Alcalase enzyme

The fermented and the non-fermented beverages used in this study were produced by Bio-K Plus International Inc., a Kerry company (Laval, QC, Canada). Organic cricket flour (60% protein content) was produced by Nexxus Foods (Montreal, QC, Canada). Pepsin (lyophilized powder from porcine gastric mucosa ≥ 3200 units/mg protein), trypsin (from bovine pancreas 10,000 BAEE units/mg protein) and alcalase enzyme (from Bacillus licheniformis ≥ 2.4 Units/g of protein) and Ellman’s reagent 5.50-dithiobis (2-nitrobenzoic acid (DTNB)) were supplied by Sigma-Aldrich (Oakville, ON, Canada). All other reagents were of analytical grade. 1-anilino-8-naphthalene sulfonate (ANS), Sodium Hydroxide (NaOH), Sulfuric Acid (H₂SO₄), Hydrochloric acid (HCl) and Kjeltabs Cu-1.5 were provided by Thermo Fisher Scientific (Saint-Laurent, QC, Canada).

Tilapia viscera were obtained from PT Aquafarm Nusantara, Semarang Industrial Estate, in Indonesia. Viscera were cleaned with water and defatted. The hydrolysis process was carried out using the Alcalase enzyme (Sigma-Aldrich No. 126741) so that TVHE rich in bioactive peptides was obtained. TVHE was optimized using the response surface methodology (RSM) to obtain TVHE with the best hydrolysis degree [31 ].

Black, red, and white kidney bean seeds (Phaseolus vulgaris L) were acquired from a local market in Cairo City, Egypt, and raw chicken meat samples were obtained from a private farm at Zagazig City, Egypt. Alcalase enzyme and DPPH were from (Sigma-Aldrich, Inc., St. Louis, MO 68178, USA), and Muller Hinton agar (Oxoid, Basingstoke, UK). Electrophoresis reagents were from Bio-Rad laboratories (Sigma-Aldrich, Inc., St. Louis, MO 68178, USA). All chemicals were of analytical grade. The microorganisms used in this study to fully assess the antimicrobial activity included; Bacillus cereus, Listeria monocytogenes, Staphylococcus pyogenes, Escherichia coli, Campylobacter jejuni, Salmonella typhi, Aspergillus niger, Aspergillus fumigatus, Aspergillus flavus, Penicillium chrysogenum, Fusarium exquisite, Fusarium avenaceum, Candida gelbeta, Candida tropicalis, Candida albicans, Rhodotorula rubra, Rhodotorula minuta, and Rhodotorula mucilginosa. These isolates were obtained from Agricultural Microbiology Department, Faculty of Agriculture, Zagazig University, Zagazig, Egypt.

Zein Protein (Merc, Germany), alcalase enzyme, lecithin, Tween 80, cholesterol, ethanol, RPMI, RNase A, and yellow tetrazolium 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) was purchased from Sigma-Aldrich (St Louise, IL). Fetal bovine serum and DMSO were provided by Thermo (Waltham, MA), PBS, trypsin/EDTA, propidium iodide (PI), Triton X100, and DCFDA/H2DCFDA Cellular ROS Assay Kit were obtained from Abcam (Cambridge, UK). Penicillin, streptomycin, and Tris-HCl buffer were purchased from GeneDirex, Inc. (Gongye Rd, Taiwan). Annexin V-FITC (EXBIO, Czech), Bax, Bcl-2, caspase-3, -8, -9, and p53 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

The cowpea (Vigna unguiculata, L.) seeds were provided from local market, Zagazig, Sharkia Governorate, Egypt. These seeds were identified by Dr. Samir Salem, Botany, Dept. Fa of Sci., Za. Univ, Egypt. Alcalase enzyme is a product metabolite from B. licheniformis; it was purchased from Sigma (St. Louis, MO, USA).

Generation of snail protein hydrolysates was performed according to the method of Hamid et al. [12 ] with some modifications. Briefly, 44 g of cleaned, whole by-product H. aspersa maxima snail was ground using a Pestle and Mortar and to this 130 mL of distilled deionised water was added. Native enzymes present in the mixture were heat-deactivated by heating the mixture to 85 °C for 15 min in a water bath followed by cooling to room temperature. Following this, the pH was adjusted to pH 8 using 1 M NaOH. The mixture was placed in a shaking incubator at 45 °C and 1.76 mL of Alcalase^® enzyme (Sigma Aldrich, Dublin, Ireland) was added to the mixture in an enzyme to substrate ratio of 4%. The temperature was maintained at 45 °C, with shaking at 150 rpm for 180 min. The pH was kept constant at the optimum for Alcalase^® using 0.1 M NaOH. After this time, the hydrolysates were heat-deactivated by heating to 90 °C for 15 min. The mixtures were cooled and subsequently centrifuged at 5000 rpm, 4 °C for 20 min after which time the supernatant was recovered, frozen at −80 °C and subsequently freeze-dried along with the shell fraction. The degree of hydrolysis (DH) was calculated using the pHstat technique after 180 min according to the Adler-Nissen method [13 ].