foods Article Effect of Addition of Tryptophan on Aggregation of Apo-α-Lactalbumin Induced by UV-Light Zichen Zhao 1 , Renjie Li 1 , Mahesha M. Poojary 1 , Søren B. Nielsen 2 and Marianne N. Lund 1,3, *   Citation: Zhao, Z.; Li, R.; Poojary, M.M.; Nielsen, S.B.; Lund, M.N. Effect of Addition of Tryptophan on Aggregation of Apo-α-Lactalbumin Induced by UV-Light. Foods 2021, 10, 1577. https://doi.org/10.3390/ foods10071577 Academic Editors: Mario Estévez and Youling Xiong Received: 24 May 2021 Accepted: 6 July 2021 Published: 7 July 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1 Department of Food Science, Faculty of Science, University of Copenhagen, Rolighedsvej 26, 1958 Frederiksberg, Denmark; zichen@food.ku.dk (Z.Z.); renjie@food.ku.dk (R.L.); mahesha@food.ku.dk (M.M.P.) 2 Arla Foods Ingredients Group P/S—Innovation R&D, Discover Department, Sønderupvej 26, 6920 Videbæk, Denmark; sobni@arlafoods.com 3 Department of Biomedical Sciences, Faculty of Health and Medicine, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen, Denmark * Correspondence: mnl@food.ku.dk; Tel.: +45-3533-3547 Abstract: UV-B illumination facilitates aggregation of alpha-lactalbumin (α-LA) by intramolecular disulfide bond cleavage followed by intermolecular thiol-disulfide exchange reactions. However, long term exposure to UV-B illumination may induce undesired oxidative modifications of amino acid residues in the protein. The purpose of this study was to examine the effect of UV-induced aggregation of apo-α-LA (a calcium-depleted form of α-LA) under aerobic and anaerobic conditions and by addition of tryptophan (Trp) as a photosensitizer. The addition of Trp to apo-α-LA illuminated under anaerobic conditions facilitated the highest level of free thiol release and disulfide-mediated aggregation as compared to without addition of Trp under both anaerobic and aerobic conditions. Addition of Trp under aerobic condition resulted in the lowest level of free thiols and disulfide- mediated aggregation and the aerobic conditions caused oxidation of the free Trp with formation of kynurenine and 5-hydroxy-Trp. Minor levels of the Trp oxidation product, 3-hydroxy-kynurenine (2% converted from Trp), was formed in apo-α-LA with added Trp under both aerobic and anaerobic conditions after UV-B treatment. Keywords: UV illumination; tryptophan; aggregation; photooxidation; whey protein 1. Introduction Whey proteins are the by-products of cheese and casein manufacture. They are widely used as food ingredients owing to their high nutritional value and versatile functional properties such as gelation, foam stabilization, and emulsification [1–4]. Alpha-lactalbumin (α-LA) is one of the major proteins present in whey together with beta-lactoglobulin (β- LG) [4]. Prior to application, whey proteins may be thermally treated to form protein aggregates to increase their stability in the final food or beverage product. However, the thermal treatment may induce adverse effects such as the formation of off-flavors [5,6]. UV treatment has, therefore, been examined as an alternative tool to produce whey protein aggregates [7]. As opposed to β-LG, native α-LA does not contain any free thiol group, since all cysteine (Cys) residues are present as disulfides [8]. α-LA is, therefore, less prone to undergo thermal aggregation via thiol-disulfide exchange reactions compared to β-LG. It is an excellent model protein to study the influence of UV light without interference from thiol-disulfide exchange reactions initiated from free Cys residues. By UV-B illumination, intramolecular disulfide bonds in α-LA are cleaved and free thiol groups released, which may then undergo thiol-disulfide exchange reactions and facilitate aggregation through the formation of intermolecular disulfide bonds [7]. It has been reported that UV-B light can induce aggregation of apo-α-LA (a calcium-depleted form of α-LA), with 98% of the Foods 2021, 10, 1577. https://doi.org/10.3390/foods10071577 https://www.mdpi.com/journal/foods