IGF 01IF21916N - Proteinhydrolysates

Plant proteins and their hydrolysates are already widely used in the food industry. Due to their smaller ecological footprint, plant proteins are increasingly preferred to animal proteins. In addition to an approx. 5-10 times lower energy and water consumption, plant proteins are also characterized by an approx. 80 % reduced need for agricultural land compared to animal proteins. Proteins and their hydrolysates also offer numerous advantages in terms of nutritional physiology compared to carbohydrates and fats: human studies have shown that isocaloric administration of a protein-rich meal induces longer satiety than the administration of other macronutrients. In addition, hydrolysates lead to faster satiety than intact proteins. It is assumed that those bioactive peptides with a satiating effect are already present in the hydrolysate and do not have to be released in the digestive tract first. In recent years, more and more peptide sequences from dietary proteins have been identified that are responsible for a satiating effect. Increasing the feeling of satiety, which leads to reduced energy intake, thus supports the maintenance of a healthy body weight. In view of the increasing numbers of overweight and obese people worldwide and the associated secondary diseases, preventive measures are of great importance for the health of the world's population. However, one disadvantage of protein-rich products is that they are often accompanied by a bitter off-taste, which limits their use depending on the food application. This bitter taste can be caused on the one hand by flavor-active peptides or L-amino acids and on the other hand by plant secondary metabolites such as saponins and polyphenols. There is evidence that encapsulated bitter-tasting substances induce satiety in healthy adults via activation of extra-oral bitter receptors (TZRs). In order to achieve greater consumer acceptance of foods based on potato and pea protein hydrolysates, it is therefore necessary to reduce the bitter taste typical of vegetable protein hydrolysates as far as possible without losing the positive effect of bitter substances on the feeling of satiety.
The aim of the research project was to identify and quantify the compounds that cause the bitter taste on a molecular level.

As part of the project, the most bitter pea or potato protein hydrolysate (EH5/KH4) and the least bitter hydrolysate (EH1/KH1) within the sample set (five EH and four KH) were identified using comparative profiling sensors. After quantification of the free amino acids as well as fatty acids known from literature and their oxidation products, reconstitution experiments showed that the bitter taste of the least bitter hydrolysates (EH1/KH1) was caused by the bitter amino acids (leucine, histidine, arginine, tyrosine, valine, isoleucine, phenylalanine, tryptophan and lysine) as well as palmitic acid and the oxidation products 9,10,11-THOA and 9,10,13-THOA, whereas the bitter taste for the most bitter hydrolysates (EH5/KH4) is caused by other bitter substances not yet described in the literature.
The sensoproteomics concept was used to elucidate the unknown bitter substances. The most bitter and least bitter hydrolysates (EH1, EH5, KH1 and KH4) were separated by ultrafiltration, subsequent solid phase extraction and preparative high-performance liquid chromatography. With the help of taste dilution analysis, the fractions obtained were weighted in terms of their bitterness and the subsequent analyses were focused on the most bitter fractions. These were then analyzed using LC-TOF-MS and the peptides contained were sequenced using protein databases and MaxQuant software. By comparing the peptides in the most and least bitter hydrolysates and using the two bitter prediction tools Q-Value and iBitter-SCM, 17 and 12 potentially bitter peptides were found in EH5 and KH4, respectively. With the help of targeted LC-MS/MS measurements and synthetic reference peptides, these peptides were not only verified, but the significance of different leucine/isoleucine variants was also worked out, so that a total of 38 target peptides were identified.
The human threshold values of the 38 target peptides were between 48 and 707 μmol/l, with 35 peptides causing a bitter taste. Quantification of the peptides by LC-MS/MS and calculation of the dose-over-threshold (DoT) factors confirmed that the peptides EVY, LEPDNRIE, LLE, AIL, LTL and ITL had DoT factors above one and thus contribute to the bitter taste of EH5. In KH4, the peptides EVY, PAF, AIL, LLE, LTL and ITL contribute to the bitter taste, and the greatest influence on bitterness was due to PAF.
The most bitter (EH5/KH4) and least bitter pea and potato protein hydrolysates (EH1/KH1) were subjected to in vitro digestion in order to subsequently identify the resulting bitter peptides. For this purpose, the peptides were measured using LC-MS, identified with MaxQuant, screened using various bitterness prediction tools and the bitterness verified by human sensory studies. Finally, the bitter-tasting peptides YPYPR, YNDQDTPVI and ALEPDN were identified for the most bitter pea protein hydrolysate EH5, while the peptides EELEK, VPE and EWR were detected for the less bitter pea protein hydrolysate EH1 after in vitro digestion. For the potato protein hydrolysates, the peptides GWPY-EPF, DKDFLPF from the less bitter KH1 were identified as bitter, for the more bitter tasting KH4 the bitter peptides EPWWPEM, VNDEKDFIPF were detected.
Subsequently, these peptides were tested for their influence on gastric proton secretion as the main mechanism of gastric acid secretion, which is essential for digestive processes, as well as their effect on the cellular secretion of serotonin as a cellular and systemic satiety signal in human parietal cells (HGT-1 cells). All six peptides from the pea protein hydrolysates similarly stimulated cellular proton secretion via functional involvement of the bitter receptors TAS2R4 and TAS2R43. In contrast, the release of the satiety hormone serotonin (ELISA) was significantly higher in HGT-1 cells at the same concentrations after incubation with the peptides from the less bitter-tasting hereditary protein hydrolysate EH1 EELEK, VPE and EWR compared to the peptides from EH5 YPYPR, YNDQDTPVI and AL-EPDN. Functional involvement of the two bitter receptors TAS2R4 and TAS2R43 was also demonstrated here.
A similar picture emerged in the experiments investigating the saturation potential of potato protein hydrolysates (KHs). Here too, the bitter peptides from the less bitter-tasting KH1 induced a stronger release of serotonin than the peptides from the more bitter-tasting KH4.
A comparative, single-blind, randomized human intervention study was then conducted with the pea protein hydrolysates EH1 and EH5 in male, slightly overweight men. Nineteen participants from a total of 23 screened subjects were included in the study and completed all three interventions (control, less bitter-tasting, more hydrolyzed EH1 and more bitter-tasting, less hydrolyzed EH5) on different days, during which measurements of gastric emptying, hunger and blood plasma concentrations of glucose, insulin, GLP-1, DPP-4, ghrelin, CCK, and serotonin as satiety indicators were performed. An increased feeling of hunger after the control intervention was prevented by the administration of EH1 and EH5, while there were no differences in energy intake. When comparing the effect of the less bitter-tasting, more hydrolyzed EH1 with the more bitter-tasting, less hydrolyzed EH5, no differences could be detected for the parameters glucose, insulin, GLP-1, ghrelin and CCK. However, reduced plasma concentrations of DPP-IV were measured 120 min after administration of EH1 compared to EH5. A reduced DPP-IV level indicates a longer lasting satiety. Increased plasma concentrations of the satiety hormone serotonin were found after administration of EH1. This also indicates an increased feeling of satiety. The study of gastric emptying showed slower gastric emptying after intervention with the more bitter-tasting, less hydrolyzed EH5 compared to EH1. A slower gastric emptying leads to a longer lasting feeling of fullness and thus to a longer lasting satiety. In summary, it can therefore be said that both pea protein hydrolysates had a positive influence on the feeling of satiety. On the one hand, the less bitter-tasting, more hydrolyzed EH1 had a stronger influence on the satiety hormones in the blood. This could be due to the fact that EH1 is more hydrolyzed and therefore bioactive peptides can pass into the blood faster, as they are not formed in the stomach during gastric digestion. In contrast, the more bitter-tasting, less hydrolyzed EH5 had a stronger influence on gastric emptying, which could be due to the lower degree of hydrolysis. These results highlight the need for designer proteins in the food industry. For future protein hydrolysate products, more hydrolytes should be selected which, in addition to slowing gastric emptying, also contain bioactive peptides that release more satiety hormones into the blood.

BMWK - IGF - DLR - FEI

Industrielle Gemeinschaftsforschung (IGF)