Alcalase from bacillus licheniformis

Fresh crickets (Gryllus bimaculatus) were purchased from a local cricket farm in Chaing Mai, Thailand. Chemical reagents used for Osborne fractionation such as sodium chloride (NaCl), sodium hydroxide (NaOH), hydrochloric acid (HCl), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid; ABTS), ferrozine, Trolox, ethylenediaminetetraacetic acid (EDTA), ferrous sulfate (FeSO₄·7H₂O)], and Alcalase from Bacillus licheniformis (2.4 U/g, EC 3.4.21.14) were all purchased from Sigma Aldrich Chemical Company. Protamex (1.5 AU-A/g) and Flavourzyme (500–1000 LAPU/g) from Novozymes were supported by Brenntag Ingredients (Bangkok, Thailand) Public Company Limited.

The wet grape pomace samples from seven grape cultivars, including Muscadine Carlos, organic Muscadine Noble, organic Cabernet Franc, Cabernet Sauvignon, Merlot, Sangiovese and Chardonnay were obtained from two North Carolina wineries. They were collected right after press, packed in gallon-size plastic bags separately and stored at −20 °C until use. Purified ochratoxin A (lyophilized powder) from Aspergillus ochraceus, carboxypeptidase A from bovine pancreas, lipase and protease from A. niger, alcalase from Bacillus licheniformis, and pepsin from porcine gastric mucosa were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetic acid, citric acid, lactic acid and hydrochloric acid were purchased from Fisher Scientific (Suwanee, GA, USA).

The enzymatic hydrolysis was performed as described in Hjellnes et al. [27 (link)] with modification. Freeze-dried C. frondosa powder (5.0 ± 0.0 g) was mixed with preheated water (50 °C), a ratio between sample/water of 1:6.5 for 60 min at 50 °C with magnetic stirring (300 rpm). Bromelain (Ananas comosus, 3 U/mg, EC 3.4.22.32, Merck Life Science AS, Oslo, Norway), and Papain (Carica papaya, 1.5–10 U/mg, EC 3.4.22.2, Merck Life Science AS, Oslo, Norway) were added in a 1:1 ratio, and Alcalase (from Bacillus licheniformis, ≤2.4 U/g, EC 232.752.2, Sigma-Aldrich, Steinheim, Germany) accounted for 0.36% of the total mixture. The enzymatic hydrolysis mixture was added in glass bottles with a blue cap in a water bath (Heto-Holten, Allerød, Denmark). The enzymatic reaction was stopped by applying heat (90 °C, 20 min). The mixture was then distributed into 15 centrifuge tubes (50 mL) and subjected to centrifugation (3500 g, 30 min, 20 °C). After this, the samples were frozen (−20 °C). The frozen samples were then divided into three fractions: oil, hydrolysate, and sludge, using a scalpel. The protein hydrolysates from C. frondosa were subsequently freeze-dried using a Labconco FreeZone 12 (Labcono Corporation, Kansas City, MO, USA, at −50 °C, <13.3 Pa). These samples were then stored at −80 °C for future analysis. The enzymatic hydrolysis was performed in duplicate.