Amylase activity assay kit

We used EBC lactate as a marker for EBC sample dilution and calculated EBC ATP-to-EBC lactate ratio. In one occasion, insufficient sample material was collected and median EBC lactate was used. Lactate in capillary blood gas and EBC was performed on a RapidPoint 500 System (Siemens, Germany, detection limit 180 μmol/L). Subsequently, EBC was stored at − 80 °C for amylase assay. A colorimetric (405 nm) amylase assay was performed to detect possible saliva contamination. Amylase activity was assessed using an Amylase Activity Assay Kit (MAK009, Sigma-Aldrich, USA) and a Varioskan LUX multimode microplate reader (Thermo Fischer Scientific, USA) according to manufacturer protocol.

The α-amylase activity was performed on the most promising extract (considering the results obtained in the previous assays) using an Amylase Activity Assay Kit (Sigma, MAK009) and according to the manufacturer’s protocol. This assay was carried out using a coupled enzymatic method, which results in a colorimetric (405 nm) product. Nitrophenol (2 mM) was used as standard. The results were expressed as a percentage of α-amylase inhibition.

B. subtilis strain 168 was struck out on LB agar plate overnight. One colony was inoculated in LB medium for 16 h at 37 °C, pelleted and resuspended in M9 medium. For amylase production testing, the cell resuspension was inoculated into M9, M9 + 0.2% starch (through 0.22uM filter), and M9 + 0.2% starch + amino acid in a 96-well plate at the starting inoculum of OD600 = 0.07 and final volume of 200ul. All amino acid concentrations were at 20 mM when possible, based on solubility data provided by the manufacturer (Millipore Sigma). Exceptions are Asp (3.4 mM), Glu (5.8 mM), Ile (14 mM), Leu (3.8 mm), Phe (16.3 mM), Trp (6.6 mM), Try (0.3 mM). All growth conditions were set up in triplicates. After 24 h at 37 °C, amylase activity and production were determined using the Sigma-Aldrich® Amylase Activity Assay Kit (MAK009). The protocol was followed exactly as suggested by the assay kit manufacturer. OD₄₀₅ was measured at 24 h, and the value of the starch-only sample was subtracted from the value of each amino acid- supplemented sample. Amylase activity was reported as nmole/min/mL (milliunits), considering that one unit of amylase is the amount of amylase that cleaves ethylidene-pNP-G7 to generate 1.0 mmole of p-nitrophenol per minute at 25 °C.

All solvents and chemicals used were of analytical grade. Methanol, Folin-Ciocalteu reagent, concentrated hydrochloric acid sodium carbonate, gallic acid, vanillin, catechin, 1 M triethylammonium bicarbonate (TEAB) buffer, sodium chloride, acetone, urea, iodoacetamide, tris(2-carboxyethyl)phosphine hydrochloride (TECP), acetonitrile (ACN), trifluoroacetic acid (TFA), β-lactoglobulin, phosphate-buffered saline, 4-octanol used in the GC analysis and amylase activity assay kit were purchased from Sigma-Aldrich (Castle Hill NSW, Australia). Micro bicinchoninic acid (BCA) protein assay kit was purchased from Thermo Fisher Scientific (Waltham, MA). Purified water was obtained from a Milli-Q system (Millipore Australia, Bayswater, Victoria, Australia). Eight Shiraz wines produced in Victoria, Australia, were purchased from local wine producers. Due to the same wine samples being no longer available on the market when the spiking tests were conducted, Wine 8 of vintage 2019 was used for the spiking tests.