Identification of a TLR2 Inhibiting Wheat Hydrolysate.

SCOPE: Wheat hydrolysates are used in medical nutrition to provide undernourished patients a readily digestible protein source, for instance to recover from chemotherapy-induced intestinal mucosal inflammation. Since many hydrolysates of different sources can modulate the immune system, likely via Toll-like receptors (TLRs), it is hypothesized that also wheat hydrolysates might interact with TLR signaling, which could be a way to prevent intestinal inflammation and damage. METHODS AND RESULTS: The capacity of three wheat hydrolysates to modulate immunity by interfering with TLR signaling is determined. All wheat hydrolysates have TLR modulating effects but only one has strong TLR2 inhibiting effects, attenuating both TLR2/1 and TLR2/6 signaling in a reporter cell system. This is likely induced by direct TLR2-ectodomain binding, as confirmed by ELISA. Furthermore, this TLR2 blocking hydrolysate reduces IL-6 production in human dendritic cells. Application of reversed-phase-ultra HPLC combined with MS reveals that the presence of peptide WQIPEQSR is associated with the observed TLR2 inhibiting capacity. CONCLUSION: The study demonstrates TLR2-inhibiting capacities of a wheat hydrolysate. The findings provide a good start for further research to investigate whether this hydrolysate might contribute to the management of intestinal mucosal inflammation in cancer patients receiving chemotherapy.

The epithelial barrier-protecting properties of a soy hydrolysate.

Enhancing the epithelial barrier function could be a possible strategy to prevent food allergy or reduce its symptoms. Soy hydrolysates containing bioactive peptides could be instrumental in this. In this study, the protective effects of pretreatment with 6 soy hydrolysates on calcium ionophore A23187-induced TEER reduction were studied in T84 cells. The effects of the most potent soy hydrolysate on tight junction gene expression were studied. In order to identify the underlying pathways involved, the barrier disruptor specificity of the effect was studied by comparing the protective effects on TEER and Lucifer Yellow flux after the exposure to barrier disruptors that work via different intracellular pathways, i.e. the disruptors A23187, mellitin, and deoxynivalenol (DON). Preincubation with one of the six hydrolysates protected the epithelial cells from a decrease in TEER induced by A23187 (restored to 105% of the starting point, while A23187 alone decreased to 53% of the starting value) and mellitin (restored to 11% of the starting point, while mellitin alone decreased to 3.8% of the starting value). This soy hydrolysate was found to increase claudin-1 and decrease claudin-2 expression. The protective effect of the hydrolysate on TEER was specific for the barrier disruptors A23187 and mellitin, but was not observed for DON. This observation suggests that the soy hydrolysate may act via PKC isoforms, which are known to lead to changes in the expression of claudin-1 and 2. Our data suggest that specific soy hydrolysates may be designed to strengthen the epithelial barrier which might be instrumental in the management of the barrier function in individuals at risk of developing food allergy.

Immunomodulating protein aggregates in soy and whey hydrolysates and their resistance to digestion in an in vitro infant gastrointestinal model: new insights in the mechanism of immunomodulatory hydrolysates.

Hydrolysates, which are used in hypoallergenic infant formulas, have been found to possess immune modulating effects. For an optimal utilization of hydrolysates, the working mechanisms and responsible proteins underlying the effects should be elucidated. In this study, the immunomodulating activity of whey and soy hydrolysates was studied by quantifying TLR activation and assessing cytokine production in hydrolysate stimulated dendritic cells. The responsible protein fraction was identified and characterized by gel electrophoresis. The immune effects under gastrointestinal conditions were studied by digesting the hydrolysates in an in vitro infant digestion model, after which the digests were analyzed. In both soy and whey hydrolysates, TLR activation and cytokine production in dendritic cells were induced by a fraction containing protein aggregates larger than 1000 kDa, which were formed by electrostatic interactions and disulfide bonds. Only soy aggregates remained intact during duodenal digestion, and maintained the TLR activating capacity. Soy and whey protein aggregates larger than 1000 kDa possess immunomodulatory properties, but only soy aggregates remain under intestinal digestion conditions. This knowledge is important for a better understanding of the effects of hydrolysates.

Toll-like receptor mediated activation is possibly involved in immunoregulating properties of cow's milk hydrolysates.

Immunomodulating proteins and peptides are formed during the hydrolysis of cow's milk proteins. These proteins are potential ingredients in functional foods used for the management of a range of immune related problems, both in infants and adults. However, the mechanism behind these effects is unknown. We hypothesize that the interaction of peptides with Toll-like receptors (TLRs) can induce immune effects, since these receptors are known to sample many dietary molecules in the intestine in order to regulate immune effects. To investigate this, we compared the immune effects and TLR activation and inhibition by whey and casein hydrolysates with different hydrolysis levels. We first measured cytokine production in primary peripheral blood mononuclear cells stimulated with either whey, casein, or their hydrolysates. IL-10 and TNFα were induced by whey hydrolysates (decreasing with increasing hydrolysis level), but not by casein hydrolysates. Next, the activation of TLR 2, 3, 5 and 9 receptors were observed by intact and mildly hydrolysed whey proteins only and not by casein hydrolysates in TLR reporter cell lines. Many casein hydrolysates inhibited TLR signaling (mainly TLR 5 and 9). These results demonstrate that the effects of cow's milk proteins on the immune system are protein type and hydrolysis dependent. TLR signaling is suggested as a possible mechanism for differences in effect. This knowledge contributes to a better understanding of the immune effects of hydrolysates and the design of infant formula, and nutrition in general, with specific immunoregulatory effects.

Zinc Absorption from Milk Is Affected by Dilution but Not by Thermal Processing, and Milk Enhances Absorption of Zinc from High-Phytate Rice in Young Dutch Women.

Background: Milk has been suggested to increase zinc absorption. The effect of processing and the ability of milk to enhance zinc absorption from other foods has not been measured directly in humans.Objective: We aimed to assess zinc absorption from 1) milk undergoing various processing and preparatory steps and 2) from intrinsically labeled high-phytate rice consumed with milk or water.Methods: Two randomized crossover studies were conducted in healthy young women [age:18-25 y; body mass index (in kg/m2): 20-25]: 1) a milk study (n = 19) comparing the consumption of 800 mL full-fat ultra-high temperature (UHT) milk [heat-treated milk (HTM)], full-fat UHT milk diluted 1:1 with water [heat-treated milk and water (MW)], water, or unprocessed (raw) milk (UM), each extrinsically labeled with 67Zn, and 2) a rice study (n = 18) comparing the consumption of 90 g intrinsically 67Zn-labeled rice with 600 mL of water [rice and water (RW)] or full-fat UHT milk [rice and milk (RM)]. The fractional absorption of zinc (FAZ) was measured with the double-isotope tracer ratio method. In vitro, we assessed zinc extraction from rice blended into water, UM, or HTM with or without phytate.Results: FAZ from HTM was 25.5% (95% CI: 21.6%, 29.4%) and was not different from UM (27.8%; 95% CI: 24.2%, 31.4%). FAZ from water was higher (72.3%; 95% CI: 68.7%, 75.9%), whereas FAZ from MW was lower (19.7%; 95% CI: 17.5%, 21.9%) than HTM and UM (both P < 0.01). FAZ from RM (20.7%; 95% CI: 18.8%, 22.7%) was significantly higher than from RW (12.8%; 95% CI: 10.8%, 14.6%; P < 0.01). In vitro, HTM and UM showed several orders of magnitude higher extraction of zinc from rice with HTM than from rice with water at various phytate concentrations.Conclusions: Milk enhanced human FAZ from high-phytate rice by 62% compared with water. Diluting milk with water decreases its absorption-enhancing proprieties, whereas UHT processing does not. This trial was registered at the Dutch trial registry as NTR4267 (http://www.trialregister.nl/trialreg/admin/rctview.asp?TC=4267).

Vitamin B2, vitamin B12 and total homocysteine status in children and their associations with dietary intake of B-vitamins from different food groups: the Healthy Growth Study.

PURPOSE: To examine the associations between the dietary intakes of certain B-vitamins from different food sources with the relevant plasma status indices in children. METHODS: A representative subsample of 600 children aged 9-13 years from the Healthy Growth Study was selected. Dietary intakes of vitamins B2, B12, B6 and folate derived from different food sources were estimated. Plasma levels of vitamin B2 (or riboflavin), methylmalonic acid (MMA) and total homocysteine (tHcy) were also measured. RESULTS: Plasma concentrations of vitamin B2 below 3 µg/L were found in 22.8 % of the children. Children in the lower quartile of dietary vitamin B2 intake were found to have the lowest plasma vitamin B2 levels compared to children in the upper three quartiles (5.06 ± 7.63 vs. 6.48 ± 7.88, 6.34 ± 7.63 and 6.05 ± 4.94 µg/L respectively; P = 0.003). Regarding vitamin B12 children in the lower quartile of dietary intake had higher mean plasma tHcy levels compared to children in the upper two quartiles, respectively (6.00 ± 1.79 vs. 5.41 ± 1.43 and 5.46 ± 1.64 µmol/L; P = 0.012). Positive linear associations were observed between plasma vitamin B2 levels and dietary vitamin B2 derived from milk and fruits (ß = 0.133; P = 0.001 and ß = 0.086; P = 0.037). Additionally, nonlinear associations were also observed between plasma vitamin B2 levels and vitamin B2 derived from red meat, as well as between tHcy levels and vitamins B12 and B6 derived from milk; vitamins B12, B6 and folate derived from cereal products and folate derived from fruits. CONCLUSION: A considerably high prevalence of poor plasma vitamin B2 status was observed in children. The intake of milk, fruits and cereals was associated with more favorable tHcy levels, while the intake of milk and fruits with more favorable plasma B2 levels. However, these findings need to be further confirmed from controlled dietary intervention studies examining the modulation of biomarkers of B-vitamins.