Degree of hydrolysis is a poor predictor of the sensitizing capacity of whey- and casein-based hydrolysates in a Brown Norway rat model of cow's milk allergy.

The use of infant formulas (IFs) based on hydrolyzed cow's milk proteins to prevent cow's milk allergy (CMA) is highly debated. The risk of sensitization to milk proteins induced by IFs may be affected by the degree of hydrolysis (DH) as well as other physicochemical properties of the cow's milk-based protein hydrolysates within the IFs. The immunogenicity (specific IgG1 induction) and sensitizing capacity (specific IgE induction) of 30 whey- or casein-based hydrolysates with different physicochemical characteristics were compared using an intraperitoneal model of CMA in Brown Norway rats. In general, the whey-based hydrolysates demonstrated higher immunogenicity than casein-based hydrolysates, inducing higher levels of hydrolysate-specific and intact-specific IgG1. The immunogenicity of the hydrolysates was influenced by DH, peptide size distribution profile, peptide aggregation, nano-sized particle formation, and surface hydrophobicity. Yet, only the surface hydrophobicity was found to affect the sensitizing capacity of hydrolysates, as high hydrophobicity was associated with higher levels of specific IgE. The whey- and casein-based hydrolysates exhibited distinct immunological properties with highly diverse molecular composition and physicochemical properties which are not accounted for by measuring DH, which was a poor predictor of sensitizing capacity. Thus, future studies should consider and account for physicochemical characteristics when assessing the sensitizing capacity of cow's milk-based protein hydrolysates.

Partially Hydrolysed Whey Has Superior Allergy Preventive Capacity Compared to Intact Whey Regardless of Amoxicillin Administration in Brown Norway Rats.

Background: It remains largely unknown how physicochemical properties of hydrolysed infant formulas influence their allergy preventive capacity, and results from clinical and animal studies comparing the preventive capacity of hydrolysed infant formula with conventional infant formula are inconclusive. Thus, the use of hydrolysed infant formula for allergy prevention in atopy-prone infants is highly debated. Furthermore, knowledge on how gut microbiota influences allergy prevention remains scarce. Objective: To gain knowledge on (1) how physicochemical properties of hydrolysed whey products influence the allergy preventive capacity, (2) whether host microbiota disturbance influences allergy prevention, and (3) to what extent hydrolysed whey products influence gut microbiota composition. Methods: The preventive capacity of four different ad libitum administered whey products was investigated in Brown Norway rats with either a conventional or an amoxicillin-disturbed gut microbiota. The preventive capacity of products was evaluated as the capacity to reduce whey-specific sensitisation and allergic reactions to intact whey after intraperitoneal post-immunisations with intact whey. Additionally, the direct effect of the whey products on the growth of gut bacteria derived from healthy human infant donors was evaluated by in vitro incubation. Results: Two partially hydrolysed whey products with different physicochemical characteristics were found to be superior in preventing whey-specific sensitisation compared to intact and extensively hydrolysed whey products. Daily oral amoxicillin administration, initiated one week prior to intervention with whey products, disturbed the gut microbiota but did not impair the prevention of whey-specific sensitisation. The in vitro incubation of infant faecal samples with whey products indicated that partially hydrolysed whey products might confer a selective advantage to enterococci. Conclusions: Our results support the use of partially hydrolysed whey products for prevention of cow's milk allergy in atopy-predisposed infants regardless of their microbiota status. However, possible direct effects of partially hydrolysed whey products on gut microbiota composition warrants further investigation.

Cow's milk allergy prevention and treatment by heat-treated whey-A study in Brown Norway rats.

BACKGROUND: Food processing, including heat-treatment, can affect protein structure and stability, and consequently affect protein immunogenicity and allergenicity. A few studies have shown that structural changes induced by heat-treatment impact the intestinal protein uptake and suggest this as a contributing factor for altered allergenicity. OBJECTIVE: To investigate the impact of heat-treatment of a whey-based protein product on allergenicity and tolerogenicity as well as on intestinal uptake in various animal models. METHODS: Immunogenicity and sensitizing capacity of the heat-treated whey product were compared to that of the unmodified product by intraperitoneal and oral exposure studies, while tolerogenic properties were assessed by oral primary prevention and desensitization studies in high-IgE responder Brown Norway rats. RESULTS: Heat-treatment of whey induced partial protein denaturation and aggregation, which reduced the intraperitoneal sensitizing capacity but not immunogenicity. In contrast, heat-treatment did not influence the oral sensitizing capacity, but the heat-treated whey showed a significantly reduced eliciting capacity compared to unmodified whey upon oral challenge. Heat-treatment did not reduce the tolerogenic properties of whey, as both products were equally good at preventing sensitization in naïve rats as well as desensitizing already sensitized rats. Results from inhibitory ELISA and immunoblots with sera from sensitized rats demonstrated that heat-treatment caused an altered protein and epitope reactivity. Protein uptake studies showed that heat-treatment changed the route of uptake with less whey being absorbed through the epithelium but more into the Peyer's patches. CONCLUSION AND CLINICAL RELEVANCE: These results support the notion that the physicochemical features of proteins affect their route of uptake and that the route of uptake may affect the protein allergenicity. Furthermore, the study highlights the potential for heat-treatment in the production of efficient and safe cow's milk protein-based products for prevention and treatment of cow's milk allergy.

Pellet-free isolation of human and bovine milk extracellular vesicles by size-exclusion chromatography.

Studies have suggested that nanoscale extracellular vesicles (EV) in human and bovine milk carry immune modulatory properties which could provide beneficial health effects to infants. In order to assess the possible health effects of milk EV, it is essential to use isolates of high purity from other more abundant milk structures with well-documented bioactive properties. Furthermore, gentle isolation procedures are important for reducing the risk of generating vesicle artefacts, particularly when EV subpopulations are investigated. In this study, we present two isolation approaches accomplished in three steps based on size-exclusion chromatography (SEC) resulting in effective and reproducible EV isolation from raw milk. The approaches do not require any EV pelleting and can be applied to both human and bovine milk. We show that SEC effectively separates phospholipid membrane vesicles from the primary casein and whey protein components in two differently obtained casein reduced milk fractions, with one of the fractions obtained without the use of ultracentrifugation. Milk EV isolates were enriched in lactadherin, CD9, CD63 and CD81 compared to minimal levels of the EV-marker proteins in other relevant milk fractions such as milk fat globules. Nanoparticle tracking analysis and electron microscopy reveals the presence of heterogeneous sized vesicle structures in milk EV isolates. Lipid analysis by thin layer chromatography shows that EV isolates are devoid of triacylglycerides and presents a phospholipid profile differing from milk fat globules surrounded by epithelial cell plasma membrane. Moreover, the milk EV fractions are enriched in RNA with distinct and diverging profiles from milk fat globules. Collectively, our data supports that successful milk EV isolation can be accomplished in few steps without the use of ultracentrifugation, as the presented isolation approaches based on SEC effectively isolates EV in both human and bovine milk.

Purification of the photosynthetic pigment C-phycocyanin from heterotrophic Galdieria sulphuraria.

BACKGROUND: The phycobiliprotein C-phycocyanin (C-PC) is used in cosmetics, diagnostics and foods and also as a nutraceutical or biopharmaceutical. It is produced in the cyanobacterium Arthrospira platensis grown phototrophically in open cultures. C-PC may alternatively be produced heterotrophically in the unicellular rhodophyte Galdieria sulphuraria at higher productivities and under improved hygienic standards if it can be purified as efficiently as C-PC from A. platensis. RESULTS: Ammonium sulfate fractionation, aqueous two-phase extraction, tangential flow ultrafiltration and anion exchange chromatography were evaluated with respect to the purification of C-PC from G. sulphuraria extracts. Galdieria sulphuraria C-PC showed similar properties to those described for cyanobacterial C-PC with respect to separation by all methodologies. The presence of micelles in G. sulphuraria extracts influenced the different procedures. Only chromatography was able to separate C-PC from a second phycobiliprotein, allophycocyanin. CONCLUSION: C-PC from heterotrophic G. sulphuraria shows similar properties to cyanobacterial C-PC and can be purified to the same standards, despite initial C-PC concentrations being low and impurity concentrations high in G. sulphuraria extracts.