No 4 filter paper

Commiphora myrrha resin (25 g) was ground into a fine powder and macerated separately in glass bottles with 150 mL of methanol. The bottles were shaken for 24 h at room temperature in an orbital shaker (Heidolph^® Unimax Orbital Shakers 1010, Schwabach, Germany). Whatman No. 4 filter paper was used to filter the extracts. With new aliquots of the same solvent, residues were extracted twice. To obtain methanolic extract (3.86 g, 15.44%, w/w), supernatants from each solvent were combined and evaporated under vacuum (Heidolph^TM Instruments Hei-VAP) at 40 °C. The obtained crude extracts were utilized for further analysis after being re-dissolved in methanol at a concentration of 1 μg/μL [74 (link)].

Seven different varieties of mustard were used in this study—two varieties of Sinapis alba (AC Pennant and Andante) and five varieties of Brassica juncea (Duchess, Centennial Brown, AC Vulcan, Cutlass, and Dahinda). All mustard seed samples were generously offered by Dr. Janitha Wanasundara of the Saskatoon Research and Development Centre of Agriculture and Agri-Food Canada (Saskatoon, SK, Canada). The seeds were frozen in liquid nitrogen, ground to a fine powder using an analytical mill (IKA A11, IKA, Staufen, Germany), and defatted with hexane (1:5 w/v) under constant magnetic stirring. The slurry was filtered using a Whatman No. 4 filter paper, and extractions were repeated three times. Defatted samples were dried overnight (~10–12 h) in a fume hood in order to remove all traces of residual solvent. Defatted flours were then homogenized for 30 s in a coffee grinder (Custom Grind Deluxe, Hamilton Beach, Washington, WA, USA) and stored in screw-capped plastic tubes at −80 °C until further use. Protein content in the defatted flour samples was determined by Dumas combustion (Leco FP-428, Leco Corporation, St Joseph, MI, USA). Percent of protein was calculated from protein nitrogen using a conversion factor of 6.25.

To extract the lipids from the sausage samples [34 (link)], a 30 g sample was homogenized at 4 °C using a high-shear homogenizer (IKA homogenizer, Model T25 digital ULTRA-TURRAX, Germany), along with 210 mL of chloroform: methanol: distilled water mixture in a 60:120:30 ratio. The homogenizer was set to a speed of 10,000 rpm for a span of 1 min. Subsequently, 60 mL of chloroform was added to dilute the homogenate, which was then re-homogenized under the same conditions, but for a shorter period of 30 s. This dilution and homogenization step was repeated twice. Post homogenization, the mixture was centrifuged using a centrifuge (Sorvall, model RC 5C Plus, Asheville, NC, USA) at 4 °C for 10 min at 5000× g. The supernatant was carefully decanted into a separating flask. The chloroform layer was then transferred into a separate 250 mL Erlenmeyer flask and combined with 2–5 g of anhydrous sodium sulfate. After vigorous mixing, the solution was then decanted into a round-bottom flask using a Whatman No. 4 filter paper. Subsequently, the solvent was evaporated at 40 °C with a rotary evaporator (Eyela, Model N-100, Tokyo, Japan), and any remaining solvent was purged with nitrogen. The extracted lipid was then subjected to both TBARS and PV analyses.

The proteolysis activity was evaluated by measuring the free amino groups using the o-phthaldialdehyde (OPA) method of Church et al. [38 (link)]. Briefly, 2 g of fermented milk, and 1 mL of deionized water were mixed with 5 mL of 0.75 mol/L trichloroacetic acid (TCA) for 10 min, and filtered using Whatman No. 4 filter paper. Aliquots of 200 μL of the TCA filtrate and 4 mL of the OPA reagent were vortexed and reacted at room temperature (20 ± 2 °C) for 10 min. The absorbance at 340 nm was measured by a spectrophotometer (Aoyi, Shanghai, China). The free amino groups were calculated against the L-leucine standards (0–10 mmol/L).

Fusarium cultured for 4–6 days was inoculated into 20 ml of PDB liquid culture medium, and single C3 colonies of approximately 0.4 cm were added to the medium (the control group was not inoculated with single C3 colonies). The culture was shaken at 180 r/min for 24 h and then transferred to an incubator at 30°C for 4–5 days. The supernatant was filtered with Whatman No. 4 filter paper to obtain hyphae. The hyphae were washed with distilled water and dried in a Petri dish at 80°C for 24 h. Then, we determined the dry weight of the mycelia (m = m₁−m₂−m₃, m is the weight of the mycelia, m₁ is the weight of petri dish and filter paper after drying, m₂ is the weight of the Petri dish, and m₃ is the weight of the filter paper) and calculated the inhibition rate. Each treatment was repeated three times. We picked hyphae for observation under a microscope (Olymous BX53).

The embryo axes (0.5 g) of each variant were homogenized with 75 mL of acetonitrile-methanol-water (16:3:1, v/v/v) and filtered (Whatman no. 4 filter paper). Moniliformin (MON) was extracted and purified according to the methods described by Waśkiewicz et al. [24 (link)]. MON was quantified by high-performance liquid chromatography (HPLC) using a Waters 2695 apparatus (Waters, Milford, MA, USA) with a C-18 Nova Pak column (3.9 × 300 mm, 4 µm) and a Waters 2996 Array Detector (λ_max = 229 nm). Acetonitrile: water (15:85, v/v) buffered with 10 mL of 0.1 M K₂HPO₄ in 40% t-butyl-ammonium hydroxide in 1 L of solvent used as the mobile phase (flow rate  =  0.6 mL min⁻¹) [68 (link)]. Quantification of MON was performed by measuring the peak areas at the MON retention time according to the relevant calibration curve (the correlation coefficient for MON was 0.9990). The recovery for MON was 90%, the limit of detection was 1.0 ng g^−1, and the standard deviation was below 7%. All samples were injected in triplicate.