formulas.19, 20 While the

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present study was in progress, a consensus paper was released presenting a standardized protocol

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for simulated gastrointestinal digestion,21 which mainly differs by the concentration of the

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inorganic salts and the absence of organic additives in the digestion matrices. Since the proteases

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were the same, however, it can be assumed that the differences in the protocols would rather

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affect the absolute concentrations of the peptides than the composition of the peptide profiles.



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No common signals were detected in the MALDI-TOF spectra of kefir digests and blank control,

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which underwent the complete digestion process in the absence of kefir, so that autohydrolysis

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processes were assumed to be negligible in the applied in vitro digestion model. Peptide profiles

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were first recorded by direct MALDI-TOF-MS measurement in the m/z range of 600−5000 Da

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after C18 stage-tip extraction. Although direct MALDI analysis does not generate a

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comprehensive overview of the entire peptide fraction,6, 22 it provides a fast fingerprint of

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predominant peptides, which can be used to monitor changes in the peptide profile in different

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samples. In particular, changes in the size distribution of the peptides can be easily recorded. In

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total, four different samples were analyzed: kefir and the three digests that were sequentially

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released by in vitro digestion (Figure 1). Most of the signals in kefir and digest 1 were detected

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in the range of 1000−2200 Da, corresponding to the peptide length of approximately 7−20 amino

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acids. In these two samples, MALDI-TOF-MS displayed 25 signals for each sample. No major

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changes in the peptide profile were detected in digest 1. This result was expected, because the

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saliva model contained only α-amylase, which should not cause protein or peptide degradation.

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In contrast, the spectrum of digest 2 showed clear differences compared to the peptide profile of

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kefir and digest 1. The relative intensities of the two most abundant peptides in kefir and digest 1

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with m/z 1700.7 and m/z 1718.1 decreased substantially. Furthermore, the action of pepsin led to

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a series of new signals in the m/z-range of 2000−3000 Da (Figure 1C). Most likely, these

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peptides originate from peptic proteolysis of milk proteins or larger protein fragments. Figure 1D

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shows that the pancreatic enzymes almost completely degraded the medium-sized peptides from

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the gastric digest. Instead, short-sized peptides arose. Compared to the native kefir sample, more

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signals of digest 3 appeared in the range of 800−2000 Da. However, the peptide with the m/z

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1881.1 was always one of the most intensive peaks.


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Identification of Predominant Peptides by UPLC˗ESI-MS/MS

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To identify the peptides that were detected by MALDI-TOF-MS in the native kefir and the

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digests, EMS and EPI spectra of these peptides were recorded by targeted UPLC˗ESI-MS/MS. In

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addition, untargeted UPLC˗ESI-MS/MS analysis was performed for a comprehensive analysis of

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remaining peptides. The application of two complementary ionization methods can increase the

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overall number of detected peptides.8 In total, 92 peptide sequences were identified in kefir and

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the digests as shown in Table 1. All of the 25 most abundant peptides in kefir represented β-

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casein (19) and κ-casein (6) fragments. Fourteen of these kefir peptides have been identified

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previously in kefir samples by untargeted LC˗ESI-MS/MS.8 After salivary digestion, three

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additional fragments were detected in digest 1 and confirmed as RDMPIQAFL,

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SLSQSKVLPVPQ, and EMPFPKYPVEPF, corresponding to the amino acid sequences 183˗191,

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164˗175, and 108˗119 of β-casein. After pepsin hydrolysis, 43 peptides were identified in digest

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2, 18 of which arose from β-casein, 14 from αS1-casein and αS2-casein, 4 from κ-casein, and 7

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from β-lactoglobulin. Three peptides, corresponding to the C-terminal region of β-casein (β-

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casein199-209, β-casein194-209, and β-casein193-209) and one fragment of κ-casein (κ-casein33-41) from

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digest 1 were not degraded during the pepsin hydrolysis, whereas 39 peptides were newly

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generated. To determine the mechanism of peptide generation, an in silico digest of the milk

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proteins with pepsin was carried out with mMass. However, only one of the peptides detected in

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digest 2, αS1-casein146-149, could be assigned to a typical pepsin cleavage product. The other

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peptides could be generated by the combined activity of pepsin and proteinases from the kefir

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microflora.



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A total of 30 peptide fragments were present after the complete three-step digestive processes,

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including five αS1-casein fragments, twenty-four β-casein fragments, and one β-lactoglobulin

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fragment. Three fragments derived from the C-terminal region of β-casein (β-casein199-209, β-

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casein196-209, and β-casein193-209) and two fragments from αS1-casein (αS1-casein146-149 and αS1-

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casein24-31) in digest 2 also survived during the hydrolysis of pancreatic enzymes. Only one of

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these peptides, β-casein193-209, has also been detected after the simulated gastrointestinal

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digestion of intact milk proteins, whereas the other 29 peptides are specific for kefir.22 Sanchez-

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Rivera et al. compared the peptide profile of Spanish blue cheese before and after the simulated

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gastrointestinal digestion and revealed a total peptide homology of 12.1%. Jin et al. studied the

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diversity of the peptide profiles after gastric and pancreatic digest of yoghurt and found only less

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than 5% of the peptides present in all three samples.23

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The most abundant proteins in milk are αS1-casein (34%), β-casein (25%), κ-casein (9%), β-

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lactoglobulin (9%), αS2-casein (8%), and α-lactalbumin (4%).24 In contrast to this composition,

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76% of the peptides detected in kefir derived from β-casein and 24% from κ-casein. Although

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αS1-casein is the most abundant milk protein, and β-lactoglobulin as well as αS2-casein are

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present in similar concentrations as κ-casein, no peptides from one of these three proteins were

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detected in kefir. These results indicate preferential proteolysis of β-casein and κ-casein by

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starter cultures during milk fermentation, which is in accordance with a previous study.8 Neither

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were any peptides detected from α-lactalbumin, but this observation could also be caused by its

234

lower abundancy in milk. In a similar way, 56% of the detectable peptides that were newly

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generated in digest 2 or digest 3 derived from β-casein. Thus, it can be concluded that the

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proteases in the simulated gastrointestinal digestion also preferentially attack β-casein, which is

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consistent with a previous study showing that pancreatic hydrolysis of bovine casein mainly


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released peptides from β-casein.25 Particularly in digest 2, several peptides derived from β-

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lactoglobulin were detected. It can, therefore, be assumed that β-lactoglobulin is of relevance for

240

the digestive formation of peptides, whereas whey proteins are not important as peptide

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precursors during fermentation or indigenous proteolysis.6, 8 Caseins are extremely sensitive to

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proteolysis due to their flexible and open structures.23 During gastrointestinal digestion, the

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globular structure of β-lactoglobulin is gradually denatured, which may promote its proteolysis.

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To determine the exact proteolysis rate of each milk protein at different steps, however, in

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addition to the number of specifically released peptides, their concentration must be measured.

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Interestingly, the identified peptides were not randomly distributed along the total protein

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sequences. While specific cleavage sites in the parent protein sequence are particularly prone to

248

proteolysis and release a large number of peptides during fermentation and digestion, some

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protein regions, however, are not susceptible to peptide formation. These observation indicates

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the action of site-specific microbial and digestive enzymes: in kefir, several peptides from β-

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casein originate from the regions between N132 and W143 and from Y193 to C-terminus. The

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cleavage sites of N132˗L133 and Y193˗Q194 can be explained by the activity of lactococcal

253

proteinase deriving from the starter culture of kefir production.8 A detailed comparison of casein

254

cleavage sites detected after kefir fermentation and the specificity of lactococcal proteinases has

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been given by Ebner et al..8 In digest 2 and 3, peptides also mainly originate from the C-terminal

256

region of β-casein. The cleavage site of R202˗G203 can be explained by the activity of trypsin

257

added for the simulated intestinal digestion. Several cleavage sites in the peptides of digest 2 and

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3 can be attributed to the activity of the gastric protease pepsin, e.g. F23˗F24 and Y91˗L92 in αS1-

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casein, and L139˗L140, F190˗L191, and L192˗Y193 in β-casein.26 Therefore, pepsin, which cleaves



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peptide bonds between hydrophobic and preferably aromatic amino acids, is probably

261

responsible for the activity at a part of the remaining cleavage sites.

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Fate of Bioactive Peptides from Kefir during the Simulated Digestion Process

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Kefir-derived peptides are of particular nutritional relevance when they exert some kind of

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bioactivity. Therefore, the peptides that were already present in kefir or generated during

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digestion were searched for components with known bioactivity. Biofunctional properties have

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been recorded before for 16 of the 92 identified peptides. Among the bioactive peptides, three

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were found in kefir and digest 1, seven in digest 2, and ten peptides in digest 3. The major part of

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these milk protein-derived bioactive peptides are ACE inhibitors. ACE increases blood pressure

270

by converting the inactive angiotensin I to the potent vasoconstrictor angiotensin II and by

271

inactivating the vasodilator bradykinin. Inhibition of ACE may exert an antihypertensive effect

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by decreasing the concentration of angiotensin II and increasing the concentration of

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bradykinin.27 Eleven peptides in kefir and digests have been reported as ACE inhibitors.27-37 The

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prevalence of ACE-inhibiting peptides may be biased by the fact that these type of bioactive

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milk peptides are very well studied and many ACE-inhibitory sequences have been identified,

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whereas other biofunctions of milk peptides are not so well investigated. One of the identified

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peptides causes a bitter taste.38 Besides the direct impact of bitter compounds on the food taste,

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they may also interact with extra-orally expressed taste receptors influencing thus, for example,

279

appetite or gastrointestinal motility.39 Two antimicrobial peptides, casecidin 15 and casecidin 17,

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with sequences YQEPVLGPVRGPFPI (β-casein193-207) and YQEPVLGPVRGPFPIIV (β-

281

casein193-209), were identified. These two antimicrobial peptides have identical inhibitory


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concentrations (MICs) of 0.4 mg/mL against E. coli DPC6053 and 0.5 mg/mL against E. coli

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DH5α.40 Two fragments, VYPFPGPIPN (β-casein59-68) and YPVEPF (β-casein114-119), are

284

capable of binding opioid receptors and, thus, may have an impact on food intake and

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gastrointestinal motility.36, 41 Additionally, the fragments YPEL (αS1-casein146-149) and

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VYPFPGPIPN (β-casein59-68) are reported to have antioxidant effects, whereas PGPIPN (β-

287

casein63-68) and LYQEPVLGPVRGPFPIIV (β-casein192-209) can affect the immune system. The

288

former has been found to inhibit the proliferation of human ovarian cancer cell lines and decrease

289

tumor growth rate in xenograft ovarian cancer model mice in a dose-dependent manner,42

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whereas they had a stimulatory effect on primed lymph node cells and enhanced the proliferation

291

of rat lymphocytes.31 In addition to these known bioactive peptides, the function of other

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peptides may still be unrevealed. Bioactive peptides may exert their function locally in the

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gastrointestinal tract. For example, antioxidative or antimicrobial peptides may directly improve

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gut health. Moreover, opioid and bitter peptides can interact with the µ-receptor or bitter

295

receptors in the intestine, thus controlling gastrointestinal function.5 ACE inhibitors and

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immunomodulatory peptides, in contrast, exclusively function systemically. Therefore, their

297

absorption behavior and stability in vivo need to be considered in further studies to fully evaluate

298

their physiological potential.

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To evaluate if the bioactive peptides can be of nutritional relevance, changes of their

300

concentrations during in vitro digestion were monitored. For this purpose, a scheduled

301

UPLC˗ESI-MS/MS-MRM method was developed for each of the 16 bioactive peptides and

302

relative quantification was performed using angiotensin I as internal standard.

303

Two bioactive peptides, namely β-casein183-190 and β-casein169-175 were primarily present in kefir

304

and digest 1 as shown in Figure 2A, but were considerably degraded during the gastric and 15 ACS Paragon Plus Environment


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intestinal digestion. Therefore, it can be concluded that they are probably of minor nutritional

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relevance. Seven peptides, αs1-casein146-149, αs2-casein89-95, β-casein130-140, β-casein192-209, β-

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casein132-140, β-casein193-209, and αs1-casein24-31 were released from precursors during the gastric

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digest. Among those were four ACE inhibitors, one immunomodulator, one peptide with radical

309

scavenging activity, and one peptide with multiple functions. Six of these peptides were strongly

310

degraded during the following hydrolysis step (Figure 2A). Thus, it can be concluded that they

311

may be of rather local relevance, such as antimicrobial activity of β-casein193-209 or radical

312

scavenging activity of αs1-casein146-149 in the stomach. The fragment αs1-casein24-31, in contrast,

313

was formed during gastric digest, but was not degraded any further during small intestinal

314

hydrolysis. This peptide was also identified in the gastric and pancreatic digest of yoghurt, and

315

showed DPP-IV inhibition and ACE inhibition with IC50 of 2.52 µM and 35.76 µM.23

316

Additionally, seven other bioactive peptides, namely β-casein108-113, β-casein59-68, β-casein203-209,

317

β-casein114-119, β-casein63-68, β-casein193-207, and β-casein6-14 were largely released during

318

intestinal digestion and remained stable during this step (Figure 2B). Among those, β-casein108-

319

113,

320

in Spanish blue cheese and/or yoghurt after the simulated gastrointestinal digestion,23, 43 whereas

321

one immunomodulating peptide (β-casein63-68), which suppresses ovarian cancer cell growth in

322

vitro and in vivo, was uniquely released during the digestion of kefir.42 Bioactive peptides

323

present after intestinal digestion can be assumed to exert their activity in the small intestine. For

324

example, β-casein193-207 may act as an antimicrobial compound in the digestive tract.

325

Additionally, intestinal absorption of peptides may take place by different mechanisms leading to

326

systemic activity.12 This mechanism is of particular importance for the clinical effects of ACE

327

inhibitory peptides.

β-casein59-68, β-casein203-209, β-casein114-119, β-casein193-207, and β-casein6-14 were also detected


Page 17 of 34


328

Compared to the two bioactive peptides originally predominant in the kefir sample,

329

gastrointestinal in vitro digestion released –in some cases transiently− 14 additional peptides

330

with known bioactivity. Although both major bioactive peptides in kefir were considerably

331

degraded during the entire digestion process, six peptides were transiently released and another

332

eight were predominant after the complete digestion process. These results suggest that

333

physiological digestion may rather lead to an increase of bioactive peptides by their release from

334

precursor proteins or larger peptides than to a decrease by the degradation of originally present

335

kefir peptides.

336

However, where and how these bioactive peptides exert beneficial effects in humans will be

337

subject to further investigation. In particular, the bioavailibility of peptides addressing systemic

338

targets must be investigated. Additionally, the concentrations of each of the bioactive peptides

339

need to be studied in order to evaluate their relevance. Boutrou et al. quantified the opioid β-

340

casomorphin-7 in the jejunum of healthy humans after the consumption of milk proteins and

341

determined a concentration in the IC50 range of its activity, suggesting biological relevance.14

342

Similar studies are now required for other functional peptides.

343

344

Abbreviations

345

ACE, angiotensin-converting enzyme; MALDI-TOF-MS, matrix-assisted laser

346

desorption/ionization time-of-flight mass spectroscopy; UPLC˗ESI-MS/MS, ultra-performance

347

liquid chromatography electrospray ionization tandem mass spectrometry; sMRM, scheduled

348

multiple reaction monitoring; EMS, enhanced MS spectrum; EPI, enhanced product ion

349 17 ACS Paragon Plus Environment


350

Acknowledgments

351

We thank Dr. Xiang from the Institute of Biochemistry, Friedrich-Alexander-Universität

352

Erlangen-Nürnberg (FAU), for providing the MALDI-TOF mass spectrometer and Christine

353

Meissner for proofreading the manuscript.

Page 18 of 34

354

355

Funding Sources

356

Financial support by the China Scholarship Council (CSC) to YL is gratefully acknowledged

357

(No. 201206790009).

358

359

Supporting Information (Table of sMRM parameters for the internal standard (angiotensin I)

360

and targeted bioactive peptides in kefir and digests) is available online.


Page 19 of 34


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477


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Figure Captions

479

Figure 1. Kefir was subjected to simulated subsequent oral (digest 1), gastric (digest 2), and

480

small intestinal (digest 3) digestion and the peptide profiles were analyzed by MALDI-TOF‒MS.

481

The mass spectra of kefir (A), digest 1 (B), digest 2 (C), and digest 3 (D) in the mass range of

482

600˗5000 Da are depicted. The peaks were assigned by UPLC‒ESI‒MS/MS to peptide

483

sequences as indicated.

484

Figure 2. Relative quantification of bioactive peptides in kefir and oral (digest 1), gastric (digest

485

2) and small intestinal (digest 3) digests using angiotensin I as reference: A) peptides

486

predominantly present in kefir, digest 1 and 2; B) peptides predominantly present in digest 3.

487

The mean values of an independent triplicate ± standard deviation are shown (*p

Identification and Relative Quantification of Bioactive Peptides

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