Application of in vitro protein solubility for selection of microalgae biomass as protein ingredient in animal and aquafeed G. Venkata Subhash 1 & Neera Chugh 1 & Supriya Iyer 1 & Ashish Waghmare 1 & Amar S. Musale 1 & Rajesh Nandru 1 & Rakhi Bajpai Dixit 1 & Mahadev S. Gaikwad 1 & Deepthi Menon 1 & Rakesh Thorat 1 & G.Raja Krishna Kumar 1 & Vinod Nagle 1 & Uma Shankar Sagaram 1 & Santanu Dasgupta 1 Received: 29 June 2020 /Revised and accepted: 17 August 2020 # Springer Nature B.V. 2020 Abstract Microalgae when grown under certain conditions can be rich sources of protein and can complement conventional protein sources like fishmeal and soymeal, in the aquaculture and animal feed industry. In this study, evaluation of four marine microalgae strains (Picochlorum sp., Nannochloris sp., Nannochloropsis sp., and Chlorella sp.) revealed that in vitro protein solubility and digestibility may serve as key indicators in determining the suitability of microalgae as a protein ingredient in feed. The greenhouse areal biomass productivities, protein and lipid concentrations of these strains ranged between 9–17 g m -2 day -1 , 30–38% and 22–24%, respec- tively. Preliminary in vitro assays using undisrupted biomass of Picochlorum sp. revealed that its protein solubility was 47% and 67% less and digestibility was 28% and 22% less compared with fishmeal and de-oiled soy flour (DOSF), respectively. However, disruption of Picochlorum sp. biomass resulted in 2.5- and 1.5-fold increase in protein solubility and digestibility, respectively, as compared with undisrupted biomass. Further in vitro studies indicated that the soluble protein fractions differed significantly among the four experimental microalgae. The highest in vitro protein solubility (%) and soluble protein fractions (g kg -1 biomass) recorded in the four strains were the following: Picochlorum sp. (53%; 176 g kg -1 ), Nannochloris sp. (57%; 217 g kg -1 ), Nannochloropsis sp. (71%; 214 g kg -1 ), and Chlorella sp. (53%; 197 g kg -1 ). In addition, extracts from all these four strains were tested for the presence of trypsin inhibitors and found that all these strains have significantly lower trypsin inhibiting activity (TIA) compared with DOSF. The methodology presented in this study combines growth, biochemical composition, protein solubility, in vitro protein digestibil- ity, and TIA and thus provides a reliable strategy in selection of microalgae as protein feed ingredient. Keywords Alternative feed ingredient . Microalgae protein . Microalgae cell disruption . Protein solubility and digestibility . Trypsin inhibition activity Introduction Microalgae are considered to be the next generation feed stocks for biofuels, as they can be produced all year round with higher oil yields compared with conventional crops (Mofijur et al. 2019). Apart from serving as feedstock for biofuels and chemicals, microalgae are good sources of vita- mins, minerals, proteins, carotenoids, and omega-3 fatty acids that can have wide applications in the food and the feed sector (Becker 2004; Pulz and Gross 2004; Wijffels et al. 2010; Enzing et al. 2014; Chua and Schenk 2017; García et al. 2017; Schiano di Visconte et al. 2019). For example, Chlorella and Arthrospira (Spirulina) are used as additives in food and animal feed (Moore 2001; Olaizola 2003). The feed industry is evolving constantly and the requirement for protein ingredients is also steadily increasing. Currently, fishmeal and soymeal are the major sources of protein for the feed industry. Around 70% of the global supplies of fishmeal are being consumed by the aquaculture industry (Chauton et al. 2015; FAO 2016). With such high levels of usage and exploitation of limited natural resources, fishmeal G. Venkata Subhash and Neera Chugh contributed equally to this work. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10811-020-02235-9) contains supplementary material, which is available to authorized users. * Uma Shankar Sagaram Uma.Sagaram@ril.com * Santanu Dasgupta Santanu.Dasgupta@ril.com 1 Reliance Technology Group, Reliance Industries Limited, Reliance Corporate Park, Ghansoli, Thane-Belapur Road, Navi Mumbai 400701, India Journal of Applied Phycology https://doi.org/10.1007/s10811-020-02235-9