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Summary Demineralized whey, whey protein concentrates and, to a lesser extent, some other whey protein fractions are key raw materials in infant formula manufacture. An estimate of the amount of whey protein used in infant formulae annually is in the order of 30–40 000 tons. Infant formula development has been a long lasting effort to approach the nutrient composition of human breast milk while using cow's milk as the raw material. This required extensive fractionation of the bovine milk followed by subsequent recombination of specific fractions. Criteria to judge protein quality and define adequate protein quantity in infant formulae have also evolved. Early criteria for protein adequacy were based on Protein Efficiency Ratio (PER) testing in rats and/or nitrogen balance studies. More recently, the approach of essential amino acid scores, based on the amino acid pattern of mature human milk has been put forward. Regulatory authorities such as CODEX, EC Commission and more recently LSRO in its suggestions to FDA in the USA, have issued reference amino acid profiles which are based on the average amino acid composition of human milk. The same authorities have also regulated protein density in infant formulae, accounting for an observed protein mean density of 1.5–1.6 g /100 kcal in mature breast milk. The minimum protein level of cow's milk based infant formulae was fixed at 1.8 g/100 kcal by these authorities. Current infant formulae have however protein densities significantly higher than the minimum of 1.8 g/100 kcal. The higher protein density compensates for the likeliness that the protein in the best of the formulae, is not as ideal for the infant as the protein from breast milk. Even if a complete equivalence may be difficult to reach, efforts continue in the direction of further optimizing the protein quality in terms of essential and semi-essential amino acid profiles. This is the only way to be able to lower the protein content in a formula to levels that are nearer to the low protein density of human milk. Among several possibilities, the increase in the mass proportion of bovine α-lactalbumin is a particularly promising way to achieve this goal.

International audience; Despite great homology with the equivalent hum an protein, bovine a-lactalbum in (Ba-La),a major com ponent of whey, has been identified as a major milk allergen. The aim of this study was to investigate the relationship between structure and IgE binding capacity in a-Las : the importance of three-dimensional structure using native vs disulfide bridge-reduced B a-La; and the incidence of amino acid sequence divergence on specific IgE cross-reactivity to human vs bovine a-La. Purified native, reduced and S-carboxymethylated Ba-La and human a-La (Ha-LA) were prepared. Specific IgE of 20 sera from patients with clinically recognized cow’s milk protein allergy and positive RAST‡ tests to B a-La were measured in original direct and competitive ELISA inhibition tests. All sera containin specific anti-native B a-La IgE also reacted with denatured protein, but the IgE levels were generally lower, showing that three-dimensional structure is an important feature in B a-La allergeni city but that sequential epitopes are also exposed after protein denaturation. Despite lower IgE levels, allsera also gave significant IgE responses to H a-La. Competitive ELISA inhibition confirmed results obtained by direct ELISA. The demonstrated IgE cross-reactivity between B a-LA and H a-La could be related to the high degree of sequence homology between the two proteins but did not prove to have a clinical significance. However, it is of great interest for a study of the relationship between structure, IgE binding capacity and allergeni city in a-Las.

Background: Oral tolerance against food proteins has been achieved in different animal models with use of native or moderately hydrolyzed proteins as inducers. However, native proteins remain highly allergenic, although it has been demonstrated that protein hydrolyzates and resulting peptides can lose their allergenicity. Objective: This study was designed to evaluate the ability of β-lactoglobulin hydrolyzate and peptides to induce oral tolerance to native β-lactoglobulin and to identify tolerogenic β-lactoglobulin peptides with low allergenicity. Methods: β-Lactoglobulin was hydrolyzed by trypsin and fractionated by ion exchange chromatography. Peptide enrichment of fractions was evaluated. Balb/c mice were fed β-lactoglobulin hydrolyzate or fractions by single gavage at day 1. Five days later animals were challenged intraperitoneally with native β-lactoglobulin. At day 27 delayed-type hypersensitivity was performed. Twenty-four hours later mice were bled, and intestinal contents and spleens were collected. Oral tolerance was measured by titrating specific IgE in sera and intestinal samples. Specific T-cell responses were analyzed by splenocyte proliferation. Antigenicity of hydrolyzate and fractions was evaluated by specific ELISA inhibition. Results: Mice fed either β-lactoglobulin hydrolyzate or 2 fractions of the hydrolyzate were tolerized against β-lactoglobulin. Specific serum and intestinal IgE were suppressed. Delayed-type hypersensitivity and proliferative responses were inhibited. One tolerogenic fraction was found to be 50 times less antigenic than the total β-lactoglobulin hydrolyzate was. Conclusion: These findings support the strategy of inducing oral tolerance in "at-risk" patients by means of tolerogenic cow's milk peptides or hydrolyzate. (J Allergy Clin Immunol 2000;105:514-21.)

The specific IgE binding capacity of native bovine alpha-lactalbumin (alpha-La), a globular whey protein, and tryptic peptides was investigated using 19 sera from patients with cow's milk protein allergy. The specific anti-bovine alpha-La IgE titers ranged from 0.6 to 125 IU/ml. Highly purified tryptic peptides from native and disulfide-bond-reduced alpha-La were obtained by reverse phase chromatography. By ELISA technique using immobilized native protein or peptides, 11 of the 19 sera reacted exclusively with intact protein while 8 of them also presented a specific IgE response to different tryptic peptides. Polyclonal IgE population specificity was not related to anti-bovine alpha-La IgE levels. Sequence (17G-K58) and larger peptides sharing this sequence were most strongly and frequently recognized. Competitive ELISA inhibition tests confirmed this IgE-specific response and gave also clear evidence for IgE binding to smaller peptides corresponding to sequences (6C-R10):S-S:(115L-L123) and (109A-L123). IgE binding to native alpha-La and large peptides confirmed the importance of conformational epitope(s). However, in some sera reduced and S-alkylated peptide (59I-K94) exhibited a similar or higher IgE binding capacity than the native corresponding fragment, suggesting the existence of sequential epitope(s) exposed through protein denaturation. Moreover, IgE binding sequences were also located within hydrophobic regions of alpha-La and/or within parts with high sequence homology to human alpha-La.

α-Lactalbumin (α-La) is a protein found in the milk of all mammalian species and is involved in lactose biosynthesis in association with galactosyltransferase (Brodbecket al.1967). The primary structure of α-La from several species such as bovine (Brewet al.1970), human (Findlay & Brew, 1972), and goat (McGillivrayet al.1979) has been determined. Hallet al.(1982) have established the nucleotide sequence and the organization of the human α-La gene (Hallet al.1987).