Rolf Jost

Tryptic phosphopeptides from whole casein. II. Physicochemical properties related to the solubilization of calcium

Casein phosphopeptides (CPP) were produced by tryptic hydrolysis of sodium caseinate and further purified by precipitation and chromatography on QAE-Sephadex A-25. Their physico-chemical properties were compared with the properties of an enzymically dephosphorylated equivalent preparation (DPP). Binding of Ca2+ to the peptides was measured using a Ca selective electrode and was found to increase with pH and to show 1/1 stoicheiometry Ca/Porg in CPP at pH 6.5 and 7.6. Klotz plots indicated equivalent binding sites at these two pH values, but some heterogeneity was seen at pH 3.5. In contrast, DPP did not bind significant amounts of Ca2+. CPP effectively inhibited the formation of insoluble calcium phosphates at different Ca/P ratios. The effective CPP concentration was 10 mg/l and complete stability of calcium phosphate solutions was obtained at about 100 mg/l. This stabilizing effect was dependent on the presence of organic P.

Allergenicity of whey protein: its modification by tryptic in vitro hydrolysis of the protein

Enzymatic in vitro hydrolysis was evaluated as a possible treatment to abolish the allergenicity of whey proteins in view of their use in infant formulas. Guinea pigs without prior immunological contact (including fetal life) with cows milk were fed various preparations of cows milk proteins. Oral exposure to milk or untreated whey protein led to anaphylactic sensitization of the animals. In contrast, trypsin-hydrolyzed whey protein and a peptide preparation produced from the tryptic hydrolysate by ultrafiltration were devoid of sensitizing capacity by the oral route. The hydrolysate (crude or purified) was also ineffective in triggering local or systemic anaphylaxis in previously sensitized animals.

Human IgE binding capacity of tryptic peptides from bovine α-lactalbumin

The specific IgE binding capacity of native bovine α-lactalbumin (α-La), a globular whey protein, and tryptic peptides was investigated using 19 sera from patients with cow’s milk protein allergy. The

Tryptic phosphopeptides from whole casein. I. Preparation and analysis by fast protein liquid chromatography

Tryptic phosphopeptides were obtained from whole bovine casein by chromatography on the anion exchange resin QAE-Sephadex A 25. Salt gradient elution of the column allowed separation of non-phosphorylated peptides from phosphorylated species. The preparations obtained contained at least seven distinct phosphopeptides of which the following casein fragments were identified: α s1 (43–58):2P, α s1 (59–79): 5P, α s2 (46–70): 4P, β(1–28): 4P, β(2–28): 4P, and β(33–48): 1P. Fast protein liquid chromatography (FPLC) on Mono Q HR 5/5 resin showed that the phosphopeptides were eluted in the same order as from the QAE-Sephadex resin. However, on the analytical column HR 5/5 the fragments α s1 (59–79): 5P and β(2–28): 4P, having the same net charge under the conditions of chromatography, co-eluted, whereas they were at least partly separated on the preparative column HR 16/10. Following enzymic dephosphorylation, the peptides eluted at lower salt strength in the gradient. FPLC on Mono Q resin thus permitted dephosphorylation to be monitored and intermediates between the parent species and the fully dephosphorylated peptide to be identified.

Protein composition of micellar casein obtained by cross-flow micro-filtration of skimmed milk

In this study of the products of cross-flow micro-filtration of unheated skim milk, it was found that some lactoferrin, serum albumin and immunoglobulins were retained with the casein fraction. An acid-soluble casein, identified as proteose peptone component 5, also remained associated with the micellar fraction during micro-filtration.

Analysis of the peptide sweetener aspartame by liquid chromatography

ZusammenfasssungFür die Analyse des Peptidsüß stoffes Aspartam (l-Aspartyl-l-phenylalanin methyl-ester) in Lebensmitteln entwickelten wir 2 Methoden, beide basierend auf der Flüssigchromatographie.Mit Hilfe des Beckman PA-35 Ionenaustauscherharzes und eines Aminosäureanalysators waren wir in der Lage, den Dipeptidester von einem seiner Zersetzungsprodukte,l-Aspartyl-l-phenylalanin, sowie von möglicherweise in der Analyse interferierenden Aminosäuren zu trennen. Durch Hochdruckflüssigchromatographie mittels einer Octadecylsilan-Phasenumkehr Kolonne gelang zudem auch die Trennung und der Nachweis des aus Aspartam durch Cyclisierung entstehenden Diketopiperazins.Beide Techniken gestatteten es, Aspartam auf direkte Weise in verschiedenen flüssigen Lebensmitteln (Getränken auf Molken-, Frucht-, Kolabasis, Tee oder Kaffee) zu messen. Es war überdies möglich, sich ein Bild vom Zerfallsspektrum des Dipeptidesters in Lösung unter bestimmten Temperatur- und pH-Bedingungen zu machen.Dabei fanden wir bei saurem pH und Raumtemperatur ein nahezu äquimolares Verhältnis der beiden Zersetzungsprodukte freies Dipeptid und Diketopiparazin. Demgegenüber führte Exposition von gelöstem Aspartam bei saurem pH und hoher Temperatur vornehmlich zur Bildung des Diketopiperazins und außerdem zu einer geringfügigen Hydrolyse der Peptidbindung.SummaryWe developed two methods, based up on liquid chromatography, for the analysis of the peptide sweetener aspartame (l-aspartyl-l-phenylalanine methylester). Ion-exchange chromatography by means of a Beckman PA-35 resin and post-column ninhydrin detection in an amino acid analyzer enabled us to resolve the dipeptide ester from one of its main decomposition products,l-aspartyl-l-phenylalanine, as well as from several possibly interfering amino acids.High performance liquid chromatography on a reversed-phase octadecylsilane column with gradient elution, was capable of resolving aspartame, the free dipeptide and in addition the diketopiperazine, which may result from cyclization of aspartame.Both techniques were succesfully applied to measure this peptide sweetener in beverages based on whey, fruit juice, cola, tea or coffee.We have been able to obtain in addition a picture of the decomposition products of aspartame under particular pH and temperature conditions. In acidic solution and at room temperature, we observed the slow formation of both free dipeptide and diketopiperazine in an almost equimolar ratio. In contrast, exposure of aspartame in acidic enviroment at a high temperature led mainly to the diketopiperazine, together with a low extent of peptide-bond hydrolysis.