Alcalase 2.4 l fg

The enzymatic recovery of BG from antler-type fruiting bodies of G. lucidum was performed according to some modifications in Vaithanomsat et al. [9 (link)]. Water was added (1:15 w/v), the reaction was stirred with heating to 90 °C for 3 h and cooled down to room temperature. After that, ethanol solution was added (1:15 w/v) and the reaction was incubated at 4 °C for 15 h. The precipitate was harvested by centrifugation and immediately frozen in liquid nitrogen. Protease enzyme (2.4 AU/mg; Alcalase^® 2.4 L FG, Novozymes A/S, Kalundborg, Denmark) was applied at concentration of 1.0% (w/v). The reaction was incubated at 55 °C for 2 h, subjected to centrifugation, and the precipitates were washed with water. Ethanol (precipitate:ethanol) at a ratio of 1:4 was added. The mixture was centrifuged to obtain the BG precipitate, which then was dried at 40 °C for 48 h under vacuum. G. lucidum BG was ground into powder using a multi-purpose grinder (BL-T70-PR2, Toshiba, Tokyo, Japan), subjected to a stainless-steel sieve mesh No. 40, and kept in aluminum foil bags at 4 °C until use.

The enzymatic recovery of BG from yeast biomass was performed according to Vaithanomsat et al. with some modifications [12 ]. The yeast pellet from the aforementioned broth culture was collected via centrifugation. To obtain a cell content of 15% (w/v), water was added and incubated at 55 °C for 24 h with shaking at 120 rpm. After that, the cells were heated at 80 °C for 15 min, then cooled down to room temperature, and harvested via centrifugation. Protease enzyme (Alcalase^® 2.4 L FG, Novozymes A/S, Kalundborg, Denmark) with a declared activity of 2.4 AU/mg was added at concentrations of 0.25, 0.5, 0.75, 1.0% (w/v), and the mixture was incubated at 55 °C for 5 h. The cells were then separated by centrifugation, washed with water, and the precipitate was separated by centrifugation. Ethanol was added at a ratio of 1:4 (precipitate:ethanol). The BG precipitate was collected by centrifugation and dried at 40 °C under vacuum for 48 h. BG extract was ground into fine powder using a multi-purpose grinder (BL-T70-PR2, Toshiba, Tokyo, Japan), then passed through a stainless steel sieve (mesh No. 40), and stored in aluminum foil bags at 4 °C for further analysis.

Cephalothorax of King prawn (Penaeus vannamei) and (Illex argentinus) were obtained as byproducts from the industrial processing of both species and kindly provided by Pescanova S.A. (Vigo, Spain) and Cabomar S.A. (Marín, Spain). In the case of shells of prawn, the production of chitin was performed according to Vázquez et al. [39 (link)]. Briefly, shells were ground (0.5–1.5 cm), washed to eliminate the rest of the crustacean body, deproteinized by Alcalase^® 2.4 L FG (Novozymes, Novodirsk, Bagsværd, Denmark) hydrolysis, demineralized with HCl twice, purified with NaOH treatment, neutralized with an intense water wash, depigmented using NaClO wash, and the α-chitin obtained was finally dried in an oven. By contrast, pens were ground and sieved (0.5 cm), deproteinized with Alcalase, and the recovered β-chitin was also dried in an oven [40 (link)].
The raw biomass was placed inside a tubular furnace at 1000 °C for 1 h with a 0.3 L h⁻¹ N₂ flow-controlled environment to further collect the ashes (39 wt.% recoveries). The variation of time and temperature of carbonization was applied, and preliminary results were obtained. The temperature and time of 1000 °C and 1 h presented the best results, so these parameters were chosen to proceed with this study. The results are presented in Table S1 in Supporting Information.

Helix aspersa maxima by-product snails were supplied by Mr Stephen Ryan, Tuam, Co. Galway (Ireland) who farms these snails. By-product snails are those considered to be under-sized and not suitable for market. By-product Helix aspersa maxima were carefully washed using running water prior to placement on ice and subsequent freezing at −80 °C. Alcalase^® 2.4 L FG was kindly supplied by Novozymes (Bagsvaerd, Denmark). The ACE-I inhibition assay kit was supplied by NBS Biologicals Ltd. (Cambridgeshire, England, UK). The positive control Captopril© was purchased from Sigma-Aldrich (Sigma-Aldrich, Dublin, Ireland). All other chemicals used were of analytical grade.

All enzymes, including a cellulase Celluclast^® 1.5 L, the xylanase Shearzyme^® 500 L, the protease (endo-peptidase) Alcalase^® 2.4 L FG, and the multi-enzyme mixture Viscozyme^® L which contains a wide range of carbohydrases, including arabinase, cellulase, β-glucanase, hemicellulase, and xylanase, were provided by Novozymes A/S (Bagsværd, Denmark). All chemicals used in this study were from Merck (St. Louis, IL, USA). All solvents used were HPLC grade and purchased from Lab-Scan (Dublin, Ireland). The standards for amino acids analysis were purchased from Sigma-Aldrich (St. Louis, MO, USA). HPLC grade water was prepared by a Milli-Q^® Advantage A10 water deionizing system from Millipore Corporation (Billerica, MA, USA).