Gastric digestion of the sweet-tasting plant protein thaumatin releases bitter peptides that reduce H. pylori induced pro-inflammatory IL-17A release via the TAS2R16 bitter taste receptor

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Food Chemistry 448 (2024) 139157

Available online 28 March 2024

Gastric digestion of the sweet-tasting plant protein thaumatin releases

bitter peptides that reduce H. pylori induced pro-inammatory IL-17A

release via the TAS2R16 bitter taste receptor

Phil Richter

, Karin Sebald

, Konrad Fischer

, Angelika Schnieke

, Malek Jlilati

Verena Mittermeier-Klessinger

, Veronika Somoza

TUM School of Life Sciences Weihenstephan, Technical University of Munich, Alte Akademie 8, 85354 Freising, Germany

Leibniz Institute for Food Systems Biology at the Technical University of Munich, Lise-Meitner-Str. 34, 85354 Freising, Germany

Livestock Biotechnology, TUM School of Life Sciences, Technical University of Munich, Liesel-Beckmann-Str. 1, 85,354 Freising, Germany

Food Chemistry and Molecular Sensory Science, Technical University of Munich, Lise-Meitner-Str. 34, 85354 Freising, Germany

Nutritional Systems Biology, TUM School of Life Sciences, Technical University of Munich, Lise-Meitner-Str. 34, 85,354 Freising, Germany

Department of Physiological Chemistry, Faculty of Chemistry, University of Vienna, Josef-Holaubek-Platz 2 (UZA II), 1090 Wien, Austria

ARTICLE INFO

Keywords:

Thaumatin

Bitter peptides

Gastric digestion

Gastric acid secretion

Bitter taste receptors

Cytokines

ABSTRACT

About half of the world's population is infected with the bacterium Helicobacter pylori. For colonization, the

bacterium neutralizes the low gastric pH and recruits immune cells to the stomach. The immune cells secrete

cytokines, i.e., the pro-inammatory IL-17A, which directly or indirectly damage surface epithelial cells. Since (I)

dietary proteins are known to be digested into bitter tasting peptides in the gastric lumen, and (II) bitter tasting

compounds have been demonstrated to reduce the release of pro-inammatory cytokines through functional

involvement of bitter taste receptors (TAS2Rs), we hypothesized that the sweet-tasting plant protein thaumatin

would be cleaved into anti-inammatory bitter peptides during gastric digestion. Using immortalized human

parietal cells (HGT-1 cells), we demonstrated a bitter taste receptor TAS2R16-dependent reduction of a H. pylori-

evoked IL-17A release by up to 89.7 ±21.9% (p ≤0.01). Functional involvement of TAS2R16 was demonstrated

by the study of specic antagonists and siRNA knock-down experiments.

1. Introduction

The sweet-tasting protein thaumatin is naturally occurring in Thau-

matococcus daniellii, a plant found mainly in Africa. First described and

isolated in 1972, its sweet intensity was demonstrated to be 1600 times

that of sucrose on a weight basis (van der Wel & Loeve, 1972). Due to its

intense sweetness, thaumatin (E 957) is added as sugar substitute to

foods and beverages in concentrations between 0.5 and 400 mg/kg

(Younes et al., 2021). Although the pharmacological characteristics of

thaumatin have been extensively studied before the FDA has given GRAS

notice (Gleba, 2017), peptides released after gastric digestion of thau-

matin have so far not been characterized, neither for their taste quality

nor for their (taste quality-associated) effects on the stomach's

physiology.

Humans can perceive ve recognized taste qualities: sweet, bitter,

salty, sour and umami. The perception of these taste qualities after food

intake is primarily mediated by taste receptors located on taste cells of

the tongue. In extra-oral tissues, the expression of taste receptors,

namely bitter taste receptors (TAS2Rs), have been demonstrated to play

central roles in various physiological functions (Behrens & Meyerhof,

2013). While intestinal TAS2Rs modulate mechanisms regulating the

control of food intake and the innate immune system, activation of

gastric TAS2Rs stimulates proton secretion from parietal cells and are

thereby involved in digestive mechanisms of gastric acid secretion (Liszt

et al., 2017; Liszt et al., 2022).

However, an oral bitter taste perception of a given food constituent

does not necessarily result in the activation of gastro-intestinal TAS2Rs.

Digestive degradation might change the chemical structure and, in turn,

the TAS2R activation prole and associated physiological functions of

the parent compound. In one of our previous works, we have

* Corresponding author at: Leibniz Institute for Food Systems Biology at the Technical University of Munich, Lise-Meitner-Str. 34, 85354 Freising, Germany.

E-mail addresses: p.richter.leibniz-lsb@tum.de (P. Richter), k.sebald.leibniz-lsb@tum.de (K. Sebald), konrad.scher@tum.de (K. Fischer), angelika.schnieke@

wzw.tum.de (A. Schnieke), verena.mittermeier@tum.de (V. Mittermeier-Klessinger), v.somoza.leibniz-lsb@tum.de (V. Somoza).

Contents lists available at ScienceDirect

Food Chemistry

journal homepage: www.elsevier.com/locate/foodchem

https://doi.org/10.1016/j.foodchem.2024.139157

Received 23 December 2023; Received in revised form 8 March 2024; Accepted 25 March 2024

Food Chemistry 448 (2024) 139157

demonstrated gastric digestion of the non-bitter tasting milk protein

casein to release bitter peptides that stimulated mechanisms of gastric

acid secretion by immortalized human parietal cells (HGT-1) via func-

tional involvement of TAS2R16 and TAS2R38 (Richter et al., 2022).

Besides digestive processes, TAS2Rs have also been shown to play a role

in preventing immune competent cells from a stimulus-evoked release of

pro-inammatory cytokines (Liszt et al., 2022;Tiroch et al., 2021; Tiroch

et al., 2023). In immortalized human gingival broblasts (HGF-1 cells),

the release of the pro-inammatory interleukin 6, evoked by treatment

with lipopolysaccharides (LPS) from Porphyromonas gingivalis, was

reduced by various TAS2R agonists and TAS2R50 siRNA knock-down

(Tiroch et al., 2021; Tiroch et al., 2023).

From a mechanistic perspective, activation of TAS2R16 by its agonist

salicin (Bufe, Hofmann, Krautwurst, Raguse, & Meyerhof, 2002) sup-

presses NF-κB p65 nuclear translocation and intracellular cAMP in HGF-

1 cells, after treatment with LPS. In addition, down-regulation of gene

expression of many pro-inammatory cytokines occurred upon co-

incubation of cells with LPS and salicin, which resulted in a decreased

release of the pro-inammatory cytokine IL-8 (Zhou et al., 2021). In the

context of innate immunity, it has been shown that the activation of

TAS2R38 by corresponding bitter substances leads to calcium mobili-

zation, which might trigger the formation of NO as well-known anti-

bacterial agent (Bethineedi et al., 2023).

Inammatory processes in the stomach that are associated with the

release of various cytokines are often caused by an infection with the

bacterium Helicobacter pylori (H. pylori) (Bauditz et al., 1999; Beales &

Calam, 1997; Lv et al., 2019; Yamauchi et al., 2008). Worldwide, about

half of the population is infected with H. pylori (Suerbaum & Michetti,

2002). To protect itself from the human immune system, H. pylori has

developed specic adaptations in order to be able to settle in the gastric

mucosa. One of them is aimed at resisting the low pH in the stomach by

producing the enzyme urease. This enzyme catalyzes the breakdown of

urea into CO

and ammonia, neutralizing gastric acid (Salama, Hartung,

& Müller, 2013).

Among the pro-inammatory interleukins IL-17 is known to play a

crucial role in H. pylori infections. In infected mice IL-17 protein

expression was increased and associated with a strong neutrophil inl-

tration, which was signicantly reduced in IL-17 knock-out mice.

Moreover, the number of pathogens present in the stomach of the knock-

out mice was signicantly lower than in wild-type mice after 6 months

of infection (Shiomi et al., 2008). In humans, sustained secretion of IL-

17A, an important representative of the IL-17 family, by gastric cells

has been hypothesized to promote the development of gastric tumors

(Kang et al., 2023). Whether dietary peptides released during gastric

digestion may counteract a H. pylori-evoked release of pro-inammatory

cytokines and whether TAS2Rs are involved herein, has not been studied

so far.

We, therefore, hypothesized that gastric digestion of the sweet-

tasting protein thaumatin generates bitter peptides that reduce an

H. pylori-induced release of pro-inammatory interleukins with func-

tional involvement of TAS2Rs.

2. Material and methods

2.1. Chemicals

Thaumatin (mixture of the two forms thaumatin I and II, 99.4%

purity) was purchased from TCI Chemicals (Portland, USA), 1,5-car-

boxy-seminaphtorhodauor acetoxymethylester (SNARF-1-AM) and

Dulbecco's modied Eagle's medium GlutaMAX (DMEM) were pur-

chased from Thermo Fisher Scientic (USA). Fetal bovine serum (FBS

Supreme), trypsin/ethylenediaminetetraacetic acid, and pen-

icillin−streptomycin were obtained from PANBiotech GmbH (Aiden-

bach, Germany). Phosphate buffered saline was bought from Biozym

Scientic GmbH (Hessisch Oldendorf, Germany). Dimethyl sulfoxide

(DMSO) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide (MTT) were purchased from Carl Roth (Karlsruhe, Germany).

All other compounds were ordered from Merck KGaA (Darmstadt, Ger-

many). Custom peptides (DAGGRQLNSGES, FNVPMDF, WTIN-

VEPGTKGGKIW, and AAASKGDAAL) were synthesized by Genscript

Biotech with a purity of >95% (New Jersey, USA). Double-distilled

water (ddH

O) from Elga Purelab Classic (Veolia Water Solutions &

Technologies, France) was used for all experiments. The composition of

Krebs-Ringer-HEPES buffer (KRHB), simulated salivary uid (SSF), and

simulated gastric uid (SGF) is based on previously published work

(Brodkorb et al., 2019; Richter et al., 2022).

2.2. In vitro digestion

For digestion of the intact plant protein thaumatin, 100 mg were

dissolved in SSF as described before (Brodkorb et al., 2019; Richter

et al., 2022) and incubated for 5 min at 37 ◦C and 5% CO

(standard

conditions) in four biological replicates. After the rst sampling, SGF

was added and the pH was adjusted to 3. Pepsin was added to initiate

simulated gastric digestion. The reaction was incubated under standard

conditions and samples were taken at 0.25, 0.5, 0.75, 1, 2, 3, 4, 5, and 6

h. The samples were stored at −80 ◦C until peptide identication by LC-

ToF-MS or quantitation by LC-MS/MS.

2.3. In vivo digestion

To carry out the in vivo digestion experiments, 1 g thaumatin was

dissolved in 5 mL of water and administered to pigs (German Landrace,

German Landrace ×minipig, age: about 20 weeks) in 6 biological rep-

licates. Two hours after thaumatin administration (Richter et al., 2022),

the animals were euthanized and the stomach contents were collected

and immediately frozen in liquid nitrogen. The following purication

and desalting was carried out as previously published (Richter et al.,

2022). The samples were stored at −80 ◦C until identication by LC-

ToF-MS or quantitation by LC-MS/MS.

2.4. Peptide identication by means of Ultra-High-Performance Liquid

Chromatography–Time-of-Flight Mass Spectrometry (UHPLC-ToF-MS)

Separations and measurements of peptides released from thaumatin

digestion were performed using a Sciex ExionLC AC (Sciex, Darmstadt,

Germany) coupled to a Sciex TripleTOF 6600 mass spectrometer (Sciex,

Darmstadt, Germany) adapted from the previously published protocol

(Richter et al., 2022). Data acquisition and instrumentation control were

performed with AnalystTF software (v 1.7.1; Sciex, Darmstadt, Ger-

many). The used gradient of 0.1% aqueous formic acid and acetonitrile

containing 0.1% formic acid was only slightly adjusted according to the

following scheme: 0 min, 5% B; 0.5 min, 5% B; 14 min, 60% B; 15 min,

98% B; 16 min, 98% B; 17 min, 5% B; and 20 min, 5% B. MaxQuant

software (Cox & Mann, 2008) was used for the identication of the

peptide sequences (version 1.6.3.4; unspecic digestion; variable mod-

ications: oxidation M, acetyl protein N-term, carbamidomethyl C,

phospho STY; peptide length between 4 and 25 amino acids; andromeda

score >10; max. Score: 202.27).

2.5. Quantitation of the thaumatin peptides via LC-MS/MS

Separations and measurements for peptide quantication were per-

formed using a Sciex ExionLC AC (Sciex, Darmstadt, Germany) coupled

to a 6500+QTrap LC-MS/MS system (Sciex, Darmstadt, Germany)

operating in the positive electrospray ionization mode. This was also

based on the previously published methodology (Richter et al., 2022).

While all other parameters were adopted, the gradient of 0.1% aqueous

formic acid and acetonitrile containing 0.1% formic acid was modied

as follows: 0 min, 10% B; 8 min, 40% B; 8.5 min, 98% B; 11 min, 98% B;

11.5 min, 10% B; and 15 min, 10% B. The optimal ionization parameters

(DP, CE, and CXP; see supplementary data Table S1) for each peptide

P. Richter et al.

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Gastric digestion of the sweet-tasting plant protein thaumatin releases bitter peptides that reduce H. pylori induced pro-inflammatory IL-17A release via the TAS2R16 bitter taste receptor

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