E: Food Engineering & Materials Science Distribution of Cathepsin D Activity between Lysosomes and a Soluble Fraction of Marinating Brine Mariusz Szymczak Abstract: This paper is the first ever to describe the phenomenon of bimodal distribution of cathepsin D in the lysosomal and soluble fractions of brine left after herring marinating. Up to 2 times higher cathepsin D activity was observed in the lysosome fraction. Activity of cathepsin D in brine increased according to the logarithmic function during low frequency- high power ultrasounds treatment or according to the linear function after multiple freezing-thawing of brine. Activity enhancement was achieved only in the brine devoid of lipids and suspension. Study results show also that measurement of lysosomal cathepsin D activity in the marinating brine requires also determining cathepsin E activity. Decreasing pore size of microfilter from 2.7 to 0.3 μm significantly reduced the lysosome content in the brine. The presence of lysosomes and the possibility of their separation as well as the likely release of cathepsins shall be considered during industrial application of the marinating brine, as new cathepsins preparations in fish and meat technology. Keywords: aspartyl proteases, cathepsin D, lysosomes, marinating brine Practical Application: The marinating brine with enzymes or the enzymes alone may be reused for fish and meat ripening. The proteolytic activity may be increased in the brine even several times through damage of the membranes of lysosomes. Cathepsin D and other cathepsins may be acquired from the brine by separating lysosomes. Disintegration of lysosomes for example by ultrasound or multiple freezing-thawing should be first-essential step in production of cathepsins preparations from marinating brine-waste. Introduction Herring marinades are commonly consumed across Europe. Today, the fish industry faces problems with appropriate ripen- ing and with achieving desired sensory quality of marinated her- rings. The ripening process of fish meat proceeds in a marinat- ing brine (a solution of salt and acetic acid) and is principally based on proteolytic transformations. The incorporation of or- ganic acids (for example, acetic, lactic and citric acid) and sodium chloride (NaCl) are employed for ripening of fish muscle in the fish marinating process (Topuz 2016). Acid proteases, referred to as cathepsins, hydrolyze proteins, which causes a decrease in tex- ture hardness and simultaneously increases the content of protein hydrolysis products (PHB). Our previous investigations demon- strated that not only PHB (Szymczak and Kolakowski 2012), but also active cathepsins in the dissolved form (Szymczak and Lepczy´ nski 2016) diffuse to the brine during herring marinat- ing. Losses of aspartyl and cysteine cathepsins diminish the prote- olytic activity in the ripening meat. Lower activity of cathepsins contributes to deterioration of the quantitative and qualitative composition of PHB in semi-marinades and in the end-product (Szymczak and others 2013; Szymczak and others 2015; Szymczak 2016). The aforementioned investigations showed also that cathep- sin D-like usually predominates in the total proteolytic activity of marinades and initiates their ripening process. This protease MS 20160365 Submitted 3/8/2016, Accepted 5/27/2016. Author is with Dept. of Food Science and Technology, Faculty of Food Science and Fisheries, West Pomeranian Univ. of Technology in Szczecin, 71–459 Szczecin, Papie˙ za Pawla VI 3, Poland. Direct inquiries to author Szymczak (E-mail: mariusz.szymczak@zut.edu.pl). determines textural properties and releases other proteases, whereas products of its reactions are substrates for other proteases (Makinodan and others 1983; An and others 1994). Cathepsin D occurs in lysosomes that are present in most of cells, also these of the muscle tissue of fish (Sricet 2014). Lysosomes are “single membrane bounded organelles containing numerous hydrolytic enzymes to digest materials ingested by endocytosis and recy- cle cellular components” (Voet and Voet 1990). Cathepsin D, acid phosphatase, β -glucuronidase, and β -N-acetylglucosaminidase are commonly acknowledged as lysosomal markers (Karvinen and others 1982; Ueno and others 1986). In marinating brine, cathep- sin D occurs in the form of dissolved proteins and probably also in lysosomes. The presence of lysosomes in the brine may in- dicate that the activity of cathepsin in the brine left after mar- inating is in fact higher than reported in our earlier studies (Szymczak and Lepczy´ nski 2016; Szymczak 2016). However, the contribution of cathepsin D activity in both fractions remains unknown. Slow freezing or low frequency-high power ultra- sounds can damage lysosomes (McGann, and others 1988; Weiss and others 2011), and increase cathepsin-D concentration in the brine. Thus far conducted investigations on the proteolytic activity of the marinating brine were focused only on enzymes occurring exclusively in the dissolved form. The occurrence of cathepsins in lysosomes in the brine is important as it enhances the prote- olytic potential; therefore consideration should be given to this phenomenon in attempts of exploiting proteases from the brine. Considering that, the aim of this study was to confirm the presence of lysosomes in the marinating brine left after herring marinating and to determine cathepsin D activity distribution in the soluble and lysosomal fractions. C  2016 Institute of Food Technologists R  doi: 10.1111/1750-3841.13375 Vol. 00, Nr. 0, 2016  Journal of Food Science E1 Further reproduction without permission is prohibited