Peptidase

The bacteriocin A3 was tested for stability in terms of inhibition on target microorganisms by varying its production in different carbon sources namely glucose, lactose and sucrose. This was repeated for thermal and pH stabilities. For accessing the thermal stability test, the bacteriocin was exposed to temperatures 40, 60, 80 and 100°C for 20 minutes. The heated bacteriocin was cooled to room temperature and tested for inhibition on Bacillus cereus. For the pH stability test, the bacteriocin was adjusted to a pH value ranging from 2 to 10 by using concentrated NaOH (Merck, Germany) and HCl (Merck, Germany). This was incubated for 2 hours at 25°C before determining the antimicrobial activity by agar well diffusion method. The vulnerability of the bacteriocin to breakdown by different enzymes namely proteinase K, lysozyme, lipase, catalase, lyticase, trypsin and peptidase sourced from Sigma- Aldrich was also tested and then checked for subsequent inhibition. 500 μl of the bacteriocin was treated with the enzymes with 1 mg/ml final concentration and a control without treatment was prepared. All preparations were incubated at 37°C for 1 hour before antimicrobial measurement with B. cereus.

Semolina (Sun Valley Foods, Christchurch, New Zealand) was obtained from Foodstuffs, New Zealand (Christchurch, New Zealand). Red cod (Pseudophycis bachus) fish were bought in ice condition from Christchurch Wholesale Seafood (Christchurch, New Zealand). Folin-Ciocalteau reagent, Fluorescein, 2,2′-azobis (2-amidino-propane) dihydrochloride (AAPH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carbixylic acid (Trolox), pepsin, pancreatin, trypsin, α-chymotrypsin, peptidase, Amyloglucosidase, and 3.5-dinitrosalicylic acid (DNSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

To generate artificial metabolites of the ⁶⁸Ga-labelled trimeric conjugates the radiolabelling solution was neutralized with sodium carbonate solution (Na₂CO₃, 1 M), diluted with PBS to a final concentration of 4.4 μM and after adding 10 mU of peptidase (protease type 1 from bovine pancreas, Sigma Aldrich, Vienna, Austria) the mixture was maintained at 37°C. Samples (25 μL) were taken at different time points (5, 10, 60 and 120 min), 0.1% TFA/methanol (1:1) was added to quench the enzymatic reaction followed by subsequent radio-RP-HPLC analysis.