Millex gs

Bromelain (E.C. 3.4.22.32) lot No. B4882 from Ananas comosus, TMAO, D₂O, acrylamide and ANS were purchased from Sigma Aldrich, USA. Anhydrous sodium phosphate monobasic and sodium phosphate dibasic dihydrate of highest purity and analytical grade purchased from Sisco Research Lab (SRL), India. For the study of proteolytic activity, casein (Hammarsten) was also obtained from the SRL, India. All other chemicals used were of analytical grade with highest purity. The enzyme samples were prepared in 0.2 M sodium phosphate buffer at pH 7 with 0.5 mg/mL concentration of BM for all measurements. For all gravimetric measurements, Mettler Toledo balance with a precision of ±0.0001 g was used. All mixture samples were prepared using distilled deionized water with resistivity of 18.3 Ωcm. After completely dissolving the enzyme in the solution, all samples were filtered with 0.22 μm disposable filter (Millipore, Millex-GS) through syringe and were incubated for 15 min at 25 °C in order to obtain complete equilibrium before performing experiments. The stability and activity of BM were studied in absence and presence of 0.1, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 M of TMAO for all experiments.

A total of 100 mL of 0.1, 1.0, 2.5, and 5.0 mg/mL solutions of atropine sulfate were prepared by diluting 1, 10, 25, and 50 mL of 10 mg/mL atropine sulfate solution (Nitten Atropine Ophthalmic Solution 1%; Lot number L1779K; expiration August 2020; Nitten Pharmaceutical Co., Ltd., Nagoya, Japan) in 99, 90, 75, and 50 mL of isotonic sodium chloride solution (0.9% NaCl; Hikari Pharmaceutical Co., Ltd., Tokyo, Japan) to obtain 0.1, 1.0, 2.5, and 5 mg/mL solution (0.01, 0.1, 0.25, and 0.5% atropine sulfate solution). These solutions were sterilely dispensed at 5 mL per one bottle into sterilized white opaque polyethylene eyedropper squeezable bottles (Lot number 344161 J109; MI Chemical Co., Ltd., Hyogo, Japan) using an electron beam sterile syringe equipped with a 0.22 μm pore size filter (Millex-GS; Lot number R8JA9816; Millipore, Darmstadt, Germany) in a laminar air flow microbiological safety cabinet.

L. gasseri strain ATCC 9857 was obtained from the American Type Culture Collection (Manassas, VA, USA). The human vaginal microbiota-associated L. gasseri strain KS 120.1 was kindly provided by ProbioSwiss (Zurich, Switzerland). All Lactobacillus strains were grown in De Man, Rogosa and Sharpe (MRS) broth (Gibco, Thermo Fisher Scientific, 67403 Illkirch, France) for 18 h at 37 °C, under an atmosphere containing 5% CO₂-95% air. Isolated Lactobacillus cells and cell-free culture supernatants (CFCSs) were obtained by centrifuging the Lactobacillus cultures (18 h, 10⁹/CFU/mL) at 10,000× g, for 30 min at 4 °C. The separated bacterial cells were washed three times with sterile MRS and resuspended in fresh MRS. CFCSs were passed through a sterile 0.22 mm-pore Millex GS filter unit (Millipore, Molsheim, France), and a colony count assay was performed to check that there were no bacterial cells in the CFCSs.

Typically, a 200–300 μL sample of [¹²⁴I]sodium iodide (555–600 MBq at the start of the radiosynthesis) in 0.2 M sodium hydroxide was evaporated to dryness in a V-shaped Wheaton Vial (2.0 mL) under a stream of helium gas at 100 °C. (drying time = 20 min). The dried residue was cooled to 25 °C followed by the addition of a solution of mTMSBG (0.25 mg, 0.92 μmol) in trifluoroacetic acid (250 μL). A 20 μL aliquot of 32 % v/v peracetic acid in acetic acid was then added and the mixture was stirred at 25 °C for 5 min. The resultant mixture was then diluted with 1.5 mL of 0.6 M potassium phosphate (monobasic) and injected onto an HPLC column (Phenomenex Bondclone C18, 250 × 4.6 mm i.d.). The column was eluted at a flowrate of 2.0 mL min⁻¹ using a mixture of 0.1 % phosphoric acid:ethanol (85:15 v/v). The mobile phase was monitored continuously for both radioactivity and UV absorbance at 254 nm. The fraction eluting at 14 min, having the same retention time as reference mIBG was collected into a vial containing 16 mL of 0.9 % saline and 100 μL of 8.4 % sodium bicarbonate. Under aseptic conditions the resultant solution was then dispensed 3 ways via a Millipore filter (0.22 µm, Millex GS, Millipore) into sterile vials as 2.0 and 6.0 mL samples for quality control analysis and a 10.0 mL sample for PET imaging application. The total radiosynthesis time was 75 min.

The 1,3-PD, glycerol and organic acids were assayed by high-performance liquid chromatography. Samples for chemical analysis were first centrifuged at 10,000 g for 10 min at 4°C (Multifuge 3SR, Germany), filtered through a 0.22 μm membrane filter (Millex-GS, Millipore, USA), and then analyzed on an HPLC system (Agilent Technologies 1200 series).
An Agilent Technolgies 1200 series system equipped with a refractive index detector was used. Analyses were performed isocratically at a flow rate of 0.6 mL/min on an Aminex HPX-87H 300 × 7.8 column (Bio-Rad, CA, USA) at a constant temperature of 65°C. H₂SO₄ (0.5 mN) was the mobile phase. External standards were applied for identification and quantification of peak areas. Retention times (Rt) determined for the target compounds were as follows: 1,3-PD - 17.17 min; glycerol - 13.03 min; butyric acid - 20.57 min; acetic acid - 14.4 min and lactic acid - 11.19 min.

Determinations of methacrylic acid were carried out on Agilent 1260 Infinity II system consisting of autosampler (model G7129A), pump (model G7111A) and DAD (model G7115A) set at 210 nm. Analysis was performed isocratically at a flow rate 0.8 mL/min, at 40 °C, on column Rezex ROA-Organic Acid H+, 300 × 7.8 mm (Phenomenex). 10 mM sulphuric acid as a mobile phase was used. Samples were filtered (0.22 µm, Millex-GS, Millipore, Burlington, MA, USA), and the injection volume was set at 20 µL. Standard was used to identify peaks in chromatograms, and peak area was used to determine the concentrations of the samples. It was done by computer integration operated in the mode of external standards.