Julio Cesar Monti

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.

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.