Short communication Elements for optimizing a one-step enzymatic bio-refinery process of shrimp cuticles: Focus on enzymatic proteolysis screening R. Baron a, *, M. Socol a , R. Kaas b , A. Arhaliass c , J. Rodriguez del Pino a , K. Le Roux d , C. Donnay-Moreno a , J.P. Bergé a, 1 a Ifremer, BRM, Rue de l’Ile d’Yeu, BP21105, 44311 Nantes cedex 03, France b Ifremer, PBA, Rue de l’Ile d’Yeu, BP21105, 44311 Nantes cedex 03, France c Université de Nantes, GEPEA UMR CNRS 6144, CRTT, Boulevard de l’Université, 44600 Saint-Nazaire cedex, France d Ynsect, Rue de l’Ile d’Yeu, BP21105, 44311 Nantes cedex 03, France A R T I C L E I N F O Article history: Received 22 August 2016 Received in revised form 14 December 2016 Accepted 6 January 2017 Available online 13 June 2017 Keywords: Bio-refinery Shrimp cuticles Acidic enzymatic proteolysis A B S T R A C T This article complements an earlier work published in 2015 Baron et al. (2015) that showed the interest of a shrimp shells bio-refining process. We compare here the effect of eleven commercial proteases at pH 3.5 or 4.0 on a residual amount of shrimp shells proteins after 6 h at 50  C. The two pH are obtained when respectively 40 and 25 mmol of formic acid are added to 5 g of mild dried shell. Deproteinisation yield above 95% are obtained. Residual amino acids profile in the solid phase was identical for the eleven proteases except for pepsin which was similar to the raw material profile. A significant relative increase in the proportion of Glycine is observed for the ten other cases. Likewise, shapes of size exclusion chromatograms of the dissolved phase are similar except with pepsin. © 2017 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). 1. Introduction Purification of crustacean chitin shells has been studied by many authors [1–5,8–22] [6,7] and today represents an important economic activity particularly in the context of shrimp shells value-enhancing schemes [23]. In fact the applications of chitin and its derivatives are more and more widespread. However, the process used is purely chemical and allows only an enhancing value of a small portion of the biomass. Efforts were therefore made to limit the use of chemicals and make this type of purification more sustainable. Bio-refining of crustacean shells, especially shrimp, is an economic, technical and scientific objective already described by some authors [1,2,4,5,10,15–17,20,22] [6,7]. Two biotechnological ways are found in literature: fermentation [5,10,16,17] or enzymatic hydrolysis [1,2,4,15,16,20,22] [6,7]. A bio- refining process in a single step by an exogenous proteolysis in acidic media would enable us to perform chitin purification and deproteination in the same time. Recently, we have shown [1] the promising potential of the bio-refining in a single step of Litopenaeus vannamei shrimp shells. The authors have mainly focused on the kinetics of demineralization and the choice of a suitable acid that could ensure a high demineralization yield (>98%) for a pH value close to 4.0 (classical preservation value). Formic acid best fits the selected target criteria. This acid achieves a demineralization yield of 99% at pH 3.5 and 98% at pH 4.0, depending on the selected volume. An increase in solution volume promotes final demineralization. In 6 h, a combination of formic acid and ASP enzyme (Acid Stable Protease), in sufficient concentration, allowed to go beyond the 95% protein removalyield, at pH 3.5 or 4.0. The purity of the obtained chitin is respectively 92% at pH 3.5 and 90% at pH 4.0. The resulting chitin purity over 90%, for a single stage process working in 3.5–4 pH range avoids the additional steps of neutralization of both the solid and dissolved phases. Here we focus on determining the effectiveness of ten other commercial proteases compared to the ASP enzyme working in 3.5–4.0 pH range. The determination of an enzyme reaching a maximum deproteination yield after 6 h of hydrolysis in 3.5–4.0 pH range, and preferably at pH 4.0 needing less amount of acid, was first sought. The amount of residual proteins was determined using the sum of the quantitative analysis of 16 amino acids. The amino acid profile was also analyzed. The study of size exclusion chromatographs in conjunction with the molecular weight distribution of the generated peptides was conducted on the * Corresponding author. E-mail address: rbaron@ifremer.fr (R. Baron). 1 Present adress: Idmer, 2 Rue Batelière, 56100 Lorient. http://dx.doi.org/10.1016/j.btre.2017.01.003 2215-017X/© 2017 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Biotechnology Reports 15 (2017) 70–74 Contents lists available at ScienceDirect Biotechnology Reports journal homepage: www.elsevier.com/locate/btre