2.5. Chemical and microbiological analyses

The protease activity was determined following the method of Chakravarty et al. (2018), with casein used as a substrate. Enzymatic reactions were monitored at 660 nm using a 96-well microplate reader (Model Synergy HT, BioTek Instruments, Inc., Winooski, VT, USA) with BioTek Gen5 software. One unit of protease activity was defined as the amount of enzyme required to release 1 μmol of L-tyrosine per minute. Results were expressed as units (U) per mL of liquid hydrolysate produced during SPW fermentation. The pH of the hydrolysate was measured using a pH meter (Model Starter 2100, OHAUS Corporation, Parsippany, NJ, USA). The degree of hydrolysis (DH) was determined following the method of Benjakul and Morrissey (1997) based on the comparison of free amino groups in the hydrolysate to the total amino groups in the raw material after acid hydrolysis with 6 M HCl (100 °C, 24 h). The amount of free amino groups was spectrophotometrically analyzed at 420 nm using a microplate reader, with L-leucine employed as a standard.

The nutrient analysis of SPW samples and their hydrolysates was conducted following the methods of the Association of Official Analytical Chemists (AOAC, 2019). Briefly, total fat content was determined after acid hydrolysis and petroleum ether extraction using a Soxtec system. The total nitrogen and chitin nitrogen were analyzed by the Dumas combustion method. Corrected protein content was obtained by subtracting chitin nitrogen from total nitrogen and multiplying by a conversion factor of 6.25 (Tshinyangu and Hennebert, 1996). The ash content was determined by burning samples in a muffle furnace at 550 °C. The moisture content was determined by drying samples at 105 °C. The total carbohydrate content was calculated by subtracting the contents of the aforementioned nutrients from 100, and the energy was calculated using multiplication factors of 9 for fat and 4 for carbohydrate and protein. The calcium content was determined using a flame atomic absorption spectrometer.

The efficiencies of protein and mineral recovery from the SPW samples were reported as percentages of deproteinization (DP) and demineralization (DM) (also decalcification, DC), taking into account the masses of the nutrients before and after fermentation (Chakravarty et al., 2018; Ghorbel-Bellaaj et al., 2012). The calculations were performed using the following equations

where PO and PR are protein contents before and after fermentation, and O and R are masses (g DW) of the original sample and fermented residue, respectively

where AO and AR are ash contents before and after fermentation, and O and R are masses (g DW) of the original sample and fermented residue, respectively and

where CO and CR are calcium contents before and after fermentation, and O and R are masses (g DW) of the original sample and fermented residue, respectively.

The number of bacterial cells was determined by viable counts on culture media using the standard plate count method (BAM, 2001), and the total bacterial count was determined using plate count agar. The Bacillus count was determined on LB agar, and the Lactobacillus count was determined on MRS agar. For all microbial assays, culture incubation times were 24–48 h at 37 °C. Bacterial colonies that appeared on the agar media were counted and reported as CFU per mL of sample.