Variable Glycemic Responses to Intact and Hydrolyzed Milk Proteins in Overweight and Obese Adults Reveal the Need for Precision Nutrition.

Background: Dietary modifications can contribute to improved pancreatic ß cell function and enhance glycemic control. Objectives: The objectives of this study were as follows: 1) to investigate the potential of milk protein hydrolysates to modulate postprandial glucose response; 2) to assess individual responses; and 3) to explore the inter- and intraindividual reproducibility of the response. Methods: A 14-d randomized crossover study investigated interstitial glucose levels of participants in response to 12% w/v milk protein drinks (intact caseinate and casein hydrolysate A and B) consumed in random order with a 2-d washout between treatments. Milk protein drinks were consumed immediately prior to study breakfast and evening meals. Twenty participants (11 men, 9 women) aged 50 ± 8 y with a body mass index (in kg/m2) of 30.2 ± 3.1 were recruited. Primary outcome was glucose levels assessed at 15-min intervals with the use of glucose monitors. Results: Repeated-measures ANOVA revealed that for breakfast there was a significant difference across the 3 treatment groups (P = 0.037). The ability to reduce postprandial glucose was specific to casein hydrolysate B in comparison with intact caseinate (P = 0.039). However, despite this significant difference, further examination revealed that only 3 out of 18 individuals were classified as responders (P < 0.05). High intraclass correlation coefficients were obtained for glucose response to study meals (intraclass correlation coefficient: 0.892 for breakfast with intact caseinate). The interindividual CVs were higher than the intraindividual CVs. Mean inter- and intraindividual CVs were 19.4% and 5.7%, respectively, for breakfast with intact caseinate. Conclusion: Ingestion of a specific casein hydrolysate successfully reduced the postprandial glucose response; however, at an individual level only 3 participants were classified as responders, highlighting the need for precision nutrition. Exploration of high interindividual responses to nutrition interventions is needed, in combination with the development of precision nutrition, potentially through an n-of-1 approach. This clinical trial was registered as ISRCTN61079365 (https://www.isrctn.com/).

Peptide composition and dipeptidyl peptidase IV inhibitory properties of ß-lactoglobulin hydrolysates having similar extents of hydrolysis while generated using different enzyme-to-substrate ratios.

ß-Lactoglobulin hydrolysates (ßlgHs) were generated using elastase at enzyme-to-substrate ratios (E:S) of 0.5, 1.0 and 1.5% in order to reach target degree of hydrolysis (DH) values of 9 and 13%. The impact of different E:S during manufacture on hydrolysates having similar DHs was assessed. Samples with similar DHs generated with different E:S showed comparable molecular mass distribution profiles and in vitro dipeptidyl peptidase IV (DPP-IV) inhibitory activities (p>0.05). Liquid-chromatography tandem mass spectrometry (LC-MS/MS) analysis showed that 62 and 84% of the peptides identified were common within hydrolysates having a similar DH of 9 or 13%, respectively. Differences in the peptides identified within hydrolysates having similar DHs may be due to E:S dependent modifications in specificity and enzyme kinetics. Overall, this study showed that reduction in E:S while targeting the development of a similar DH for ßlgHs may be employed to reduce the cost of hydrolysate production without having an adverse impact on the bioactivity and physicochemical properties studied herein.

Strategies for the release of dipeptidyl peptidase IV (DPP-IV) inhibitory peptides in an enzymatic hydrolyzate of α-lactalbumin.

Bovine α-lactalbumin (α-La) contains numerous dipeptidyl peptidase IV (DPP-IV) inhibitory peptide sequences within its primary structure. In silico analysis indicated that the targeted hydrolysis of α-La with elastase should release DPP-IV inhibitory peptides. An α-La isolate was hydrolysed with elastase under different conditions using an experimental design approach incorporating 3 factors (temperature, pH and enzyme to substrate ratio (E : S) ratio) at 2 levels. The hydrolyzate generated at pH 8.5, 50 °C, E : S 2.0% (w/w) (H9) displayed the highest mean DPP-IV inhibition value at 3.1 mg mL(-1) of 75.8 ± 3.7% and had a half maximal DPP-IV inhibitory concentration (IC50) value of 1.20 ± 0.12 mg mL(-1). Five α-La-derived DPP-IV inhibitory peptides (GY, GL, GI, NY and WL) predicted to be released in silico were identified by liquid-chromatography tandem mass spectrometry (LC-MS/MS) within H9 and its simulated gastrointestinal digestion (SGID) sample. This preliminary study demonstrated the benefit of using a targeted approach combined with an experimental design in the generation of dietary protein hydrolyzates with DPP-IV inhibitory properties.

Enzymatic generation of whey protein hydrolysates under pH-controlled and non pH-controlled conditions: Impact on physicochemical and bioactive properties.

Enzymatic hydrolysis of whey protein (WP) was carried out under pH-controlled and non pH-controlled conditions using papain and a microbial-derived alternative (papain-like activity). The impact of such conditions on physicochemical and bioactive properties was assessed. WP hydrolysates (WPH) generated with the same enzyme displayed similar degree of hydrolysis. However, their reverse-phase liquid chromatograph mass spectrometry peptide profiles differed. A significantly higher oxygen radical absorbance capacity (ORAC) value was obtained for WP hydrolysed with papain at constant pH of 7.0 compared to the associated WPH generated without pH regulation. In contrast, there was no significant effect of pH regulation on dipeptidyl peptidase IV (DPP-IV) properties. WP hydrolysed with papain-like activity under pH regulation at 7.0 displayed higher ORAC activity and DPP-IV inhibitory properties compared to the associated WPH generated without pH regulation. This study has demonstrated that pH conditions during WPH generation may impact on peptide release and therefore on WPH bioactive properties.

Improved short peptide identification using HILIC-MS/MS: retention time prediction model based on the impact of amino acid position in the peptide sequence.

Short peptides can have interesting beneficial effects but they are difficult to identify in complex mixtures. We developed a method to improve short peptide identification based on HILIC-MS/MS. The apparent hydrophilicity of peptides was determined as a function of amino acid position in the sequence. This allowed the differentiation of peptides with the same amino acid composition but with a different sequence (homologous peptides). A retention time prediction model was established using the hydrophilicity and peptide length of 153 di- to tetrapeptides. This model was proven to be reliable (R(2)=0.992), it was validated using statistical methods and a mixture of 14 synthetic peptides. A whey protein hydrolysate was analysed to assess the ability of the model to identify unknown peptides. In parallel to milk protein database and de novo searches, the retention time prediction model permitted reduction and ranking of potential short peptides, including homologous peptides, present in the hydrolysate.

Bovine ß-lactoglobulin/fatty acid complexes: binding, structural, and biological properties.

Ligand-binding properties of ß-lactoglobulin (ß-lg) are well documented, but the subsequent biological functions are still unclear. Focusing on fatty acids/ß-lg complexes, the structure-function relationships are reviewed in the light of the structural state of the protein (native versus non-native aggregated proteins). After a brief description of ß-lg native structure, the review takes an interest in the binding properties of native ß-lg (localization of binding sites, stoichiometry, and affinity) and the way the interaction affects the biological properties of the protein and the ligand. The binding properties of non-native aggregated forms of ß-lg that are classically generated during industrial processing are also related. Structural changes modify the stoichiometry and the affinity of ß-lg for fatty acids and consequently the biological functions of the complex. Finally, the fatty acid-binding properties of other whey proteins (α-lactalbumin, bovine serum albumin) and some biological properties of the complexes are also addressed. These proteins affect ß-lg/fatty acids complex in whey given their competition with ß-lg for fatty acids.

Complexes between linoleate and native or aggregated ß-lactoglobulin: interaction parameters and in vitro cytotoxic effect.

The dairy protein ß-lactoglobulin (ßlg) is known to form a complex with fatty acids (FA). Due to industrial processing, ßlg is often in its non-native form in food products, which can modify the FA/ßlg complex properties. We investigated the interaction of bovine ßlg, in selected structural forms (native ßlg, a covalent dimer and as nanoparticles), with linoleate (C18:2). Using fluorescence and Isothermal Titration Calorimetry, linoleate was found to bind ßlg at two different binding sites. Regardless of the structural state of ßlg, association constants remained in the same order of magnitude. However, the stoichiometry increased up to 6-fold for nanoparticles, compared to that of native ßlg. The impact of these structural changes on linoleate uptake in vitro was measured by cytotoxicity assays on Caco-2 cells. The order of cytotoxicity of linoleate was as follows: free>complexed to dimers>complexed to nanoparticles>complexed to native ßlg. Therefore, the in vitro cytotoxicity of linoleate could be modulated by altering the state of ßlg aggregation, which in turn affects its binding capacity to the FA.

ß-Lactoglobulin-linoleate complexes: In vitro digestion and the role of protein in fatty acid uptake.

The dairy protein ß-lactoglobulin (BLG) is known to bind fatty acids such as the salt of the essential longchain fatty acid linoleic acid (cis,cis-9,12-octadecadienoic acid, n-6, 18:2). The aim of the current study was to investigate how bovine BLG-linoleate complexes, of various stoichiometry, affect the enzymatic digestion of BLG and the intracellular transport of linoleate into enterocyte-like monolayers. Duodenal and gastric digestions of the complexes indicated that BLG was hydrolyzed more rapidly when complexed with linoleate. Digested as well as undigested BLG-linoleate complexes reduced intracellular linoleate transport as compared with free linoleate. To investigate whether enteroendocrine cells perceive linoleate differently when part of a complex, the ability of linoleate to increase production or secretion of the enteroendocrine satiety hormone, cholecystokinin, was measured. Cholecystokinin mRNA levels were different when linoleate was presented to the cells alone or as part of a protein complex. In conclusion, understanding interactions between linoleate and BLG could help to formulate foods with targeted fatty acid bioaccessibility and, therefore, aid in the development of food matrices with optimal bioactive efficacy.

ß-Lactoglobulin as a molecular carrier of linoleate: characterization and effects on intestinal epithelial cells in vitro.

The dairy protein ß-lactoglobulin (ßlg) is known to bind hydrophobic ligands such as fatty acids. In the present work, we investigated the biological activity in vitro of linoleate once complexed to bovine ßlg. Binding of linoleate (C18:2) to bovine ßlg was achieved by heating at 60 °C for 30 min at pH 7.4, resulting in a linoleate/ßlg molar binding stoichiometry of 1.1, 2.1, and 3.4. Two types of binding sites were determined by ITC titrations. Binding of linoleate induced the formation of covalent dimers and trimers of ßlg. The LD(50) on Caco-2 cells after 24 h was 58 µM linoleate. However, cell viability was unaffected when 200 µM linoleate was presented to the Caco-2 cells as part of the ßlg complex. The Caco-2 cells did not increase mRNA transcript levels of long chain fatty acid transport genes, FATP4 and FABPpm, or increase levels of the cAMP signal, in response to the presence of 50 µM linoleate alone or as part of the ßlg complex. Therefore, it is proposed that ßlg can act as a molecular carrier and alter the bioaccessibility of linoleate/linoleic acid.

Emulsion-based delivery systems for tributyrin, a potential colon cancer preventative agent.

Tributyrin, a short-chain triglyceride oil used as a food additive, has been reported to be a potential preventive agent against colon cancer. The purpose of this study was to develop tributyrin delivery systems based on food-grade oil-in-water emulsions that could potentially be incorporated into foods. Emulsions containing only tributyrin as the lipid phase were highly unstable to droplet growth due to Ostwald ripening (OR) because of the relatively high water solubility of this low molecular weight triacylglycerol. The stability of the emulsions to OR could be greatly improved by incorporating >or=15-25% corn oil (a food-grade oil with a low water solubility) into the lipid phase. In addition, the tendency for droplet sedimentation to occur was reduced because the density contrast between the lipid and water phases was reduced in the mixed tributyrin/corn oil systems. The potential anticarcinogenic ability of the tributyrin emulsions was demonstrated using a cell culture model. Treatments with emulsions containing tributyrin significantly inhibited the viability of HT29 colon carcinoma cells. These results have important implications for the development and testing of nutraceuticals encapsulated in food-grade delivery systems as anticancer agents.