Milk Protein Hydrolysates Research Articles

Detection by competitive enzyme-linked immunosorbent assay of a bovine serum albumin peptide (ABBOS) in infant formulas based on hydrolyzed cow's milk protein.

Our aim was to determine whether currently available products based on hydrolyzed milk protein and a small peptide (ABBOS) in bovine serum albumin (BSA) (positions 126-144) share common antigenic sites. The commercial products are primarily developed to reduce cow's milk protein-associated allergenicity, but whether they also could be used in intervention trials to elucidate the role of BSA in the etiology of IDDM is not known. A sensitive competitive enzyme-linked immunosorbent assay (ELISA) using a rabbit polyclonal antibody raised against the ABBOS peptide in BSA was developed. With this method, we determined cross-reactivity between the ABBOS peptide and commercially available products: four milk protein hydrolysates and eight infant formulas based on hydrolyzed milk protein. The products were further characterized by physicochemical techniques. Hydrolyzed milk protein products are found to inhibit competitively the binding of ABBOS peptide to antibody. The number of residual reactive sites varied considerably among products and was not strongly related to the degree of hydrolysis (DH) or the molecular mass distribution of the hydrolysates. The presence of possible immunoreactive peptides in infant formulas based on hydrolyzed cow's milk protein cannot be adequately predicted by the DH or molecular mass distribution of the hydrolysates. Its specific determination is needed to ensure infant formulas free of cross-reactive ABBOS antibody binding sites for use in ongoing and forthcoming intervention trials to elucidate the role of BSA as a possible environmental triggering agent of IDDM.

Enzymatic Hydrolysis of Milk Proteins Used for Emulsion Formation. 2. Effects of Calcium, pH, and Ethanol on the Stability of the Emulsions

Oil-in-water emulsions were prepared with native milk proteins (caseinate and β-lactoglobulin) or the same proteins after different extents of breakdown by treatment with trypsin. The effect of trypsin treatment of the emulsions after formation was also investigated. The stability of emulsions was measured by determining the particle sizes with light scattering. Different destabilization conditions were used; namely, decreasing pH, adding calcium ions, or incorporation of ethanol. Comparisons were made between emulsions containing modified and unmodified proteins. Trypsinolysis rendered caseinate emulsions less stable to decreasing pH and increasing concentrations of ethanol, but the stability to calcium increased and correlated with the surface composition of individual types of casein molecules and their calcium sensitivity. Emulsions containing β-lactoglobulin, on the other hand, showed improved stabilities with hydrolysis under all conditions compared with emulsions containing unhydrolyzed protein. Ke...

Enzymatic Hydrolysis of Milk Proteins Used for Emulsion Formation. 1. Kinetics of Protein Breakdown and Storage Stability of the Emulsions

Hydrolysis of β-lactoglobulin and sodium caseinate in solution or while adsorbed at oil-water interfaces in emulsions formed from unmodified proteins was carried out with trypsin. The hydrolysis was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and dynamic light scattering. The stability during storage of the emulsions containing hydrolyzed proteins was also investigated by small-angle laser light scattering. The order of the hydrolysis of the individual caseins at the oil surface was β > αs >>> κ compared with αs > β > κ in solution. The overall rate of hydrolysis of sodium caseinate was the same whether adsorbed to the interface or in solution. Conversely, the rate of hydrolysis of β-lactoglobulin was much higher on the surface of emulsion than in solution. Emulsions formed from hydrolyzed protein solutions were less stable during storage than those formed from unmodified proteins and subsequently hydrolyzed or those formed with unmodified protein solutions. Keywords: Emulsions; casein; β-lactoglobulin; trypsin; peptides; emulsion stability

Residual antigenicity of hypoallergenic infant formulas and the occurrence of milk-specific IgE antibodies in patients with clinical allergy

Background: Milk protein hydrolysates are frequently used in milk substitutes for children with cow's milk allergy. However, cases of hypersensitivity to commercially available hypoallergenic infant formulas based on milk protein hydrolysates have been reported. Objective: Our purpose was to determine the immunologic response of milk protein–specific IgE and IgG in the serum of patients allergic to cow's milk against four commercially available hypoallergenic milk protein hydrolysates and eight infant formulas. Methods: Antibody levels in patients' serum and milk protein–specific residual antigenicity of the hypoallergenic products were determined by indirect and competitive ELISA. Results: Patients allergic to cow's milk had IgE and IgG antibodies to several protein fractions of cow's milk; intraindividual and interindividual variation in the concentrations of these antibodies was considerable. In general, IgE and IgG residual antigenicity of individual milk proteins in the hypoallergenic products was lower compared with that of the intact milk protein, but immunoreactive epitopes could still be detected in all products. Their number varied considerably among the individual milk proteins and also differed among products. Conclusions: The individual sensitization pattern of the patient allergic to cow's milk and the milk protein–specific residual antigenicity might be considered as possible laboratory predictors of adverse reactions to hypoallergenic products. Their determination could be a useful preclinical screening test for pediatricians to select a formula adapted to the individual patient. (J A LLERGY C LIN I MMUNOL 1995;96:365-74.)

REDUCTION OF ALLERGENICITY AND INCREASING THE BIOLOGICAL VALUE OF BUFFALO'S MILK PROTEINS BY ENZYMATIC MODIFICATION

Buffalo milk proteins were covalently enriched with methionine (Met) during the enzymatic peptide modification (EPM) process. The maximal Met-enrichment occurred when the α-chymotryptic hydrolysate of buffalo's milk proteins was treated with methionine ethyl ester in the presence of α-chymotrypsin during the EPM. Methionine content of this product was more than three times (5.4%) higher than that of the original buffalo milk protein (1.5%). The electrophoretic patterns of the modified proteins showed that the enzymatic reaction was mainly characterized by transpeptidation. Molecular masses of the modified protein fractions were influenced by the concentration of L-methionine ethyl esters in the reaction mixture during the EPM. EPM products from the buffalo milk protein hydrolysates with α-chymotrypsin and trypsin or with pepsin and trypsin showed low allergenic activities compared with those when buffalo milk proteins were hydrolyzed with α-chymotrypsin or with pepsin alone.

Nutritional Value of Milk Protein Hydrolysates

Nutritional Value of Milk Protein Hydrolysates

Nutritional Value of Milk Protein Hydrolysates: A Response to O'Brien

Nutritional Value of Milk Protein Hydrolysates: A Response to O'Brien

Non-Size Exclusion Effects During Gel Permeation Chromatography of Milk Protein Hydrolysates on an FPLC Superose 12 Column

Abstract Hydrophobic peptides and aromatic amino acids adsorbed to the matrix of a Superose 12 FPLC column during gel permeation chromatography (GPC) of milk protein hydrolysates. The adsorption phenomenon was most obvious when hydrolysates were prepared using enzyme mixtures which contained exopeptidases. The elution areas of peaks for tryptophan and tyrosine from a Superose 12 column was linear with concentration in the range 0 to 1 μmol. ml-1. Amino acid analysis confirmed that a strongly adsorbed peak in the elution profile, measured by absorbance at 280 nm, of an extensively hydrolysed rennet casein contained tryptophan. Individual hydrophobic peptides, used as molecular weight markers, also interacted with the column, eluting later than expected. Nitrate in a general water supply used for the production of a whey protein hydrolysate on a pilot-scale was responsible for the appearance of an additional peak in the elution profiles from a Superose 12 column, as measured by absorbance at 214 nm.

Enzymology of lactococci in relation to flavour development from milk proteins

Abstract Although the individual chemical compounds which impart flavour profiles to cheese and milk protein hydrolysates have not been defined, it is clear that the sequential breakdown of these proteins to small hydrophillic peptides and to free amino acids is a prerequisite to balanced flavour formation. This paper will review and discuss current knowledge of specificity, stability and availability of enzymes in lactococci in relation to flavour and taste compounds, which can be derived from peptide sequences and amino acid residues present in the proteins in milk.

Characteristics and Application of Milk Protein Hydrolysates

Characteristics and Application of Milk Protein Hydrolysates

Nutritional Value and Antigenicity of Two Milk Protein Hydrolysates in Rats and Guinea Pigs

Two milk protein (whey protein and casein) hydrolysates were obtained by enzymatic hydrolysis. In the casein hydrolysate, an ultrafiltration stage was performed to remove all the peptides with molecular weights > 2500. A nutritional value study of both hydrolysates was undertaken. No differences were found between the native proteins and their enzymatic hydrolysates. Neither the enzymatic hydrolysis nor the heat treatments used affected the nutritional value of the protein sources. The two hydrolysates were studied for antigenic properties. The enzymatic hydrolysis of the whey protein concentrate followed by a thermal treatment at 90 degrees C for 10 min reduced significantly its antigenicity. However, the in vivo allergenicity tests showed some positive reactions in both systemic anaphylaxis and passive cutaneous anaphylaxis. The casein hydrolysate, after being ultrafiltered, was devoid of sensitizing capacity by the oral route and was also ineffective in producing local or systemic anaphylaxis in previously sensitized animals, as shown by in vivo assays.

Effects of two protein hydrolysates on growth, nitrogen balance and small intestine adaptation in growing rats.

The effects of feeding 2 protein hydrolysates, one prepared by controlled pepsin and pancreatic protease (including elastase II) hydrolysis of milk proteins (PPPH) and the other a di- and tripeptide bacterial protease hydrolysate of bovine albumin (DTPH), on the growth, nitrogen balance and small intestine adaptation of growing rats were analyzed. Two groups of 3-week-old rats (8 rats/group) were fed the liquid diets ad libitum for 2 weeks. The diets had the same caloric, nitrogen, carbohydrate and lipid contents. The amino acid compositions fulfilled the needs of growing rats. The diet differed in the original proteins, the hydrolysis technique used and the molecular weights of the peptides. Nitrogen intakes were similar. Although there was no difference in weight gain, nitrogen balance was significantly higher in the rats fed the PPPH diet (day 4-day 6:PPPH, 60 +/- 4%, DTPH, 25 +/- 5%; day 12-day 15: PPPH, 58 +/- 3%; DTPH, 30 +/- 5%). The stool nitrogens were identical, suggesting improved nitrogen storage in the rats fed the PPPH diet. Small intestine adaptation showed that the rats on the PPPH diet had significantly more protein (mg) and DNA (microgram) per 10 cm of the jejunum (PPPH, 25.6 +/- 2, 393 +/- 20; DTPH: 15.7 +/- 2, 258 +/- 23) and sucrase-specific activity and per microgram of DNA (PPPH, 133 +/- 5.7, 9.7 +/- 0.5; DTPH, 113 v 5, 7 +/- 1). The N-aminopeptidase-specific activity was the same in both groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Free D- and L-Amino Acids from Hydrolyzed Milk Proteins by Pseudomonas fluorescens ATCC 948

Cell-associated peptidase activity of Pseudomonas fluorescens ATCC 948 was studied on hydrolyzed milk proteins. The substrate was produced by treatment of the UHT skim milk with neutral endoprotease B500. The cell-associated peptidase activity was determined by gas chromatographic analysis of free D- and L-amino acids. The total free amino acids were higher when the cell-associated peptidases acted on hydrolyzed milk proteins (202.8μg/ml) rather than on unhydrolyzed skim milk (63.9μg/ml). Glutamic acid (65.1μg/ml), Leu (36.9μg/ml), and Ala (16.5μg/ml) were the most abundant. Concentrations of D-amino acid isomers (28.3 and 3.7μg/d for D-Glu and D-Ala, respectively) also were high. Hydrolysis of the dipeptide L-leucyl-L-leu was 61% but was minimal for the other D- and L-configurational isomers of the dipeptide.

Allergenicity of milk protein hydrolysate formulae in children with cow's milk allergy.

Cow's milk protein hydrolysate formulae have been developed to lower or eliminate the allergenicity of cow's milk proteins, and to reduce the antigenic load and the risk of sensitization. Cross-reactivity between different hydrolysate formulae and cow's milk proteins has been demonstrated. We have studied 20 children (median age 31 months, range 15-76 months) with a history of IgE-mediated cow's milk allergy. All the children had immediate allergic respiratory and/or cutaneous and/or gastro-intestinal reactions to cow's milk ingestion. In addition, the children had positive prick skin tests and positive RAST to cow's milk. Prick skin test, RAST, and double-blind placebo controlled food challenges were performed with three different hydrolysate formulae: a casein hydrolysate formula and two whey formulae, one partially and one extensively hydrolyzed. All 20 children had immediate allergic reactions after the challenge test with cow's milk. Only 2/20 children had a positive challenge test with a casein hydrolysate formula (Alimentum): one developed asthma and one urticaria. Two of the 15 children challenged with an extensively hydrolysed whey formula (Profylac) developed perioral erythema. Nine out of 20 children had a positive challenge test with a partially hydrolysed whey formula (Nidina H.A.): four developed asthma, three urticaria and two lip oedema. All children had positive prick skin tests to cow's milk proteins (casein and/or lactalbumin); 9 to Nidina H.A.; 3 to Profylac, and 3 to Alimentum. Specific IgE antibodies to cow's milk were present in all children; in 13 to Nidina H.A., in 4 to Profylac, and in 3 to Alimentum.

Detection and identification of the cross-linking amino acidsN ?-andN ?-(2?-amino-2?-carboxy-ethyl)-l-histidine (?histidinoalanine?, HAL) in heated milk products

An unknown ninhydrin positive compound, X, was detected in acid hydrolysates of heated skim milk samples by amino acid analysis, eluting between phenylalanine and pyridosine in the chromatogram. The formation of X correlated with heating time and temperature. preparative ion-exchange chromatography enabled the isolation of X and a second minor compound from a milk protein hydrolysate and from a model mixture consisting of N alpha-acetylhistidine and methyl-2-acetamidoacrylate (acetyldehydroalaninemethylester), in a relative abundance of 8 to 1. By 1H-NMR spectroscopy, the two compounds could be identified as the N tau- and N pi-isomers of N-(2'-amino-2'-carboxy-ethyl)-L-histidine (histidinoalanine), a cross-link amino acid that has not been described in food proteins up to now. In a number of foods containing milk protein, the N tau-histidinoalanine contents were between 50 and 1800 mg/kg protein, which is in a concentration range comparable to the potential nephrotoxic cross-link lysinoalanine, which was determined simultaneously.

Endophyte-Host Associations in Grasses. XIX. A Systematic Study of Some Sympatric Species of Epichloë in England

Systematic studies were conducted on populations of Epichloe species on host grasses Agrostis capillaris, Dactylis glomerata, and Holcus lanatus. Three species of Epichloe are described and two, E. baconii and E. clarkii, are proposed as new, being distinguishable from the type by disarticulation of ascospores to form uniquely shaped part-spores. The type species E. typhina does not form part-spores. Reproductive compatibility between fungi was examined. Intraspecies crosses were found to yield a dense layer of perithecia that differentiated asci and ascospores internally. Interspecies crosses gave one of three results, including no reaction, necrotic browning, or perithecia that did not mature. Cultural features of these fungi are compared. Isolates are seen to exhibit differences in growth rates on sugars and hydrolysis of milk protein and corn starch. Generic limits of Epichloe are discussed and several species are excluded. It is proposed that a biological species concept be adopted to aid definition of species within this genus.

Nonclinical testing of formulas containing hydrolyzed milk protein

The allergenic potential of cow milk-based infant formula can be reduced by protein hydrolysis. Controlled clinical studies are necessary to demonstrate conclusively the biologic efficacy of these formulations in human beings. Nonclinical testing programs provide manufacturers with the opportunity to characterize various molecular and immunologic properties of these hydrolysates and their corresponding final product forms. Physicochemical analyses provide data relating to the extent of protein hydrolysis and peptide molecular weight distribution. Immunochemical analyses can semiquantitatively estimate hydrolysate reactivity with preformed antibody. The ability of hydrolysate-based products to induce an immune response can be evaluated by using animal models. It is the responsibility of the manufacturer to choose the appropriate combination of nonclinical tests and use them to document product consistency, thus helping to ensure consistent clinical performance.

Effects of prolonged exposure to partially hydrolyzed milk protein

Formulas containing proteins with reduced potential to sensitize might be effective in reducing the risk of atopic disease, but such products should be nutritionally adequate. We designed a randomized, double-blind study to evaluate nutritional adequacy and sensitization potential of a formula containing partially hydrolyzed whey-predominant milk proteins. Subjects were term infants enrolled at birth. Infants in group A were fed human milk, and group B received the partially hydrolyzed formula; group C, fed a formula containing intact whey-predominant milk proteins, served as control subjects. We recorded anthropometric measurements and symptoms of formula intolerance at monthly intervals for 4 months in 205 infants. Milk IgE and IgG antibodies were measured until 8 months of age in 63 infants. Daily weight gain was not significantly different among the groups. Gastrointestinal symptoms attributed to feeding intolerance were also comparable. Increases in serum IgG antibodies were significantly greater in group C throughout the study. There were no significant differences in IgE antibodies. Thus a formula containing partially hydrolyzed whey-predominant milk protein promoted adequate growth and induced a lesser priming effect for IgG antibody response than did an intact whey-predominant formula.

Molecular weight-related antigenicity of peptides contained in cow's milk protein hydrolysates

Molecular weight-related antigenicity of peptides contained in cow's milk protein hydrolysates

Splanchnic fluxes of amino acids after duodenal infusion of carbohydrate solutions containing free amino acids or oligopeptides in the non-anaesthetized pig.

Seven non-anaesthetized pigs (mean body-weight 64.6 kg) were used to study the intestinal absorption and hepatic metabolism of glucose and amino acids (AA) using carbohydrate solutions (maltose dextrin; 440 g/2 I), containing 110 g of either an enzymic milk-protein hydrolysate (PEP) with a large percentage of small peptides (about 50% with less than five AA residues) and very few free AA (8%) or a mixture of free AA (AAL) with an identical pattern, infused intraduodenally. Each pig was previously fitted under anaesthesia with electromagnetic flow probes around the portal vein and the hepatic artery, and with permanent catheters in the portal vein, carotid artery, one hepatic vein and the duodenum. Each solution was infused for 1 h after a fasting period (18 h) and each pig received both solutions at 8 d intervals. The observation period lasted 8 h. For most AA (his, lys, phe, thr, arg, tyr, pro) the absorption rate after infusion of PEP was significantly higher than after that of AAL during the 1st hour, but the differences quickly disappeared. After 8 h, the only differences concerned his and tyr (PEP > AAL) and met, glu and asp (AAL > PEP). There was a large uptake of blood AA by gut-wall cells, higher after AAL infusion than after PEP infusion, particularly for branched-chain AA (BCAA). The absorption of ammonia-nitrogen after both infusions was equivalent to two-thirds of urea-N passing from blood to intestinal tissues and lumen. Glucose absorbed within 8 h represented only 76% (PEP) or 69% (AAL) of the infused amounts. The cumulative hepatic total AA (TAA) uptake increased from 13 to 27% of the infused amounts between the 1st and the 8th hour after PEP infusion, and from 8 to 31% after AAL infusion. Most essential AA were largely taken up by the liver, with the exception of met (PEP) and thr and of BCAA, which were poorly retained for both solutions; there was a high uptake of ala and gly, and a release of asp, glu, and gln. Urea-N released by the liver within 8 h was equivalent to 23-25% absorbed amino-N and to around 1.5 times ammonia-N taken up by the liver within 8 h. Glucose was highly taken up by the liver during the first hours then released, the total uptake within 8 h representing about half the absorbed amount.(ABSTRACT TRUNCATED AT 400 WORDS)