Bromelain

Beef longissimus dorsi samples were bought from a local supermarket (Huarun Suguo Supermarket. Co., Ltd., Nanjing, China). The enzymes proteinase K (Pro K, 400 unit/mg), papain (Pap, 400 unit/mg) and bromelain (Bro, 500 unit/mg) were purchased from Solarbio Company (Beijing, China). Fluorescamine and leucine were purchased from Sigma-Aldrich (Shanghai, China). All the standard flavor substances, including 2-pentanone (>99.0%), 2-octanone (>99.5%), butyraldehyde (>99.5%), octanal (>99%) and methanol (>99.9%), were bought from Aladdin (Shanghai, China). The protease inhibitor Np-Tosyl-L-phenylalanine chloromethyl (TPCK) was obtained from Macklin (Shanghai, China).

Oyster (C. talienwhanensis) was purchased from a seafood market in Qingdao, China. Upon arrival, the oyster was washed, and the edible meat was separated from the shells, homogenized, and stored at −20 °C until use.
Six proteases (compound proteinase, trypsin, bromelain, papain, neutrase, and flavourzyme) were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). The ultrafiltration (UF) system and UF membranes with molecular weight cut-offs (MWCO) of 10,000 Da and 3500 Da were purchased from Laungy Co., Ltd. (Shanghai, China). The fraction collector and computer ultraviolet (UV) detector were purchased from Shanghaihuxi Analysis Instrument Factory Co., Ltd. (Shanghai, China). The water was distilled and purified using a Milli-Q Water Purification System (Millipore, Bedford, MA, USA). All other chemicals and solvents were of analytical grade.

L. plantarum strains were centrifuged (TG20KR-D, Changsha Dongwang, China) at 6500× g for 10 min, and the cells were discarded to retain the supernatants. Part of the supernatants were adjusted to pH 6.0 with NaOH to eliminate organic acids interference, and the remaining supernatants were maintained at the original pH, both of which were filtered through 0.22 µm membranes. The agar well diffusion method with BHI semi-solid medium was carried out to determine the antimicrobial activity of L. plantarum using L. monocytogenes as the indicator strain [19 (link)]. Then the L. plantarum supernatants with antibacterial activity after excluding organic acids were adjusted to the optimum pH for catalase (5000 U/mg, Solarbio, Beijing, China), papain (800 U/mg, Solarbio, Beijing, China), bromelain (600 U/mg, Solarbio, Beijing, China), trypsin (250 U/mg, Solarbio, Beijing, China), pepsin (250 U/mg, Solarbio, Beijing, China) and proteinase K (30 U/mg, Solarbio, Beijing, China). The strain supernatants were mixed with 1 mg/mL of above six enzymes at 9:1 (v/v) and incubated at 37 °C for 1 h, respectively, followed by water bath at 70 °C for 5 min. The inhibition zone diameter was determined by agar well diffusion method and the titer was measured by double-dilution method.