Beef extract

Choice of neutralizer was dependent upon virus tested. For testing against hNoVs, the products were neutralized using PBS supplemented with 10% beef extract (Thermo Fisher), 0.04% Tween 80 (Sigma, St. Louis, MO), and 0.2% sodium thiosulfate (Sigma). The neutralizer for TuV testing was M199 media (Corning, Corning, NY), supplemented with 10% FBS and 0.2% sodium thiosulfate. M199 was further modified by addition of HCl when evaluating the EtOH-based product to achieve a pH of ~7 to 8 following product neutralization.

For the seed culture, Bacillus sp. JD3-7 colonies were placed in a culture tube containing 4 mL of nutrient broth (peptone [ThermoFisher, Waltham, MA, USA] and beef extract [ThermoFisher]) and cultured for 16 h. Then, 100 mL of nutrient medium was placed in a 500 mL flask, adding 0.2% of the total volume to Bacillus sp. JD3-7 cells were added. The flask was then incubated for 16 h, shaking at 150 rpm in an incubator at 30 °C. The culture medium was then centrifuged at 3577× g for 15 min; only the bacterial pellet was recovered. The obtained pellet was washed twice with PG buffer (50 mM phosphate buffer, 2% glycerol, pH 7.2). The washed bacterial pellet, PG buffer, and 50 mg of LT were then placed in a 500 mL flask and incubated for 72 h, shaking at 150 rpm in an incubator at 30 °C. After incubation, centrifugation was performed at 4416× g for 10 min using a centrifuge, and the divided supernatant was freeze-dried and powdered. For more accurate separation and identification, ethyl acetate (EA, DAEJUNG, Busan, Korea) fractionation was performed. The sample was dissolved in purified water, and twice the amount of EA was added to separate the layers. The water layer and the EA layer were recovered separately and repeated 3 times. The separated layers were each concentrated and sampled.

For use as an inoculum, B. amyloliquefaciens KCTC 13,588 was cultured by placing colonies from agar plates in 4 mL of nutrient broth (3 g/L of beef extract (ThermoFisher, Waltham, MA, USA) and peptone (ThermoFisher) 5 g/L) in a culturing tube, and 0.2% of the inoculum of B. amyloliquefaciens KCTC 13,588 was added to a 500-mL flask containing 100 mL of nutrient medium and cultured at 37 °C and 200 rpm for 18 h. Culture broth was centrifuged at 5000 rpm for 15 min, and the supernatant was discarded. The remaining cells were washed with phosphate glycerin buffer (PG buffer) containing 2% v/v glycerin in 50 mM of sodium phosphate at pH 7.2. After adding 5 mL of PG buffer, cells were resuspended through vortexing and centrifuging at 5000 rpm for 5 min. After discarding the supernatant, the washing was repeated. Formononetin was added to a final concentration of 4 mg/mL. Media were further incubated at 30 °C and 200 rpm for 48 h. After centrifugation at 5000 rpm for 10 min, the supernatant was concentrated using an evaporator.

Feces positive for norovirus GI, norovirus GII, and HAstV (courtesy of Dr. Buesa from Hospital Clínico Universitario, University of Valencia, Spain) were resuspended (10%, wt/vol) in phosphate-buffered saline (PBS) containing 2 M NaNO₃ (Panreac, Spain), 1% beef extract (Conda, Spain), and 0.1% Triton X-100 (Thermo Fisher Scientific, Spain) (pH 7.2), vortexed and centrifuged at 1,000 × g for 5 min. The supernatants were extracted, the RNA stored at -80°C in aliquots to be used as positive amplification controls. The human RV strain Wa (ATCC VR-2018), the cytopathogenic HM-175 strain of HAV (ATCC VR-1402), and mengovirus vMC0 (CECT 100000) were propagated in MA-104, FRhK, and HeLa cell monolayers, respectively. Semipurified stocks were thereafter produced in the same cells by low-speed centrifugations of infected cell lysates (3,000 × g for 20 min). RNA extracted from infected cell lysates was used as positive amplification control and mengovirus (MgV) was used as process control as suggested in ISO 15216-2:2019 (microbiology of the food chain) for sample concentration validation (Randazzo et al., 2019 (link)).

Fecal sample containing HEV genotype 3f (kindly provided by Dr. Alcaraz, Hospital Clínico Universitario, Valencia, Spain) was suspended (10%, wt/vol) in phosphate-buffered saline (PBS) containing 2 M NaNO₃ (Panreac, Spain), 1% beef extract (Conda, Spain), and 0.1% Triton X-100 (Fisher Scientific, United States) (pH 7.2), vortexed, and centrifuged at 1000 × g for 5 min. The supernatant was stored at −80°C in aliquots.
The cytopathogenic HM-175/18f strain of HAV (ATCC VR-1402) was propagated and assayed in FRhk-4 cells (kindly provided by Prof. Bosch, University of Barcelona, Spain). HAV infectivity was calculated by determining the 50% tissue culture infectious dose (TCID₅₀) after visual inspection of cells for presence of cytopathic effect with eight wells per dilution and 20 μl of inoculum per well using the Spearman–Karber method (Spearman, 1908 (link); Kärber, 1931 (link)).