Immunogenicity and Allergenicity of Whey Protein Hydrolysates

Allergic reactions to cow's milk are common in children. The sera of allergic subjects have been found to contain specific IgE antibodies against several whey proteins such as α-lactalbumin (α-la) and β-lactoglobulin (β-lg). This work aims to study the effect of hydrolysis on the immunogenicity and the allergenicity of α-la and β-lg. Whey proteins were submitted to hydrolysis with immobilized α-chymotrypsin. High performance liquid chromatography analysis showed a decrease of native whey proteins and the appearance of low molecular weight peptides. The immunogenicity of these hydrolysates was not modified, but their allergenicity, as assessed by RAST inhibition, was decreased. The results may have important clinical implications.     

Functional and technological aspects of whey powder and whey protein products

Commercial whey powder, whey protein concentrates and whey protein isolates (WPIs) were evaluated for certain functional properties and for their application in full‐fat and nonfat yoghurts. The functional properties of whey products varied, and the highest functionality was recorded in samples with high protein levels. Whey powder had the lowest foaming performance and emulsifying capacity, while WPIs possessed the best functional properties of all the other samples. Curd tension (CT), viscosity and syneresis were improved in yoghurts made using fortified cow's milk or reconstituted skim milk with any whey products, while whey powder had no impact on CT.     

Whey Protein Antigenicity Reduction by Fungal Proteinases and a Pepsin/Pancreatin Combination

Whey protein components were hydrolyzed with Corolase 7092? (peptidases from Aspergillus strains), pepsin and Corolase PP? (a mixture of pancreatic enzymes), either individually or in combination, in trials to eliminate protein allergenicity. The hydrolysates were characterized by physico-chemical and by immunological techniques using sera from patients allergic to milk proteins. Enzyme specificity rather than degree of hydrolysis or molecular mass distribution of hydrolysates determined the residual antigenicity of the whey proteins. Ultrafiltration was a prerequisite for obtaining hypoallergenic whey protein hydrolysates.     

Ernährungsphysiologische Bedeutung von Molke und Molkenbestandteilen

Nutritional significance of whey and whey components Deriving from positive effects of whey drinking cures in antiquity, the Middle Ages and modern time, a review is given on nutritional significance of whey. The proteins are essential components of whey and belong to the proteins with highest biological value because of their amino acid composition. Besides, they show fundamental functional properties, which enable a varied application in foods, dietetic foods and beverages in form of different whey products (powder, protein concentrates and isolates). Whey proteins have found considerable usage in infant's nutrition as whey predominant formulas as well as whey protein hydrolysates in case of cow's milk protein intolerances. A recent field of research are biological active peptide sequences which become effective during digestion and are of importance for secretion of entero hormones as well as for immune enthancing effects. They may contribute to assess the biological value of whey proteins under enlarged points of view and to develop new application forms and areas. It is pointed to further fields of application (e. g. adipositas, gout, kidney insufficiency). Concerning the quantitatively most dominant lactose in whey, it is dealt with its importance for the healthy development of infants (adaptation to the increased lactose content of mother's milk) as well as with lactose intolerance and galactosaemia. In case of mineral salts of whey it is emphasized the high nutrient density of calcium (prophylaxis for osteoporosis), the beneficial Ca: P and Na: K proportions (antihypertensive in case of the last one), the promotion of absorption of mineral salts by lactose, and the high content of iodine. The whey is rich in B-vitamins, which contribute essentially for their satisfaction or requirement in case of a corresponding consumption. To be emphasized is the vitamin B₁₂ in milk and whey, which is the sole source of this indispensable nutrient for blood-formation and cell division in lacto-ovo-vegetarian nutrition. In conclusion, a summerizing dietetics valuation of whey is performed.     

The effect of supplementing goats milk with whey protein concentrate on textural properties of set-type yoghurt

Yoghurt was manufactured from goat's milk and supplemented with 30 g L^?1 of whey protein concentrate (WPC). The textural properties of the yoghurt were evaluated during the shelf‐life of the product and the textural characteristics of yoghurt made from cow's milk were used as a reference. The instrumental analyses used were the puncture test, stress relaxation test and texture profile analysis. The addition of WPC to goat's milk enhanced the textural characteristics of yoghurt. These advantageous attributes included increased firmness, hardness and adhesiveness. These attributes were quantitatively similar (P > 0.05) to those obtained from yoghurt made from cow's milk. In addition, the textural properties were maintained constant throughout the shelf‐life of the product.     

Proteolytic Activity of a Bacillus subtilis Neutral Protease Preparation upon Caseins and Whey Proteins of Cow's Milk

The proteolytic activity of an industrial preparation of neutral protease from Bacillus subtilis has been tested on caseins and whey proteins of cow's milk. Electrophoretic analysis of κ-casein hydrolysate showed that only electropositive products can be found. Hydrolysis of β-casein generated compounds with low electrophoretic mobility as did hydrolysis of γ-casein. β-Lactoglobulin and immunoglobulins were heavily damaged, whereas α-lactalbumin was slightly hydrolyzed. Because of its wide spectrum of activity and interesting thermophilic properties, this enzymatic preparation can be applied to other fields in the food industry.     

Immunomodulation by hydrolysates and peptides derived from milk proteins

Milk protein‐derived peptides have shown immunomodulatory properties either singly or in a mixture. However, information on the behaviour of these compounds is scarce, complicating their further study. This review presents current knowledge on immunomodulatory milk protein‐derived peptides, including hydrolysates, peptide fractions and single peptides. The evidence shows that hydrolysates derived from both casein and whey proteins show immunomodulatory effects; however, this bioactivity is mainly attributed to peptides derived from caseins. The identification of peptides in hydrolysates, as well as their characterisation and knowledge of the mechanisms behind their bioactivity, is necessary to further exploit their potential health benefits.     

Cow and camel milk-derived whey and casein protein hydrolysates demonstrated effective antifungal properties against selected Candida species

Bioactive peptides derived from milk proteins are widely known to possess antibacterial activities. Even though the antibacterial effects of milk-derived peptides are widely characterized, not much focus is given to their antifungal characterization. Therefore, in this study, we investigated the antifungal properties of camel and cow whey and casein hydrolysates against various species of pathogenic Candida. The hydrolysates were produced using 2 enzymes (alcalase and protease) at differing hydrolysis durations (2, 4, and 6 h) and tested for their antifungal properties. The results showed that intact cow whey and casein proteins did not display any anti-Candida albicans properties, whereas the alcalase-derived 2 h camel casein hydrolysate (CA-C-A2) displayed a higher percentage of inhibition against Candida albicans (93.69 ± 0.26%) followed by the cow casein hydrolysate generated by protease-6 h (Co-C-P6; 81.66 ± 0.99%), which were significantly higher than that of fluconazole, a conventional antifungal agent (76.92 ± 4.72%). Interestingly, when tested again Candida krusei, camel casein alcalase 2 and 4 h (CA-C-A2 and CA-C-A4), and cow whey alcalase-6 h (CO-W-A6) hydrolysates showed higher antifungal potency than fluconazole. However, for Candida parapsilosis only camel casein alcalase-4 h (Ca-C-A4) and cow casein protease-6 h (Co-C-P6) hydrolysates were able to inhibit the growth of C. parapsilosis by 19.31 ± 0.84% and 23.82 ± 4.14%, respectively, which was lower than that shown by fluconazole (29.86 ± 1.11%). Overall, hydrolysis of milk proteins from both cow and camel enhanced their antifungal properties. Camel milk protein hydrolysates were more potent in inhibiting pathogenic Candida species as compared with cow milk protein hydrolysates. This is the first study that highlights the antifungal properties of camel milk protein hydrolysates.     

Alteration in propagating colonic contractions by dairy proteins in isolated rat large intestine

Gastrointestinal conditions in which the transit of contents is altered may benefit from nutritional approaches to influencing health outcomes. Milk proteins modulate the transit of contents along different regions, suggesting that they have varying effects on neuromuscular function to alter gastrointestinal motility. We tested the hypothesis that bovine whey and casein milk protein hydrolysates could have direct modulatory effects on colonic motility patterns in isolated rat large intestine. Casein protein hydrolysate (CPH), whey protein concentrate (WPC), whey protein hydrolysate (WPH), and a milk protein hydrolysate (MPH; a hydrolyzed blend of 60% whey to 40% casein) were compared for their effects on spontaneous contractile waves. These contractions propagate along the length of the isolated intact large intestine (22 cm) between the proximal colon and rectum and were detected by measuring activity at 4 locations. Milk proteins were perfused through the tissue bath, and differences in contraction amplitude and frequency were quantified relative to pretreatment controls. Propagation frequency was decreased by CPH, increased by MPH, and unaffected by intact whey proteins. The reduced motility with CPH and increased motility with MPH indicate a direct action of these milk proteins on colon tissue and provide evidence for differential modulation by hydrolysate type. These findings mirror actions on lower gastrointestinal transit reported in vivo, with the exception of WPH, suggesting that other factors are required.     

Inulin‐type fructans and whey protein both modulate appetite but only fructans alter gut microbiota in adults with overweight/obesity: A randomized controlled trial

Scope

Independently, prebiotics and dietary protein have been shown to improve weight loss and/or alter appetite. Our objective was to determine the effect of combined prebiotic and whey protein on appetite, body composition and gut microbiota in adults with overweight/obesity.

Methods and results

In a 12 week, placebo‐controlled, double‐blind study, 125 adults with overweight/obesity were randomly assigned to receive isocaloric snack bars of: (1) Control; (2) Inulin‐type fructans (ITF); (3) Whey protein; (4) ITF + Whey protein. Appetite, body composition and gut microbiota composition/genetic potential were assessed. Compared to Control, body fat was significantly reduced in the Whey protein group at 12 wks. Hunger, desire to eat and prospective food consumption were all lower with ITF, Whey protein and ITF + Whey protein compared to Control at 12 wks. Microbial community structure differed from 0 to 12 wks in the ITF and ITF +Whey Protein groups (i.e. increased Bifidobacterium ) but not Whey Protein or Control. Changes in microbial genetic potential were seen between Control and ITF‐containing treatments.

Conclusion

Adding ITF, whey protein or both to snack bars improved several aspects of appetite control. Changes in gut microbiota may explain in part the effects of ITF but likely not whey protein.

     

Immunomodulatory properties of the milk whey products obtained by enzymatic and microbial hydrolysis

The objective of this study was to investigate the in vitro immunomodulatory effect of milk whey through enzymatic hydrolysis, microbial fermentation and two‐stage hydrolytic reactions (enzymatic and microbial reactions) by analysing cytokine profiles. Results indicated that the milk whey sample fermented by Lactobacillus kefiranofaciens M1 and two‐stage hydrolytic reactions (hydrolysed by Alcalase and then fermented by L. kefiranofaciens M1) could significantly induce the production of tumour necrosis factor (TNF)‐α and IL‐12 compared with the milk whey control and enzymatic treatments. Further characterisation of the immunomodulatory factor by membrane filtration and mutanolysin hydrolysation, the stimulatory activity for IL‐12 and TNF‐α production was found to be reduced and to be correlated positively with the cell wall components in L. kefiranofaciens M1. In addition, Th2‐polarised splenocytes revealed that L. kefiranofaciens M1 had both IL‐12 inducing and IL‐4 repressing activities. These results suggested that L. kefiranofaciens M1 could direct the Th1/Th2 balance toward Th1.     

Effects of enzymatic hydrolysis on lentil allergenicity

Enzymatic hydrolysis and further processing are commonly used to produce hypoallergenic dietary products derived from different protein sources, such as cow's milk. Lentils and chickpeas seem to be an important cause of IgE‐mediated hypersensitivity in the Mediterranean area and India. Some studies have investigated the effects of enzymatic treatments on the in vitro immunological reactivity of members of the Leguminosae family, such as soybean, chickpea, lentil, and lupine. Nevertheless, there are only a few studies carried out to evaluate the effect on IgE reactivity of these food‐hydrolysis products with sera from patients with well‐documented allergy to these foods. In this study, lentil protein extract was hydrolyzed by sequential action of an endoprotease (Alcalase) and an exoprotease (Flavourzyme). Immunoreactivity to raw and hydrolyzed lentil extract was evaluated by means of IgE immunoblotting and ELISA using sera from five patients with clinical allergy to lentil. The results indicated that sequential hydrolysis of lentil results in an important proteolytic destruction of IgE‐binding epitopes shown by in vitro experiments. However, some allergenic proteins were still detected by sera from four out of five patients in the last step of sequential hydrolyzation.     

PROTEOLYSIS OF EWE'S CASEINS AND WHEY PROTEINS DURING FERMENTATION OF YOGURT AND STORAGE. EFFECT OF THE STARTERS USED

Yogurts are mostly produced from cow's milk and to a very limited extent from ewe's milk. Changes in the caseins and whey proteins in ovine milk subjected to different thermal treatments (63C/30 min; 73C/15 min; 85C/10 min or 96C/5 min) were followed during fermentation of yogurt, using two different starters, and during their storage up to 14 days. One starter (YC‐183) contained mixed strain culture of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus at a ratio 1:1. The other starter (ABT‐3) is a well‐defined mixed‐strain culture containing Sc. thermophilus TH4, Lb. acidophilus LA5 and Bifidobacterium Bb 12 at a ratio 1:1:1. The level of free amino groups in the yogurts increased gradually during the fermentation up to a maximum after 4 or 6 h fermentation in the case of ABT‐3 and YC‐183, respectively. A large decrease in the amount of free amino groups was observed after 4–6 h fermentation. During storage of the yogurts up to 14 days, the amount of free amino groups increased with the duration of storage time up to a maximum after 7 days both in the case of YC‐183 and ABT‐3 starters. A large decrease in the concentration of free amino groups was observed in the first 24 h and between 7 and 14 days of storage in the case of yogurt made with the two starters indicating that microorganisms continue to grow in low temperatures. During the fermentation and the storage of both yogurt types, α‐lactalbumin was hydrolyzed to a slightly greater extent than β‐lactoglobulin. During fermentation and the storage, β‐casein was degraded slightly than α_S2‐ +α_S2‐caseins. Generally, a more intense heat pretreatment led to greater degradation of whey proteins and caseins during fermentation and storage. Differences in proteolytic activity between the two starters used (proteins degraded more by ABT‐3 than by YC‐183) may lead to the improvement in production and formulation of yogurts differing in their physico‐chemical and rheological properties.     

Comparison of bioactive peptides prepared from sheep cheese whey using a food‐grade bacterial and a fungal protease preparation

Novel bacterial (HT) and fungal (FPII) food‐grade protease preparations were evaluated for their ability to hydrolyse sheep cheese whey (SCW) and the generation of bioactive peptides. Both protease preparations hydrolysed the whey proteins to small peptides over 24‐h hydrolysis time, but the time course hydrolysis profiles were different as evaluated by SDS‐PAGE. The HT whey hydrolysate had considerably higher antioxidant and angiotensin‐I converting enzyme (ACE)‐inhibitor activity than the FPII hydrolysate. Neither hydrolysate was cytotoxic towards Vero cells. OFFGEL electrophoresis of the small peptide pool fraction (<15 amino acids) of each hydrolysate indicated differences in the pI distribution of the bioactive peptides. This likely reflects the diverse hydrolytic specificity of the proteases. Although the antioxidant activity of both hydrolysates was not significantly affected by simulated gastrointestinal digestion, the loss of ACE‐inhibitor activity was greater with the FPII hydrolysate.     

Evaluation of the peptide profile with a view to authenticating buffalo mozzarella cheese

Electrophoretic and chromatographic techniques were used to determine water‐soluble peptide profiles aiming to identify the adulteration of buffalo milk mozzarella cheese by the addition of cow's milk. Thus, cheeses were produced with contents of cow's milk varying from 0% to 100%, and the peptides extracted after production and after 20 days of refrigeration. Polyacrylamide gel electrophoresis under denaturing conditions in the presence of sodium dodecyl sulphate (SDS‐PAGE) identified a potential peptide marker of exclusively bovine origin with a size of about 21 kDa for the addition of cow's milk above 30%. Reverse‐phase high‐performance liquid chromatography (RP‐HPLC) indicated the existence of two potential peptides present in higher concentrations in buffalo milk and one exclusive for cow's milk, the latter making it possible to estimate the addition of cow's milk to buffalo milk. Six commercial brands of buffalo mozzarella cheese were evaluated, and indications of adulteration found in four of them.     

Impact of enzyme preparation and degree of hydrolysis on peptide profile and nitrogen solubility of sodium caseinate hydrolysates

The solubility of casein ingredients is limited under certain pH conditions. Therefore, sodium caseinate (NaCN) was hydrolysed at laboratory scale with four enzyme preparations (Alcalase, Prolyve, FlavorPro Whey and pepsin) yielding hydrolysates having degree of hydrolysis (DH) values between 0.19 ± 0.11 and 19.25 ± 0.73%. The nitrogen solubility index (NSI) over the pH range 2.0–8.0 was affected by (i) the specificity of the enzyme preparation, (ii) the DH and (iii) the presence of unhydrolysed NaCN within the hydrolysate. The trends observed at laboratory scale (<5 L) were also seen with the semi‐pilot‐scale (300 L) hydrolysates generated with Alcalase and FlavorPro Whey. Removal of unhydrolysed NaCN from the FlavorPro Whey hydrolysate using a 5‐kDa ultrafiltration (UF) membrane increased NSI. This study has highlighted the importance of carefully selecting the proteolytic preparation along with the DH to design casein‐based ingredients with enhanced technofunctional properties.     

Effects of fermentation by lactic acid bacteria on the antigenicity of bovine whey proteins

BACKGROUND: The main whey proteins α‐lactalbumin (α‐LA) and β‐lactoglobulin (β‐LG) are considered as the major allergens in cow's milk. Microbial fermentation can produce some proteolytic enzymes, which can induce the degradation of milk protein allergens. In this study, the effects of fermentation by lactic acid bacteria on the antigenicity of α‐LA and β‐LG were investigated using indirect competitive ELISA. Meanwhile, the proteolysis of milk proteins was detected by TNBS assay and SDS‐PAGE electrophoresis. RESULTS: Fermentation by lactic acid bacteria could significantly reduce the antigenicity of α‐LA and β‐LG in skim milk. Combined strains of Lactobacillus helveticus and Streptococcus thermophilus were the most effective in reducing the antigenicity of both whey proteins. In addition, α‐LA and β‐LG antigenicity decreased to a lower value at 6 h of fermentation and at 0.5 d of cold storage by fermentation with the combined strains. The results of TNBS assay and SDS‐PAGE electrophoresis showed that lactic acid bacteria strains used in this study hydrolysed whey proteins only to a limited extent. CONCLUSION: The fermentation with lactic acid bacteria is an effective way to reduce whey proteins antigenicity. Copyright © 2010 Society of Chemical Industry     

A food matrix reduces digestion and absorption of food allergens in vivo

Scope: Food allergy is caused by primary (class 1) food allergens, e.g. Bos d 5 (cow's milk) and Cor a 8 (hazelnut) or secondary (class 2) food allergens, e.g. Mal d 1 (apple). The latter cannot sensitize susceptible individuals but can cause allergy due to immunological cross‐reactivity with homologous respiratory allergens. Here, we studied the effects of food matrix on gastrointestinal proteolysis, epithelial transport and in vivo absorption of class 1 and class 2 food allergens. Methods and results: Mal d 1 lost its IgE‐reactivity immediately after simulated gastric digestion whereas Bos d 5 and Cor a 8 did not. Only Cor a 8 maintained IgE‐binding capacity after simulated intestinal proteolysis. The presence of hazelnut and peanut extracts, which served as protein‐rich model food matrices, delayed gastrointestinal degradation and reduced epithelial transport rates of all allergens through CaCo‐2 monolayers. Finally, IgE‐reactive allergens were assessed at different time points in sera from rats fed with all three allergens with or without hazelnut extract. The levels of all allergens peaked 2 h after animals were fed without matrix and increased over 8 h after feeding. Conclusions: A protein‐rich food matrix delays gastrointestinal digestion and epithelial transport of food allergens and thereby may affect their sensitizing capacity and clinical symptoms.     

Effect of heat and enzymatic treatment on the antihypertensive activity of whey protein hydrolysates

The influence of heat and enzymatic treatments on the hypotensive activity of hydrolysates derived from whey protein isolate was examined. The whey protein isolate (WPI) was previously denatured at 65 or 95 °C and hydrolyzed using the enzymes Alcalase, α-chymotrypsin or Proteomix. The hydrolysates thus obtained were characterized and studied with regard to their angiotensin converting enzyme (ACE) inhibitory activity and hypotensive activity in spontaneously hypertensive rats (SHR). The enzyme α-chymotrypsin was found to produce hydrolysates with the highest ACE inhibitory activity. The hydrolysate that most effectively reduced blood pressure in SHR was obtained from WPI previously denatured at 65 °C and treated with the enzyme Alcalase. The hydrolysate with the highest ACE inhibitory activity was able to reduce the arterial blood pressure of the animals only after intraperitoneal administration, suggesting an interference of gastrointestinal enzymes in the absorption of active peptides from this hydrolysate.     

免疫耐受在牛乳过敏中的研究进展

食物过敏是机体对过敏原产生的一种不良免疫反应,而免疫耐受是指机体对摄入的食物抗原不产生此类免疫应答.近年来,牛乳过敏的发生率越来越高,大部分牛乳过敏的儿童3岁之后出现了免疫耐受.基于此,介绍了牛乳过敏及引发免疫耐受的相关免疫细胞包括树突状细胞、调节性B细胞、调节性T细胞在免疫耐受中的作用及3种诱导免疫耐受的方法.