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In Vitro Hydrolysis by Pancreatic Elastases / and II Reduces

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In Vitro Hydrolysis by Pancreatic Elastases / and II Reduces In vitro hydrolysis by pancreatic elastases / and II reduces β-lactoglobulin antigenicity M Gestin, C Desbois, Le Huërou-Luron, V Romé, Gwenola Le Drean, T Lengagne, L Roger, F Mendy, P Guilloteau To cite this version: M Gestin, C Desbois, Le Huërou-Luron, V Romé, Gwenola Le Drean, et al.. In vitro hydrolysis by pancreatic elastases / and II reduces β-lactoglobulin antigenicity. Le Lait, INRA Editions, 1997, 77 (3), pp.399-409. hal-00929534 HAL Id: hal-00929534 https://hal.archives-ouvertes.fr/hal-00929534 Submitted on 1 Jan 1997 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Lait (1997) 77, 399-409 399 © Eisevier/INRA Original article ln vitro hydrolysis by pancreatic elastases 1 and II reduces p-Iactoglobulin antigenicity M Gestin1, C Desbois ', 1Le Huërou-Luron!" , V Romé 1, G Le Dréan ', T Lengagnc-, L Roger/, F Mendy", P Guilloteau ' 1 Laboratoire du Jeune Ruminant, INRA, 65, rue de Saint-Brieuc, 35042 Rennes Cedex; 2 Nutrinov, 85, rue de Saint-Brieuc, 35042 Rennes Cedex; 3 l, Place de Béarn, Saint-Cloud, France (Received 15 November 1996; accepted 19 December 1996) Summary - Bovine whey proteins such as œ-lactalbumin and ~-Iactoglobulin are with bovine caseins the most commonly used proteins in infant formulas owing to their high nutritional value. How- ever, these cow milk components are not always weil tolerated and can induce allergies in infants. The purpose of this study was therefore to investigate the gastric (pepsin) and pancreatic (trypsin, chy- motrypsin and elastases 1and II)enzymatic hydrolysis of ~-Iactoglobulin with the aim of finding a means to reduce its antigenicity. Elastases 1and IIwere first purified from porcine pancreatic acetone powder. After differential precipitation steps, elastases 1and IIwere separated by cation-exchange chro- matography. The conditions regarding ~-Iactoglobulin hydrolysis by gastric and/or pancreatic enzymes were similar to those used for hypoallergenic milk preparations. Elastase II and to a lesser extent elastase I, were effective in enhancing ~-Iactoglobulin hydrolysis via a mix of pepsin, trypsin and chymotrypsin and in reducing the residual antigenicity ofhydrolytic products. The sarne hydrolytic percentage was observed when elastase 1or IIwere added, while the residual antigenicity was lower in the presence of elastase IIthan in the presence of elastase 1. The introduction of elastases in the pan- creatic IIÙX can therefore be proposed to enhance the hydrolysis of cow milk components in hypoal- lergenic milk preparations. ~-Iactoglobulin hydrolysis / pancreatic elastases 1and II / residual antigenicity / hypoaller- genie milk Résumé - L'hydrolyse in vitro de la ~-lactogIobuline par les élastases 1 et II pancréatiques réduit son antigénicité. Les protéines sériques du lait de vache telles que l'a-lactalbumine et la ~-Iacto- globuline, de haute qualité nutritionnelle, sont avec les caséines bovines les plus couramment utili- sées dans les laits de remplacement. Toutefois, ces protéines laitières ne sont pas toujours tolérées et peuvent induire des réactions de nature allergique chez les nourrissons. Afin de réduire l'antigénicité * Correspondence and reprints 400 M Gestin et al de la ~-lactoglobuline,des hydrolysespar des enzymes gastrique (pepsine)et pancréatiques (trypsine, chymotrypsine et élastases 1et II) sont réalisées. Une purification des élastases est effectuée à partir de poudre acétonique de pancréas de porc. Après les étapes de précipitation fractionnée, les élastases 1et II sont séparées par chromatographie échangeuse de cations. Les conditions d'hydrolyse de la ~- lactoglobuline par les enzymes gastrique et/ou pancréatiques sont celles utilisées dans l'industrie pour la fabrication des laits hypoallergéniques. L'élastase II et dans une moindre mesure, l'élastase 1augmententl'efficacité de l'hydrolyse de la ~-lactoglobulinepar le mélangepepsine, trypsine et chy- motrypsine et réduisent l'antigénicité résiduelle de ses produits d'hydrolyse. Le même pourcentage d'hydrolyse est obtenu en additionnantl'élastase 1ou l'élastase II. Cependant,l'antigénicité résiduelle est plus faible en présence d'élastase II. Ainsi, l'adjonction d'élastases dans un mélange pancréatique pourrait améliorer l'hydrolyse des protéines dans la fabrication des laits hypoallergéniques. hydrolyse de la ~-lactoglobuline / élastases 1and II pancréatiques / antigénicité résiduelle / lait hypoallergénique INTRODUCTION enzymes to reduce in vitro allergenicity of œ-lactalbumin and ~-lactoglobulin (Pahud et Bovine whey proteins such as œ-Iactalbu- al, 1985; Asselin et al, 1988, 1989). Heat min and ~-lactoglobulin are with bovine treatment of whey proteins has been shown caseins the most commonly used proteins to reduce antigenicity, but has been associ- in infant formulas owing to their high nutri- ated with a loss of available lysine (Hep- tional value. They differ qualitatively and/or pell et al, 1984; Jost et al, 1987). Accord- quantitatively from human milk proteins. ing to Jost et al (1987), combining selective In addition to a high casein level (80 vs 35% hydrolysis by specifie proteases with pro- of total protein in human milk), cow milk cessing or subsequent heat treatments contains 50% ~-lactoglobulin in whey pro- appeared to be a promising approach in teins. This globular protein is lacking in developing a hypoallergenic infant formula. human milk. These cow milk components However, very few studies have investi- are not always weIl tolerated, since about gated the action of pancreatic proteases other 3% of children under 2 years of age present than trypsin and chymotrypsin on milk pro- allergie reactions. The allergie reactions are tein. Elastase II, which specifically cleaves promoted by rapid absorption of incom- globular protein, could improve milk pro- pletely digested milk products. This partial tein hydrolysis. The hydrolysis rate of digestion may be explained by a low gas- bovine casein, œ-Iactalbumin and ~-lac- tric acidity during infancy along with the toglobulin by human cationic elastase (elas- high buffering capacity of milk (Mason, tase II-like) exceeds the hydrolysis rate by 1962) and possibly a deficient proteolytic human anionic and cationic trypsins or response, particularly as regards elastase II, anionic elastase (elastase I-like) (Jakobsson in the digestive tract of infants suffering et al, 1983). Elastase 1 hydrolyses both from cow milk allergy (Jakobsson et al, œ-Iactalbumin and ~-lactoglobulin at a mod- 1983). erate hydrolytic rate, producing large and very small peptides, respectively (Schmidt ln order to pre vent atopic disease, pro- and PolI, 1991). tein hydrolysates are now given as of birth for several months to an increasing number The purpose of this study was therefore to of infants (Rigo et al, 1994). Pep sin asso- investigate gastric (pepsin) and pancreatic ciated with trypsin or chymotrypsin hydrol- (trypsin, chymotrypsin and elastases 1and II) ysis constitutes an efficient combination of enzymatic hydrolysis of ~-lactoglobulin as ~-Lactoglobulin hydrolysis by elastases 401 a means of reducing p-lactoglobulin anti- Enzyme activity assays genicity. The purified elastase II is not avail- able commercially; we therefore had to Protein content was measured as described by purify pancreatic elastase II simultaneously Lowry et al (1951). Elastase 1 (EC 3.4.21.36) with pancreatic elastase 1 in order to carry and II (EC 3.4.21.71) activity assays were carried out at 25 "C in 0.2 mol/L Tris-HCl buffer out our study. (pH 8.0) with 0.01 mol/L succinyl-L-alanyl- L- alany 1-L-alanine-p-nitroanilide (Suc- Ala3-pN A; L1385, Bachem AG, Budendorf, Switzerland) (Bieth et al, 1974) and 0.01 mol/L succinyl-L- MATERIALS AND METHODS alanyl-L-alanyl-L-prolyl-L-Ieucine-p-nitroanilide (Suc-Alaz-Pro-Leu-pNA; Ll390, Bachem AG, Purification of porcine elastases 1 and II Budendorf, Switzerland) (DelMar et al, 1980) as substrates, respectively. Elastase II substrate is known to be substantially hydrolyzed by both Porcine elastases 1 and II were purified as previ- chymotrypsin and elastase 1 (especially by the ously described for the isolation of human elas- latter) (DelMar et al, 1980; Largman, 1983). tases 1 and II (Largman et al, 1976) with slight Therefore, chymotrypsin (EC 3.4.21.2) assays modifications. Briefly, 4 g acetone powder, rep- were performed at 25 "C in 0.05 mollL Tris-HCI resenting 25 g pancreatie tissue, were suspended (pH 8.0) containing 0.02 mol/L CaClz using suc- in 0.01 mollL CaClz,1 mollL Tris-base (pH 8.0), ciny 1-L-alany 1-L-alany 1-L-proly 1-L-pheny lala- activated at 4 "C for 30 min with 1: JO (w:w) nine-p-nitroanilide (Suc-Alaz- Pro- Phe-pN A; trypsin (L-I tosylamide 2-phenylethyl S7388, Sigma Chemicals, St Louis, MO, USA) as chloromethyl ketone; T8642, Sigma Chemicals, substrate (DelMar et al, 1980). The resulting St Louis, MO, USA) and centrifuged for 15 min enzymatic units were expressed as u mol of at 20 000 g. The supematant was precipitated at p-nitroaniline released per min (Il.l). pH 5.1 with 6% acetie acid and centrifuged for 10 min at 33 000 g. The latter supematant was then subjected to ammonium sulfate precipita- tion up to 55% saturation. After centrifugation Sodium dodecyl sulfate (SDS) at 27 000 g for 10 min, the precipitate was resus- gel electrophoresis pended in 0.01 mollL sodium phosphate (pH 6.5) and dialyzed (retention threshold: 6000-8000 Da) Cation-exchange chromatographie samples were for 4 h against water and maintained ovemight analyzed by SDS-polyacrylamide gel elec- against the 0.05 mmol/L sodium phosphate trophoresis (SDS-PAGE) using a 14% gel under (pH 6.5) buffer.
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