GMP NPBD 0.2M stock solutions in DMSO (AnalaR®, BDH Chemicals, Leicestershire, England); Ciprofloxacin (MP Biomedicals); Media, supplements, Anaerogen™, Campygen™ (Oxoid, Cambridge, UK). Yeast Nitrogen broth, Brucella broth (BBL™). Test strains obtained from ATCC, NCTC, RMIT Culture Collection. Clinical isolates from human pathology laboratories (Tables S6–S8 ). A summary of the physicochemical properties of NPBD is provided in Table S1 .
Analar
Manufactured by Avantor
Sourced in United Kingdom
AnalaR is a premium brand of analytical reagents and solvents manufactured by Avantor. It is designed to meet the highest purity and quality standards for use in analytical and scientific applications.
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Lab products found in correlation
Anaerogen, by Thermo Fisher Scientific (1 mentions)
Campygen, by Thermo Fisher Scientific (1 mentions)
Ciprofloxacin, by MP Biomedicals (1 mentions)
N n n n tetrakis 2 pyridylmethyl ethylenediamine tpen, by Merck Group (1 mentions)
Trimesic acid, by Thermo Fisher Scientific (1 mentions)
Styrene, by Thermo Fisher Scientific (1 mentions)
Cdcl3, by Eurisotop (1 mentions)
Flavourzyme 1000 l, by Novozymes (1 mentions)
Neutrase 0.8 l, by Novozymes (1 mentions)
Papain, by Merck Group (1 mentions)
Alcalase 2.4 l fg, by Novozymes (1 mentions)
8 protocols using analar
1
Antimicrobial Susceptibility Testing Protocol
2
Proteinaceous Fractions Extraction and Antiviral Evaluation
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In order to prepare the proteinaceous fractions, 500 mg of each initial sample was firstly dissolved in sterile deionized water (ddH2O) up to a final volume of 1.5 mL. The samples remained for 1 h at room temperature with occasional stirring. Then, the samples were centrifuged for 10 min at 10,000× g, and the supernatant aqueous fraction was collected. Subsequently, solid ammonium sulfate (NH4)2SO4 (AnalaR, BDH Chemicals Ltd., Poole, England) was gradually added up to a final concentration of 60% w/v. Samples were stirred gently and left for 20 min at room temperature for equilibration. Then, they were incubated for 10 min at −20 °C and centrifuged for 30 min at 11,000× g. The supernatant was discarded, and the pellet was resuspended in 1 mL sterile ddH2O [20 ,21 (link)]. Furthermore, proteinaceous fractions were treated with 150 μg/mL proteinase-K (Blirt, Poland) for 24 h at 37 °C at 210 rpm, and then, the fractions were tested for antiviral activity.
3
Preparation of Aqueous Solutions for Bilayer Experiments
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Chemicals were AnalaR or the best equivalent grade from BDH Chemicals (Poole, UK) or Sigma-Aldrich. All solutions were made in deionized water, and those for use in bilayer experiments were filtered through a Millipore membrane filter (0.45-μm pore). 2,2′-(Ethylenedioxy)dianiline-N,N,N′,N′-tetraacetic acid (BAPTA) (Dorset, UK) and N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) were from Sigma-Aldrich.
4
Evaluating Antimicrobial Efficacy of Tsunami Sanitizer
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TsunamiTM 100 (Ecolab Inc., MN, United States) was diluted to working concentrations of 10, 20, 40, and 80 ppm, and the concentration was confirmed as peroxide equivalents with a Palintest Photometer 5000 using the hydrogen peroxide HR test at 490 nm. The cultures were exposed to the sanitizer for 1, 2, 4, 8, and 16 min following procedures adopted from Cruz and Fletcher (2012) (link). One milliliter of each culture was centrifuged (Eppendorf, 5424R) at 15,700 RCF for 5 min. The supernatant was removed, and cells washed with sterile water and re-suspended in 1 mL sterile water. Cell suspensions (50 μl) were dispensed into a 96-well plate and 50 μl of the sanitizer was added into each well. Plates were then left at room temperature for the designated contact time. To terminate the sanitizer reaction, 150 μl of neutralizer solution containing 5% egg yolk emulsion (Difco), 1% sodium thiosulfate (AnalaR, BDH, Chemicals Ltd., Poole, United Kingdom), and 0.5% Tween® 80 (Spectrum, Gardena, CA, United States) in TSBYE was added to each well containing the bacterial culture and sanitizer. Neutralized suspensions were then serially diluted in BLEB up to 10–15, and survivors quantified using the MPN method.
5
Nylon-11 Bags for Gasoline Analysis
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Two brands of nylon-11 bags were used, from CSI Equipment, UK, 460mm x 600mm and 0.04 mm thick (experiments 1, 2, 3 and 7), and BVDA International, Holland, 250mm x 500mm and 0.04 mm thick (experiments 4, 5 and 6).
Unleaded 95 RON gasoline used in experiments 1 to 6 was bought from Total and 1% of chlorobenzene (AnalaR from BDH Chemicals Ltd (England)) and 1% of decane (99+% from Jansen Chimica (Geel, Belgium)) were added as internal standards.
A sample of "Carplan" automotive topcoat thinner (Tetrosyl Ltd, Bury, England) was used for experiment 7.
Unleaded 95 RON gasoline used in experiments 1 to 6 was bought from Total and 1% of chlorobenzene (AnalaR from BDH Chemicals Ltd (England)) and 1% of decane (99+% from Jansen Chimica (Geel, Belgium)) were added as internal standards.
A sample of "Carplan" automotive topcoat thinner (Tetrosyl Ltd, Bury, England) was used for experiment 7.
6
Synthesis of Trimesic Acid Derivatives
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Trimesic acid (98%) was purchased from Acros Organics. The used sodium chloride was food grade. Copper(ii ) acetate monohydrate (>99%, AnalaR) was purchased from the British Drug Houses Ltd. Denaturated ethanol (Technisolv, 99%), methanol (HiPerSolv, CHROMANORM), dimethylformamide (HiPerSolv, CHROMANORM), dichloromethane (HiPerSolv, CHROMANORM) and HCl 37% (AnalaR, NORMAPUR) were purchased from VWR Chemicals. Copper(ii ) chloride hexahydrate (min. 99%) was purchased from Riedel-de Haën. Ethyl diazoacetate (contains ≥13% dichloromethane) was purchased from Sigma-Aldrich. Styrene (99.5%) and trimethoxybenzene (99%) were purchased from Acros Organics. CDCl3 (D > 99.8%, H2O < 0.01%) was purchased from Euriso-top. Styrene was distilled under reduced pressure to eliminate stabilizers before use. All other chemicals were used as received.
7
Enzymatic Digestion of Locust Protein
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Frozen adult migratory locusts (Locusta migratoria, LM) were purchased from NGN BV (Helvoirt, Netherlands) and stored at −30 °C until further use.
The enzyme preparations Alcalase® 2.4 L FG (2.4 AU-A/g, endoprotease from Bacillus licheniformis), Neutrase® 0.8 L (endoprotease from Bacillus amyloliquefaciens), and Flavourzyme® 1000 L (1000 LAPU/g, endo- and exoprotease from Aspergillus oryzae) were kindly provided by Novozymes A/S (Bagsvaerd, Denmark). Papain, a cysteine-protease from papaya latex (≥10 units/mg, E.C. 3.4.22.2, Sigma no P4762), was purchased from Sigma-Aldrich Inc. (St. Louis, U.S.A). For fat extraction of LM, n-hexane with a purity of ≥95.0% was used (AnalaR, VWR Chemicals, Fontenay-sous-Bois, France).
The enzyme preparations Alcalase® 2.4 L FG (2.4 AU-A/g, endoprotease from Bacillus licheniformis), Neutrase® 0.8 L (endoprotease from Bacillus amyloliquefaciens), and Flavourzyme® 1000 L (1000 LAPU/g, endo- and exoprotease from Aspergillus oryzae) were kindly provided by Novozymes A/S (Bagsvaerd, Denmark). Papain, a cysteine-protease from papaya latex (≥10 units/mg, E.C. 3.4.22.2, Sigma no P4762), was purchased from Sigma-Aldrich Inc. (St. Louis, U.S.A). For fat extraction of LM, n-hexane with a purity of ≥95.0% was used (AnalaR, VWR Chemicals, Fontenay-sous-Bois, France).
8
Chemically Acidified Bread Production
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Chemically acidified breads were produced in the same way as the sourdough breads. Instead of sourdough, LA and AA were added. Lactic acid (90.2 % AnalaR NORMAPUR, VWR) and acetic acid (glacial, ACS, 99.7+%, VWR) were added to the water phase which was corrected in order to obtain an equal amount of water phase for the dough preparation. The acid concentrations to be tested were determined after having knowledge about the intrinsic acid content of wheat flour bread and the total acid concentrations in sourdough bread determined via HPLC (C TOT AA: 57 -100 mmole/kg bread; C TOT LA: 0 -75 mmole/kg bread).
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