Current advances for in vitro protein digestibility.

Protein is an essential macronutrient in our diet, source of nitrogen and essential amino acids, but the biological utilization of dietary protein depends on its digestibility and the absorption of amino acids and peptides in the gastrointestinal tract. The methods to define the amount and the quality of protein to meet human nutritional needs, such as the Digestible Indispensable Amino Acid Score (DIAAS), require the use of animal models or human studies. These in vivo methods are the reference in protein quality evaluation, but they are expensive and long-lasting procedures with significant ethical restrictions. Therefore, the development of rapid, reproducible and in vitro digestion methods validated with in vivo data is an old demand. This review describes the challenges of the in vitro digestion methods in the evaluation of the protein nutritional quality. In addition to the technical difficulties to simulate the complex and adaptable processes of digestion and absorption, these methods are affected by similar limitations as the in vivo procedures, i.e., analytical techniques to accurately determine bioavailable amino acids and the contribution of the endogenous nitrogen. The in vitro methods used for the evaluation of protein digestibility, with special attention on those showing comparative data, are revised, emphasizing their pros and cons. The internationally harmonized digestion protocol proposed by the INFOGEST network is being adapted to evaluate protein and amino acid digestibility. The inter-laboratory reproducibility of this protocol was demonstrated for dairy products. The in vivo/in vitro comparability results obtained to date with this protocol for several plant and animal sources are promising, but it requires an extensive validation with a wider range of foods and substrates with known in vivo digestibility. These in vitro methods will probably not be applicable to all foods, and therefore, it is important to identify their limitations, not to elude their use, but to apply them within the limits, by using the appropriate standards and references, and always as a complementary tool to in vivo tests to reduce their number.

In vitro digestibility of proteins from red seaweeds: Impact of cell wall structure and processing methods.

This study aimed to assess the nutritional quality and digestibility of proteins in two red seaweed species, Gelidium corneum and Gracilaropsis longissima, through the application of in vitro gastrointestinal digestions, and evaluate the impact of two consecutive processing steps, extrusion and compression moulding, to produce food snacks. The protein content in both seaweeds was approximately 16 %, being primarily located within the cell walls. Both species exhibited similar amino acid profiles, with aspartic and glutamic acid being most abundant. However, processing impacted their amino acid profiles, leading to a significant decrease in labile amino acids like lysine. Nevertheless, essential amino acids constituted 35-36 % of the total in the native seaweeds and their processed products. Although the protein digestibility in both seaweed species was relatively low (<60 %), processing, particularly extrusion, enhanced it by approximately 10 %. Interestingly, the effect of the different processing steps on the digestibility varied between the two species. This difference was mainly attributed to compositional and structural differences. G. corneum exhibited increased digestibility with each processing step, while G. longissima reached maximum digestibility after extrusion. Notably, changes in the amino acid profiles of the processed products affected adversely the protein nutritional quality, with lysine becoming the limiting amino acid. These findings provide the basis for developing strategies to enhance protein quality in these seaweed species, thereby facilitating high-quality food production with potential applications in the food industry.

A Shared Perspective on in Vitro and in Vivo Models to Assay Intestinal Transepithelial Transport of Food Compounds.

Assessing nutrient bioavailability is complex, as the process involves multiple digestion steps, several cellular environments, and regulatory-metabolic mechanisms. Several in vitro models of different physiological relevance are used to study nutrient absorption, providing significant challenges in data evaluation. However, such in vitro models are needed for mechanistic studies as well as to screen for biological functionality of the food structures designed. This collaborative work aims to put into perspective the wide-range of models to assay the permeability of food compounds considering the particular nature of the different molecules, and, where possible, in vivo data are provided for comparison.

CCK and GLP-1 response on enteroendocrine cells of semi-dynamic digests of hydrolyzed and intact casein.

A semi-dynamic gastrointestinal device was employed to explore the link between protein structure and metabolic response upon digestion for two different substrates, a casein hydrolysate and the precursor micellar casein. As expected, casein formed a firm coagulum that remained until the end of the gastric phase while the hydrolysate did not develop any visible aggregate. Each gastric emptying point was subjected to a static intestinal phase where the peptide and amino acid composition changed drastically from that found during the gastric phase. Gastrointestinal digests from the hydrolysate were characterized by a high abundancy of resistant peptides and free amino acids. Although all gastric and intestinal digests from both substrates induced the secretion of cholecystokinin (CCK) and glucagon-like peptide-1 (GLP-1) in STC-1 cells, GLP-1 levels were maximum in response to gastrointestinal digests from the hydrolysate. The enrichment of protein ingredients with gastric-resistant peptides by enzymatic hydrolysis is proposed as strategy to deliver protein stimuli to the distal gastrointestinal tract to control food intake or type 2 diabetes.

A multi-centre peptidomics investigation of food digesta: current state of the art in mass spectrometry analysis and data visualisation.

Mass spectrometry has become the technique of choice for the assessment of a high variety of molecules in complex food matrices. It is best suited for monitoring the evolution of digestive processes in vivo and in vitro. However, considering the variety of equipment available in different laboratories and the diversity of sample preparation methods, instrumental settings for data acquisition, statistical evaluations, and interpretations of results, it is difficult to predict a priori the ideal parameters for optimal results. The present work addressed this uncertainty by executing an inter-laboratory study with samples collected during in vitro digestion and presenting an overview of the state-of-the-art mass spectrometry applications and analytical capabilities available for studying food digestion. Three representative high-protein foods - skim milk powder (SMP), cooked chicken breast and tofu - were digested according to the static INFOGEST protocol with sample collection at five different time points during gastric and intestinal digestion. Ten laboratories analysed all digesta with their in-house equipment and applying theirconventional workflow. The compiled results demonstrate in general, that soy proteins had a slower gastric digestion and the presence of longer peptide sequences in the intestinal phase compared to SMP or chicken proteins, suggesting a higher resistance to the digestion of soy proteins. Differences in results among the various laboratories were attributed more to the peptide selection criteria than to the individual analytical platforms. Overall, the combination of mass spectrometry techniques with suitable methodological and statistical approaches is adequate for contributing to the characterisation of the recently defined digestome.

Anti-Steatotic Effects of Chlorella vulgaris, Nannochloropsis gaditana and Gracilaria vermiculophylla Algae Extracts in AML-12 Hepatocytes.

Non-alcoholic fatty liver disease (NAFLD) is considered the most common chronic liver alteration whose prevalence is increasing in Western countries. Microalgae and macroalgae have attracted great interest due to the high content in bioactive compounds with beneficial effects on health. The aim of the present study is to assess the potential interest of extracts rich in proteins obtained from the microalgae Chlorella vulgaris and Nannochloropsis gaditana and the macroalga Gracilaria vermiculophylla in the prevention of lipid accumulation in AML-12 hepatocytes. Toxicity was not observed at any of the tested doses. Both microalgae and the macroalga were effective in preventing triglyceride accumulation, with Nannochloropsis gaditana being the most effective one. Although the three algae extracts were able to increase different catabolic pathways involved in triglyceride metabolism, the mechanisms underlying the anti-steatotic effect were different in each algae extract. In conclusion, the present study demonstrates that Chlorella vulgaris, Nannochloropsis gaditana and Gracilaria vermiculophylla extracts are able to partially prevent the accumulation of triglycerides induced by palmitic acid in cultured hepatocytes, a model used to mimic the steatosis induced in liver by dietary patterns rich in saturated fat.

Avocado seed by-product uses in emulsion-type ingredients with nutraceutical value: Stability, cytotoxicity, nutraceutical properties, and assessment of in vitro oral-gastric digestion.

The avocado industry obtains 20-30% of the total by-products (peels and seeds). However, byproducts can be uses as sources of economic nutraceutical ingredients with functional potential. This work developed emulsion-type ingredients from avocado seed to evaluate its quality, stability, cytotoxicity, and nutraceutical properties before/after in vitro oral-gastric digestion. Ultrasound lipid extraction achieved an extraction yield of up to 95.75% compared with Soxhlet conventional extraction (p > 0.05). Six ingredients' formulations (E1-E6) were stable for up to day 20 during storage, preserving their antioxidant capacity and displaying low in vitro oxidation compared to control. None of the emulsion-type ingredients were considered cytotoxic according to the shrimp lethality assay (LC50 > 1000 µg/mL). Ingredients E2, E3, and E4 generated low lipoperoxides' concentrations and high antioxidant capacity during the oral-gastric stage. The 25 min-gastric phase showed the highest antioxidant capacity and low lipoperoxidation. Results suggested avocado seed-derived could be used to develop functional ingredients with nutraceutical properties.

Peptidomic Characterization and Amino Acid Availability after Intake of Casein vs. a Casein Hydrolysate in a Pig Model.

It is known that casein hydrolysis accelerates gastrointestinal transit in comparison to intact casein, although the effect of the protein hydrolysis on the composition of the digests is not fully understood. The aim of this work is to characterize, at the peptidome level, duodenal digests from pigs, as a model of human digestion, fed with micellar casein and a previously described casein hydrolysate. In addition, in parallel experiments, plasma amino acid levels were quantified. A slower transit of nitrogen to the duodenum was found when the animals received micellar casein. Duodenal digests from casein contained a wider range of peptide sizes and a higher number of peptides above five amino acids long in comparison with the digests from the hydrolysate. The peptide profile was markedly different, and although ß-casomorphin-7 precursors were also found in hydrolysate samples, other opioid sequences were more abundant in the casein digests. Within the same substrate, the evolution of the peptide pattern at different time points showed minimal changes, suggesting that the protein degradation rate relies more on the gastrointestinal location than on digestion time. Higher plasma concentrations of methionine, valine, lysine and amino acid metabolites were found in animals fed with the hydrolysate at short times (<200 min). The duodenal peptide profiles were evaluated with discriminant analysis tools specific for peptidomics to identify sequence differences between both substrates that can be used for future human physiological and metabolic studies.

Comparison of in vitro digestibility and DIAAR between vegan and meat burgers before and after grilling.

Plant-based meat alternatives of high quality and digestibility could be a way to reduce meat consumption and, consequently, the environmental impact. However, little is known about their nutritional characteristics and digestion behaviour. Therefore, in the present study, the protein quality of beef burgers, known as excellent source of protein, was compared with the protein quality of two highly transformed veggie burgers, based on soy or pea-faba proteins, respectively. The different burgers were digested according to the INFOGEST in vitro digestion protocol. After digestion, total protein digestibility was determined, either based on total nitrogen (Kjeldahl) analysis, or after acid hydrolysis based on total amino groups (o-phthalaldehyde method) or total amino acids (TAA; by HPLC). The digestibility of individual amino acids was also determined, and the digestible indispensable amino acid score (DIAAS) was calculated based on in vitro digestibility. The impact of texturising and grilling on in vitro protein digestibility and the digestible indispensable amino acid ratio (DIAAR) was evaluated at the level of the ingredients and the finished products. As expected, the grilled beef burger had the highest in vitro DIAAS values (Leu 124 %), and grilled soy protein-based burger reached in vitro DIAAS values that could be rated as good (soy burger, SAA 94 %) protein source, according to the Food and Agriculture Organization. The texturing process did not significantly affect the total protein digestibility of the ingredients. However, grilling led to a decrease in digestibility and DIAAR of the pea-faba burger (P < 0.05), which was not observed in the soy burger, but led to an increase in DIAAR in the beef burger (P < 0.005).

In vitro digestibility of dietary proteins and in vitro DIAAS analytical workflow based on the INFOGEST static protocol and its validation with in vivo data.

The FAO recommends the digestible indispensable amino acid score (DIAAS) to determine protein quality in foods, preferably tested in vivo. Here, the INFOGESTin vitrodigestion protocol was applied and supplemented with an analytical workflow allowing the assessment of protein digestibility and DIAAS calculation. The protocol was applied to selected samples WPI, zein, collagen, black beans, pigeon peas, All-Bran®, and peanuts. The total protein digestibility, digestibility of individual amino acids (AA), and DIAAS values were established and compared with in vivo data for the same substrates. Total protein digestibility (total Nitrogen, r = 0.7, P < 0.05; primary amines (OPA), r = 0.6, P < 0.02; total AA, r = 0.6, P < 0.02) and digestibility of individual AA (r = 0.6, P < 0.0001) were in good agreement, between in vitro and in vivo, with a mean difference of 1.2 %. In vitro DIAAS was highly correlated with DIAAS obtained from in vivo true ileal digestibility values (r = 0.96, R2 = 0.89, P < 0.0001) with a mean difference of 0.1 %.

Food peptides as inducers of CCK and GLP-1 secretion and GPCRs involved in enteroendocrine cell signalling.

The strong effect of protein digestion products on gastrointestinal-released hormones is recognised. However, little is known about the specific peptide sequences able to induce gastrointestinal hormone secretion and the receptors involved. Our objective was to identify peptides able to induce the secretion of cholecystokinin (CCK) and glucagon like peptide-1 (GLP-1) in the enteroendocrine cell line STC-1, and to evaluate the involvement of the calcium-sensing receptor and G-protein coupled receptor-93 in this cell signalling. The key role of the amino acidic sequence on CCK and GLP-1 secretion is demonstrated. Removing Ser from the N-terminus of κ-casein 33SRYPS37, or the N-terminal Trp-Ile in lysozyme 123WIRGCRL129 decreased the secretion of both hormones. However, substituting Tyr by Ala in peptide αs1-CN 90RYLG93 enhanced the CCK secretion levels but not the GLP-1 ones. In addition, the involvement of CaSR and GPR93 was evidenced, but our results pointed to the contribution of additional receptors or transporters.

Antioxidant Potential of the Sweet Whey-Based Beverage Colada after the Digestive Process and Relationships with the Lipid and Protein Fractions.

Whey-based beverages could be an effective way of reusing a by-product of th cheese industry, mitigating environmental hazards and, at the same time, profiting a useful food with high nutritional and antioxidant properties. In this study, a traditional Ecuadorian beverage (Colada) was prepared combining sweet whey, Maracuyá and barley. Antioxidant properties before and after an in vitro digestion using the INFOGEST method were determined, and relationships with intestinal transformations of the lipid and protein fractions were analyzed. The digestive process had a positive effect on antioxidant properties based on increased values of ABTS and FRAP located in the bioaccessible fraction (BF), together with strong increments of total polyphenols. Moreover, pretreatment of Caco-2 cells with the BF of Colada significantly reduced ROS generation (p < 0.001) measured by the dichlorofluorescein assay. Substantial changes of the fatty acid profile occurred during digestion, such as a fall of saturated fatty acids and a rise of polyunsaturated. The protein profile, examined by SDS-PAGE and exclusion molecular chromatography in the BF, showed that the major part of the proteins were digested in the intestinal phase. Analysis of NanoLC-MS/MS revealed 18 antioxidant peptides originated from whey proteins, but also 16 peptides from barley with potential antioxidant properties. In conclusion, combining sweet whey with Maracuyá and barley constitutes an excellent nutritional beverage with a strong antioxidant potential.

In vitro digestion of milk proteins including intestinal brush border membrane peptidases. Transepithelial transport of resistant casein domains.

The use of enzymes from the brush border membrane (BBM) in simulated gastrointestinal digestion of milk proteins has been evaluated. With this purpose, the resistant sequences from casein and milk whey proteins after INFOGEST in vitro digestion with and without BBM have been analyzed by tandem mass spectrometry. The use of BBM revealed additional cleavages to those found with pancreatic enzymes, although the number of total identified peptides decreased due to the reduction of the peptide size. These new cleavages were mainly attributed to the activity of amino- and carboxy-peptidases, which was also reflected in the higher concentration of free amino acids found in the gastrointestinal digests with BBM. The peptidome of the simulated gastrointestinal digests was compared with that previously obtained in digests aspirated from human jejunum after oral administration of the same substrates. The addition of BBM did not change significantly the peptide profile, although it allowed the identification of peptides found in human digests. However, none of the models was able to reproduce the large variety of peptides found in vivo. In addition, in vitro transepithelial transport of six ß-casein derived peptides resistant to gastrointestinal digestion, including the opioid ß-casomorphin-7, was also evaluated. The results point to the importance of the nature of the N- and C-terminal end for the transport rate through the Caco-2 cell monolayer. Therefore, the use of BBM as a supplementary step after simulated pancreatic digestion can be considered in bioavailability studies since the final sequence can determine the absorption of peptides.

In vitro simulated semi-dynamic gastrointestinal digestion: evaluation of the effects of processing on whey proteins digestibility and allergenicity.

The effect of thermal processing on digestibility of milk proteins should be better understood as this can greatly affect their immunoreactivity. The aim of this study was to evaluate the effects of thermal processing and lactosylation on digestibility and allergenicity, by comparing non heat-treated with industrially processed whey proteins. A semi-dynamic model was used to mimic the kinetics of digestion, and ELISA inhibition tests against human specific serum IgE were performed on the mass-spectrometry characterized products. A quicker gastric digestion of the industrially treated sample produced a lower immunogenic response in comparison with the raw sample, where intact conformational epitopes remained. In later digests, greater IgE reactivity was shown in the heat treated product, probably due to the release of linear epitopes, while at intestinal level the immunogenic response was negligible. Moreover, transepithelial transport of a reported ß-lactoglobulin-derived allergen, KIDALNENVLVL, produced during digestion was assayed. It was found that the epitope-belonging peptide could be transported through the cell monolayer, both in the native and mono-lactosylated forms, with a favored passage of the native peptide.

In vitro dipeptidyl peptidase IV inhibitory activity and in situ insulinotropic activity of milk and egg white protein digests.

Dietary proteins are involved in the regulation of glucose homeostasis by different mechanisms. Food protein digestion products are reported to inhibit dipeptidyl peptidase IV (DPP-IV), induce incretin secretion or directly exert an insulinotropic effect in pancreatic ß-cells. This study illustrates the DPP-IV inhibitory activity of gastric and intestinal digests of casein, whey and egg white proteins determined in vitro, using Gly-Pro-AMC, and in situ using non-differentiated Caco-2 cells. Comparable trends in the DPP-IV inhibitory profiles were obtained by these two methods although the extent of inhibition in situ was consistently lower than the inhibition observed in vitro. Casein intestinal digests and whey protein gastric and intestinal digests showed potent DPP-IV inhibitory activities in Caco-2 cells with IC50 values ranging from 0.8 to 1.2 mg mL-1. The absorbed fraction of the intestinal digests from whey and egg white protein induced insulin secretion in BRIN-BD11 cells when determined using a two-tiered cellular model (Caco-2 and BRIN-BD11). However, the gastric digests from the same substrates showed no insulin secretion. This may be related to limited trans-epithelial transport through the Caco-2 monolayer of the gastric digestion products. However, both, gastric and intestinal digests were able to induce insulin secretion in BRIN-BD11 cells when the monolayer was composed of a co-culture of STC-1 and Caco-2 cells. This result may be attributed to the activation of STC-1 cells and subsequent incretin secretion, induced by the gastric digest, as shown by an enhanced intracellular calcium uptake.

Composition of Brazil Nut (Bertholletia excels HBK), Its Beverage and By-Products: A Healthy Food and Potential Source of Ingredients.

The consumption of plant-based beverages is a growing trend and, consequently, the search for alternative plant sources, the improvement of beverage quality and the use of their by-products, acquire great interest. Thus, the purpose of this work was to characterize the composition (nutrients, phytochemicals and antioxidant activity) of the Brazil nut (BN), its whole beverage (WBM), water-soluble beverage (BM-S), and its by-products of the beverage production: cake, sediment fraction (BM-D), and fat fraction (BM-F). In this study, advanced methodologies for the analysis of the components were employed to assess HPLC-ESI-QTOF (phenolic compounds), GC (fatty acids), and MALDI-TOF/TOF (proteins and peptides). The production of WBM was based on a hot water extraction process, and the production of BM-S includes an additional centrifugation step. The BN showed an interesting nutritional quality and outstanding content of unsaturated fatty acids. The investigation found the following in the composition of the BN: phenolic compounds (mainly flavan-3-ols as Catechin (and glycosides or derivatives), Epicatechin (and glycosides or derivatives), Quercetin and Myricetin-3-O-rhamnoside, hydroxybenzoic acids as Gallic acid (and derivatives), 4-hydroxybenzoic acid, ellagic acid, Vanillic acid, p-Coumaric acid and Ferulic acid, bioactive minor lipid components (ß-Sitosterol, γ-Tocopherol, α-Tocopherol and squalene), and a high level of selenium. In beverages, WBM had a higher lipid content than BM-S, a factor that influenced the energy characteristics and the content of bioactive minor lipid components. The level of phenolic compounds and selenium were outstanding in both beverages. Hydrothermal processing can promote some lipolysis, with an increase in free fatty acids and monoglycerides content. In by-products, the BM-F stood out due to its bioactive minor lipid components, the BM-D showed a highlight in protein and mineral contents, and the cake retained important nutrients and phytochemicals from the BN. In general, the BN and its beverages are healthy foods, and its by-products could be used to obtain healthy ingredients with appreciable biological activities (such as antioxidant activity).

Protein Profile and Simulated Digestive Behavior of Breast Milk from Overweight and Normal Weight Mothers.

Human milk proteins have shown to vary in concentration and distribution through lactation. However, while some regulatory components, such as hormones, have shown associations with regard to the mothers' body mass index, there is limited information on the possible influence of this condition on the whole protein distribution. The objective of this study was to evaluate the protein profile of human milk from normal weight and overweight or obese mothers to identify differences in protein expression in colostrum, transitional and mature milk. The mass spectrometry analysis showed the ability to class with a high degree of confidence the lactation state and the milk profile according to the mother's condition. Individual milk samples were subjected to a digestion in vitro model that takes into account the specificities of the gastrointestinal conditions of full-term newborn infants. The digestion products were compared with available data from the digestive contents in newborns. The behavior of the most abundant proteins and the overall peptide generation and survival, showed good correspondence with in vivo data.

Compared digestibility of plant protein isolates by using the INFOGEST digestion protocol.

The use of ingredients based on plant protein isolates is being promoted due to sustainability and health reasons. However, it is necessary to explore the behaviour of plant protein isolates during gastrointestinal digestion including the profile of released free amino acids and the characterization of resistant domains to gastrointestinal digestion. The aim of the present study was to monitor protein degradation of four legume protein isolates: garden pea, grass pea, soybean and lentil, using the harmonized Infogest in vitro digestion protocol. In vitro digests were characterized regarding protein, peptide and free amino acid content. Soybean was the protein isolate with the highest percentage of insoluble nitrogen at the end of the digestion (12%), being this fraction rich in hydrophobic amino acids. Free amino acids were mainly released during the intestinal digestion, comprising 21-24% of the total nitrogen content, while the percentage of nitrogen corresponding to peptides ranged from 66 to 76%. Legume globulins were resistant to gastric digestion whereas they were hydrolysed into peptides and amino acids during the intestinal phase. However, the molecular weight (MW) distribution demonstrated that all intestinal digests, except those from soybean, contained peptides with MW > 4 kDa at the end of gastrointestinal digestion. The profile of free amino acids released during digestion supports legume protein isolates as an excellent source of essential amino acids to be used in protein-rich food products. Peptides released during digestion matched with previously reported epitopes from the same plant species or others, explaining the ability to induce allergic reactions and cross-linked reactivity.

Peptidomic data of egg white gastrointestinal digests prepared using the Infogest Harmonized Protocol.

These data are related to the research article entitled "Induction of CCK and GLP-1 release in enteroendocrine cells by egg white peptides generated during gastrointestinal digestion". In this article, the peptide and free amino acid profile of egg white gastrointestinal in vitro digests is shown. Egg white proteins were digested following the INFOGEST gastrointestinal digestion protocol. Different time points of gastric and intestinal digestion were characterized regarding protein, peptide and amino acid content. Protein degradation was followed by SDS-PAGE where some electrophoretic bands were identified by MALDI-TOF/TOF after tryptic digestion. Moreover, the molecular weight distribution of egg white peptides found at different times of gastrointestinal digestion was performed using MALDI-TOF. Peptides identified from the most abundant egg white proteins by tandem mass spectrometry were represented using a peptide profile tool and raw data are given in table format. These results reveal the protein regions resistant to digestion and illustrate the free amino acid profile of egg white protein at the end of the digestion process. These data can be used for nutritional purposes and to identify allergen epitopes or bioactive sequences.

Induction of CCK and GLP-1 release in enteroendocrine cells by egg white peptides generated during gastrointestinal digestion.

The effect of dietary protein on the induction of intestinal hormones is recognised. However, little is known about the nature of the digestion products involved in this intestinal signalling. Our aim was to characterise egg white protein digestion products and study their ability to induce CCK and GLP-1 release in enteroendocrine STC-1 cells. Intestinal digests triggered GLP-1 release at a higher rate than gastric digests. Peptides, but not free amino acids, showed a potent GLP-1 secretagogue effect, while proteins only had a modest effect. CCK was released in response to peptides and free amino acids but not proteins. Two hydrophobic negatively charged peptides triggered CCK release, while the highest GLP-1 response was found with a hydrophobic positively charged peptide, pointing to the involvement of different receptors or active sites. Identifying peptide sequences and receptors involved in hormonal secretion could open up new ways to control food intake and glucose metabolism.