Peptide Characterization and Functional Stability of a Partially Hydrolyzed Whey-Based Formula over Time.

Human clinical trials have shown that a specific partially hydrolyzed 100% whey-based infant formula (pHF-W) reduces AD risk in the first yeast of life. Meta-analyses with a specific pHF-W (pHF-W1) confirm a protective effect while other meta-analyses pooling different pHF-W show conflicting results. Here we investigated the molecular composition and functional properties of the specific pHF-W1 as well as the stability of its manufacturing process over time. This specific pHF-W1 was compared with other pHF-Ws. We used size exclusion chromatography to characterize the peptide molecular weight (MW), a rat basophil degranulation assay to assess the relative level of beta-lactoglobulin (BLG) allergenicity and a preclinical model of oral tolerance induction to test prevention of allergic sensitization. To analyze the exact peptide sequences before and after an HLA binding assay, a mass cytometry approach was used. Peptide size allergenicity and oral tolerance induction were conserved across pHF-W1 batches of production and time. The median MW of the 37 samples of pHF-W1 tested was 800 ± 400 Da. Further oral tolerance induction was observed using 10 different batches of the pHF-W1 with a mean reduction of BLG-specific IgE levels of 0.76 log (95% CI = -0.95; -0.57). When comparing pHF-W1 with three other formulas (pHF-W2 3 and 4), peptide size was not necessarily associated with allergenicity reduction in vitro nor oral tolerance induction in vivo as measured by specific IgE level (p < 0.05 for pHF-W1 and 2 and p = 0.271 and p = 0.189 for pHF-W3 and 4 respectively). Peptide composition showed a limited overlap between the formulas tested ranging from 11.7% to 24.2%. Furthermore nine regions in the BLG sequence were identified as binding HLA-DR. In conclusion, not all pHF-Ws tested have the same peptide size distribution decreased allergenicity and ability to induce oral tolerance. Specific peptides are released during the different processes used by different infant formula producers.

Allergenicity, antioxidant activity and ACE-inhibitory activity of protease hydrolyzed peanut flour.

Regularly consuming peanuts has been reported to have many health benefits. Peanut flour, a by-product of peanut oil processing, has higher protein and dietary fiber contents than peanut kernels, but its application as protein source in foods and dietary supplement is limited due to the fear of peanut allergy. This study indicates that hydrolysis of peanut flour (12% lipid) up to 4 h with Alcalase lowered the immunoreactivity of both soluble and insoluble portions of peanut flour, generated peanut flour hydrolysate (PFH) with good in vitro antioxidant and ACE-inhibitory activities. Importantly, the fraction smaller than 5 kDa did not bind IgE of peanut allergic patients, but exhibited higher ACE-inhibitory activity than the crude PFH. Thus, peanut flour could be an inexpensive protein source of antioxidant and anti-hypertensive ingredient. These findings are important for the value added application of peanut flour. However, studies with food and animal/human models are needed to confirm the benefits.

Design, quality, safety and efficacy of extensively hydrolyzed formula for management of cow's milk protein allergy: What are the challenges?

Cow's milk protein allergy (CMPA) is one of the most common food allergies in infancy. Clinical food allergy guidelines recommend an extensively hydrolyzed formula (EHF) as the first-line treatment in nonbreastfed infants with CMPA. Designing and commercializing EHF poses both technical and regulatory challenges. Each manufacturing step, from sourcing of raw materials to release of the final product, needs to be managed in accordance with comprehensive quality systems. To avoid cross-contamination via externally sourced ingredients, suppliers should be carefully selected based on quality requirements. Strict zoning of the manufacturing areas according to contamination risk and air flow control are effective strategies to prevent accidental allergen contamination. Furthermore, dedicated manufacturing lines for hypoallergenic products are used to prevent potential cross-contamination from other products produced on the same line. The enzymatic hydrolysis, heat treatment and ultrafiltration used are specific to each manufacturer. Consequently, EHF are a heterogenous group of products with differences in the molecular weight profile of peptides, content of residual immunogenic cow's milk allergens, and residual in-vitro allergenicity. These differences are likely to affect clinical efficacy and safety. As not all commercialized EHF products have undergone formal testing in the laboratory and clinical trials, there is a need to develop guidelines for minimum technical and regulatory requirements for EHF products, including validated assays for ongoing quality control. Clinical trials assessing new EHF products for their hypoallergenicity and ability to support normal growth remain the definitive proof of efficacy and safety in infants and young children with CMPA.

Epicutaneous immunogenicity of partially hydrolyzed whey protein evaluated using tape-stripped mouse model.

BACKGROUND: Hydrolyzed cow's milk protein formulas are widely used for infants with a history or risk of cow's milk allergy. Based on the current theory that food allergen sensitization occurs via the skin, we investigated the epicutaneous immunogenicity of partially hydrolyzed whey proteins, which are ingredients in infant formulas. METHODS: BALB/c mice were exposed epicutaneously to whey protein concentrate (WPC) or partial whey protein hydrolysates (PWH1 or PWH2) on tape-stripped skin. Sensitization was assessed by evaluating serum ß-lactoglobulin (ß-LG)-specific antibodies, basophil activation, and cytokine production from ß-LG-stimulated lymphoid cells. The anaphylaxis reaction was evaluated by measuring the rectal temperature and plasma level of mouse mast cell protease-1 after oral ß-LG challenge. Immune cell accumulation in the skin was also analyzed. RESULTS: Substantive sensitization and ß-LG-induced anaphylaxis reaction were observed in WPC-exposed mice, whereas no significant changes were observed in PWH1- or PWH2-exposed mice. The basophil and eosinophil counts increased in WPC-exposed murine skin, not but in PWH1- or PWH2-exposed mice. CONCLUSION: The epicutaneous immunogenicity of PWH1 and PWH2 is markedly decreased, which may reduce the risk of allergen sensitization. Further studies are required to investigate the clinical value of these partial hydrolysates for high-risk infants.

Tolerogenic Effect Elicited by Protein Fraction Derived From Different Formulas for Dietary Treatment of Cow's Milk Allergy in Human Cells.

Several formulas are available for the dietary treatment of cow's milk allergy (CMA). Clinical data suggest potentially different effect on immune tolerance elicited by these formulas. We aimed to comparatively evaluate the tolerogenic effect elicited by the protein fraction of different formulas available for the dietary treatment of CMA. Five formulas were compared: extensively hydrolyzed whey formula (EHWF), extensively hydrolyzed casein formula (EHCF), hydrolyzed rice formula (HRF), soy formula (SF), and amino acid-based formula (AAF). The formulas were reconstituted in water according to the manufacturer's instructions and subjected to an in vitro infant gut simulated digestion using a sequential gastric and duodenal static model. Protein fraction was then purified and used for the experiments on non-immune and immune components of tolerance network in human enterocytes and in peripheral mononuclear blood cells (PBMCs). We assessed epithelial layer permeability and tight junction proteins (occludin and zonula occludens-1, ZO-1), mucin 5AC, IL-33, and thymic stromal lymphopoietin (TSLP) in human enterocytes. In addition, Th1/Th2 cytokine response and Tregs activation were investigated in PBMCs from IgE-mediated CMA infants. EHCF-derived protein fraction positively modulated the expression of gut barrier components (mucin 5AC, occludin and ZO-1) in human enterocytes, while SF was able to stimulate the expression of occludin only. EHWF and HRF protein fractions elicited a significant increase in TSLP production, while IL-33 release was significantly increased by HRF and SF protein fractions in human enterocytes. Only EHCF-derived protein fraction elicited an increase of the tolerogenic cytokines production (IL-10, IFN-γ) and of activated CD4+FoxP3+ Treg number, through NFAT, AP1, and Nf-Kb1 pathway. The effect paralleled with an up-regulation of FoxP3 demethylation rate. Protein fraction from all the study formulas was unable to induce Th2 cytokines production. The results suggest a different regulatory action on tolerogenic mechanisms elicited by protein fraction from different formulas commonly used for CMA management. EHCF-derived protein fraction was able to elicit tolerogenic effect through at least in part an epigenetic modulation of FoxP3 gene. These results could explain the different clinical effects observed on immune tolerance acquisition in CMA patients and on allergy prevention in children at risk for atopy observed using EHCF.

Reduced IgE and IgG antigenic response to milk proteins hydrolysates obtained with the use of non-commercial serine protease from Yarrowia lipolytica.

The aim of the study was to investigate the use of serine protease from Yarrowia lipolytica yeast for reduction of milk proteins allergenicity. Whey protein concentrate (WPC-80), αs-casein and their hydrolysates were analyzed for the capacity to bind IgE and IgG antibodies present in sera from patients with cow milk protein allergy using a competitive ELISA. The hydrolysis of αs-casein and whey protein concentrate contributed to a significant reduction of their immunoreactive epitopes. In case of IgE antibodies, the lowest binding capacity was detected in the 24 h hydrolysates of both proteins in which the inhibition of the reaction was ≤20 and ≤68% for αs-casein and whey protein concentrate respectively. One hour hydrolysis of WPC-80 reduced the protein antigenicity, while the longer time (5 h) might lead to the exposure of new IgE - reactive epitopes.

Prevention of Allergic Sensitization and Treatment of Cow's Milk Protein Allergy in Early Life: The Middle-East Step-Down Consensus.

Allergy risk has become a significant public health issue with increasing prevalence. Exclusive breastfeeding is recommended for the first six months of life, but this recommendation is poorly adhered to in many parts of the world, including the Middle-East region, putting infants at risk of developing allergic sensitization and disorders. When breastfeeding is not possible or not adequate, a partially hydrolyzed whey formula (pHF-W) has shown proven benefits of preventing allergy, mainly atopic eczema, in children with a genetic risk. Therefore, besides stimulating breastfeeding, early identification of infants at risk for developing atopic disease and replacing commonly used formula based on intact cow milk protein (CMP) with a clinically proven pHF-W formula is of paramount importance for allergy prevention. If the child is affected by cow's milk protein allergy (CMPA), expert guidelines recommend extensively hydrolyzed formula (eHF), or an amino acid formula (AAF) in case of severe symptoms. The Middle-East region has a unique practice of utilizing pHF-W as a step-down between eHF or AAF and intact CMP, which could be of benefit. The region is very heterogeneous with different levels of clinical practice, and as allergic disorders may be seen by healthcare professionals of different specialties with different levels of expertise, there is a great variability in preventive and treatment approaches within the region itself. During a consensus meeting, a new approach was discussed and unanimously approved by all participants, introducing the use of pHF-W in the therapeutic management of CMPA. This novel approach could be of worldwide benefit.

Confirmed Hypoallergenicity of a Novel Whey-Based Extensively Hydrolyzed Infant Formula Containing Two Human Milk Oligosaccharides.

BACKGROUND: We sought to determine whether an extensively hydrolyzed formula (EHF) supplemented with two human milk oligosaccharides (HMO) was tolerated by infants with cow's milk protein allergy (CMPA). METHODS: A whey-based EHF (Test formula) containing 2'fucosyl-lactose (2'FL) and lacto-N-neotetraose (LNnT) was assessed for clinical hypoallergenicity and safety. The Control formula was a currently marketed EHF without HMO. Children with CMPA, aged 2 months to 4 years, were assessed by double-blind, placebo-controlled food challenges (DBPCFC) to both formulas, in randomized order. If both DBPCFC were negative, subjects participated in a one-week, open food challenge (OFC) with the Test formula. Symptoms and adverse events were recorded. Hypoallergenicity was accepted if at least 90% (with 95% confidence intervals) of subjects tolerated the Test formula. RESULTS: Of the 82 children with CMPA that were screened, 67 (intention-to-treat [ITT] cohort-mean age 24.5 ± 13.6 months; range 2-57; 45 [67.2%] male) were randomized to receive either the Test or the Control formula during the first DBPCFC. Of these, 64 children completed at least one DBPCFC (modified intention-to-treat [mITT] cohort). Three children were excluded due to protocol deviations (per protocol [PP] cohort; n = 61). There was one allergic reaction to the Test, and one to the Control formula. On the mITT analysis, 63 out of 64 (98.4%; 95% CI lower bound 92.8%), and on the PP analysis 60 out of 61 (98.4%; 95% CI lower bound 92.5%) participants tolerated the Test formula, confirming hypoallergenicity. CONCLUSION: The whey-based EHF supplemented with 2'FL and LNnT met the clinical hypoallergenicity criteria and can be recommended for the management of CMPA in infants and young children.

Oral Immunotherapy with Egg Peptides Induces Innate and Adaptive Tolerogenic Responses.

SCOPE: The mechanism through which peptide-based immunotherapy provides effective desensitization toward food allergy is investigated. METHODS AND RESULTS: Ex vivo experiments are conducted with intestinal epithelial cells (IECs), dendritic cells (DCs), and T cells from mice sensitized to egg white (EW) and either left untreated or tolerized by the oral administration of a hydrolysate of ovalbumin with pepsin (OP). IECs from EW-sensitized mice upregulate Il33 and Tslp to a higher extent than those from tolerized mice and induce bone marrow (BM)-DCs to express Tnfsf4 and produce pro-inflammatory cytokines. On the other hand, incubation with OP upregulates Aldh1a1 in IEC cultures and BM-DCs conditioned with supernatants of OP-pulsed IECs also overexpress Aldh1a2 and Tgfb1. DCs from tolerized mice, in co-culture with CD4+ T cells from sensitized mice, reduce the secretion of IL-5, IFN-γ, and IL-17, following stimulation with EW, to a level similar than DCs from sham-sensitized mice. Furthermore, incubation with OP of DCs and CD4+ T cells, regardless of the mouse sentitization status, promotes the secretion of TGF-ß and the generation of Foxp3+ RORγt+ cells. CONCLUSION: OP induces the expression of aldehyde dehydrogenase enzymes in cells of the innate immune system and the development of Foxp3+ RORγt+ T cells.

Impact of enzymatic hydrolysis followed by transglutaminase-induced cross-linking on decreasing antigenicity and reserving partial interfacial properties of whey protein isolate.

Whey protein isolate (WPI) was hydrolyzed by alcalase and trypsin for three hydrolysis degrees (DHs), followed by transglutaminase (TGase) induced cross-linking. The prepared products were measured for surface hydrophobicity and emulsifying and foaming properties, as well as in vitro antigenicity for α-lactalbumin and ß-lactoglobulin. The results indicated that enzymatic hydrolysis of WPI mostly resulted in WPI hydrolysates with significantly decreased antigenicity of α-lactalbumin and ß-lactoglobulin, especially in the case of a higher DH value. Moreover, the TGase-induced cross-linking led to a further antigenicity decrease for these prepared products. Alcalase was always more potent than trypsin to decrease antigenicity. In comparison with WPI, the conducted enzymatic hydrolysis also brought losses to surface hydrophobicity and emulsifying and foaming properties. On the other hand, the conducted cross-linking could partially rescue these properties. It is thus concluded that the assessed enzymatic hydrolysis coupled with TGase-induced cross-linking might be an applicable process for WPI to decrease its potential antigenicity but reserve partial interfacial properties.

Microbiota and Derived Parameters in Fecal Samples of Infants with Non-IgE Cow's Milk Protein Allergy under a Restricted Diet.

Cow's milk protein allergy (CMPA) is the most common food allergy in infancy. Non-IgE mediated (NIM) forms are little studied and the responsible mechanisms of tolerance acquisition remain obscure. Our aim was to study the intestinal microbiota and related parameters in the fecal samples of infants with NIM-CMPA, to establish potential links between type of formula substitutes, microbiota, and desensitization. Seventeen infants between one and two years old, diagnosed with NIM-CMPA, were recruited. They were all on an exclusion diet for six months, consuming different therapeutic protein hydrolysates. After this period, stool samples were obtained and tolerance development was evaluated by oral challenges. A control group of 10 age-matched healthy infants on an unrestricted diet were included in the study. Microbiota composition, short-chain fatty acids, calprotectin, and transforming growth factor (TGF)-ß1 levels were determined in fecal samples from both groups. Infants with NIM-CMPA that consumed vegetable protein-based formulas presented microbiota colonization patterns different from those fed with an extensively hydrolyzed formula. Differences in microbiota composition and fecal parameters between NIM-CMPA and healthy infants were observed. Non-allergic infants showed a significantly higher proportion of Bacteroides compared to infants with NIM-CMPA. The type of protein hydrolysate was found to determine gut microbiota colonization and influence food allergy resolution in NIM-CMPA cases.

Immunomodulatory Protein Hydrolysates and Their Application.

Immunomodulatory protein hydrolysate consumption may delay or prevent western immune-related diseases. In order to purposively develop protein hydrolysates with an optimal and reproducible immunomodulatory effect, knowledge is needed on which components in protein hydrolysates are responsible for the immune effects. Important advances have been made on this aspect. Also, knowledge on mechanisms underlying the immune modulating effects is indispensable. In this review, we discuss the most promising application possibilities for immunomodulatory protein hydrolysates. In order to do so, an overview is provided on reported in vivo immune effects of protein hydrolysates in both local intestinal and systemic organs, and the current insights in the underlying mechanisms of these effects. Furthermore, we discuss current knowledge and physicochemical approaches to identify the immune active protein sequence(s). We conclude that multiple hydrolysate compositions show specific immune effects. This knowledge can improve the efficacy of existing hydrolysate-containing products such as sports nutrition, clinical nutrition, and infant formula. We also provide arguments for why immunomodulatory protein hydrolysates could be applied to manage the immune response in the increasing number of individuals with a higher risk of immune dysfunction due to, for example, increasing age or stress.

Effect of enzymatic hydrolysis on bioactive properties and allergenicity of cricket (Gryllodes sigillatus) protein.

Food-derived bioactive peptides have gained attention for their role in preventing chronic diseases. Edible insects are viable sources of bioactive peptides owing to their high protein content and sustainable production. In this study, whole crickets (Gryllodes sigillatus) were alcalase-hydrolyzed to a degree of hydrolysis (DH) ranging from 15 to 85%. Antioxidant activity, angiotensin converting enzyme (ACE), and dipeptidyl peptidase-4 (DPP-IV)- inhibition of the cricket protein hydrolysates (CPH) were evaluated before and after simulated gastrointestinal digestion (SGD). Antioxidant activity was similar among CPH, whereas ACE and DPP-IV inhibition was greater (p < 0.05) in CPH with 60-85% DH. Bioactivity improved after SGD. CPH allergenicity was evaluated using human shrimp-allergic sera. All sera positively reacted to tropomyosin in the unhydrolyzed cricket and CPH with 15-50% DH, whereas 60-85% DH showed no reactivity. In conclusion, CPH (60-85% DH) had the greatest bioactive potential and lowest reactivity to tropomyosin, compared with other CPH and the unhydrolyzed control.

Ingestion of partially hydrolyzed whey protein suppresses epicutaneous sensitization to ß-lactoglobulin in mice.

BACKGROUND: Epicutaneous sensitization to food allergens can occur through defective skin barriers. However, the relationship between oral tolerance and epicutaneous sensitization remains to be elucidated. We aimed to determine whether prior oral exposure to whey proteins or their hydrolysates prevents epicutaneous sensitization and subsequent food-allergic reaction to the whey protein, ß-lactoglobulin (ß-LG), and investigated the underlying mechanisms. METHODS: BALB/c mice were given whey protein concentrate (WPC), two kinds of partial whey protein hydrolysate (PWH1 or PWH2), or extensive whey protein hydrolysate (EWH) in drinking water for 21 days. The mice were then epicutaneously sensitized with ß-LG on tape-stripped skin. Sensitization was assessed by basophil activation tests and by measuring the level of serum ß-LG-specific antibodies and cytokines secreted from ß-LG-restimulated spleen and mesenteric lymph node (MLN) cells. Development of an allergic reaction was assessed by monitoring body temperature and by measuring mast cell protease-1 level in plasma after the ß-LG oral challenge. Activated T-cell population among ß-LG-restimulated MLN cells was also analyzed. RESULTS: In mice fed with WPC, PWH1, or PWH2, sensitization and the development of an allergic reaction were totally reduced. The acceleration of cytokine release from the spleen and MLN cells or T-cell activation was not evident after ß-LG restimulation. In EWH-fed mice, a suppressive effect, though milder than that in WPC-, PWH1-, or PWH2-fed mice, was observed during the development of the allergic reaction. CONCLUSIONS: Prior oral exposure to partially hydrolyzed whey protein prevents epicutaneous sensitization and subsequent allergic response to ß-LG in mice.

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.

Immunomodulatory and anticancer protein hydrolysates (peptides) from food proteins: A review.

Bioactive peptides are oligopeptides that consist of 2-20 amino acids that can exert beneficial effects on human health in addition to basic nutritional effects. Food derived protein hydrolysates or peptides with immunomodulatory and anticancer activities have been reported from a variety of food protein sources such as milk, egg, fish, rice, soybean, pea, chlorella, spirulina, oyster and mussel. In vitro hydrolysis of food proteins using commercial proteolytic enzymes is the most commonly employed process for the production of immunomodulatory and anticancer food protein hydrolysates. The immunomodulatory and anticancer activities of food derived protein hydrolysates or peptides are related to the amino acid composition, sequence and length. Most immunomodulatory and anticancer food protein hydrolysates or peptides were tested using cell culture and animal models, while a few involved clinical trials. This review provides a comprehensive overview of immunomodulatory and anticancer food derived protein hydrolysates or peptides, their production and mechanisms of action.

A chimeric IgE that mimics IgE from patients allergic to acid-hydrolyzed wheat proteins is a novel tool for in vitro allergenicity assessment of functionalized glutens.

BACKGROUND: Acid-hydrolyzed wheat proteins (acid-HWPs) have been shown to provoke severe allergic reactions in Europe and Japan that are distinct from classical wheat allergies. Acid-HWPs were shown to contain neo-epitopes induced by the deamidation of gluten proteins. However, products with variable rates of deamidation can be found. OBJECTIVES: In this work, we studied the effect of the extent of wheat proteins deamidation on its allergenicity. A recombinant chimeric IgE was produced and compared to patients' IgE for its capacity to assess the IgE-mediated triggering potential of acid-HWPs. METHODS: Sera from acid-HWP allergic patients were analyzed via ELISA and a functional basophil assay for their IgE reactivity to wheat proteins with different deamidation levels. A chimeric mouse/human IgE (chIgE-DG1) specific for the main neo-epitope, QPEEPFPE, involved in allergy to acid-HWPs was characterized with respect to its functionality and its reactivity compared to that of patients' IgE. RESULTS: Acid-HWPs with medium (30%) and high (50-60%) deamidation levels displayed a markedly stronger IgE binding and capacity to activate basophils than those of samples with weak (15%) deamidation levels. The monoclonal chIgE-DG1 allowed basophil degranulation in the presence of deamidated wheat proteins. ChIgE-DG1 was found to mimic patients' IgE reactivity and displayed the same ability to rank acid-HWP products in a degranulation assay. CONCLUSION: Increasing the deamidation level of products from 15% to 60% resulted in an approximately 2-fold increase in their antigenicity and a 100-fold increase in their eliciting potential. The chimeric ChIgE-DG1 may be a useful tool to evaluate functionalized glutens for their allergenic potential. By mimicking patient sera reactivity, chIgE-DG1 also provided data on the patients' IgE repertoire and on the functionality of certain repeated epitopes in gluten proteins.

Proteomic analysis of Lupinus angustifolius (var. Zeus and Bojar) and Lupinus luteus (var. Lord and Parys) seed proteins and their hydrolysates.

BACKGROUND: Proteins enzymatic digestion is a very complex process, during which some components are degraded, whereas others remain in an unchanged form. Moreover, enzymatic hydrolysis is one of the most popular methods used to reduce the allergenicity of food proteins. In the present study, the efficiency of enzymatic hydrolysis of lupin seed proteins was assessed by proteomic analysis as performed by two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry identification. Two digestion systems were used: oriented digestion carried out by trypsin and model in vitro digestion mimicking the conditions present in the gastrointestinal tract. RESULTS: The comparisons of 2-DE maps of proteins isolated form different lupin seed species revealed that the differences in proteins expression were observed mainly in the central parts of gels (i.e. in the molecular weight range from 20 to 70 kDa, and the pH range 5-7). In total, 27 differentially expressed proteins spots were successfully identified by mass spectrometry analysis. An important reduction in the number of proteins spots on 2-DE maps was observed when trypsin and the in vitro digestion model were applied. The protein spot insensitive to digestion in both hydrolysis systems was identified as ß-conglutin. CONCLUSIONS: The results of the present study provide insight into the nature of the digestion process that may take place after lupin seed protein intake and highlight the important fact that some of the proteins are insensitive to digestive enzyme activity. Moreover, evaluation of digestion activity of trypsin towards lupin seed proteins may be used for the development of specific processes with respect to hypoallergenic food production. © 2017 Society of Chemical Industry.

Enzymatic characterisation of the immobilised Alcalase to hydrolyse egg white protein for potential allergenicity reduction.

BACKGROUND: This study examined technique characteristics of the immobilised Alcalase to hydrolyse egg white protein for potential allergenicity reduction. Alcalase was immobilised covalently on carboxyl-functionalised magnetic beads by carbodiimide activation. The technique characteristics of the immobilised Alcalase were investigated, followed by determining the degrees of hydrolysis (DH), immunoglobulin G (IgG) binding, and IgE binding of the digested egg white protein by immobilised Alcalase. RESULTS: Enzymatic activity, enzyme loading, and immobilisation yield of the prepared immobilised Alcalase were 20.55 U mg-1 , 925 mg g-1 , and 45%, respectively. Immobilised Alcalase showed maximum activity at pH 8.0 and 60 °C. Compared with free Alcalase, immobilised Alcalase exhibited better thermal and storage stability. Moreover, immobilised Alcalase can be reused 10 times and still maintained 55% of its initial activity. Partial hydrolysis of egg white protein by immobilised Alcalase can effectively reduce IgG and IgE binding of the hydrolysates. CONCLUSION: This study indicates that the immobilised Alcalase can be used to hydrolyse continuously egg white protein for potential allergenicity reduction. © 2016 Society of Chemical Industry.