Benchtop freeze dryer

We used β-propiolactone (BPL, Sigma-Aldrich, St. Louis, MO, USA) for RABV inactivation. In brief, BPL was diluted in a chemical hood using cold, sterile water to 10% (v/v), and added to RABV ERA-2GnRH (4.0 × 10⁸ ffu/mL) to a final concentration of 0.04% (v/v). After incubation on ice for 18 h, the virus solution was further incubated in a water bath at 37 °C for 2 h. The BPL-killed virus was used immediately for lyophilization, or stored at −80 °C. For lyophilization of inactivated ERA-2GnRH virus, a benchtop freeze dryer from Labconco was used (Labconco, Fort Scott, KS, USA). One part stabilizer BSA at 10% (Sigma-Aldrich, St. Louis, MO, USA) was added to 19 parts of ERA-2GnRH, and the formula was divided into 2 mL aliquots per 5 mL vial, and was freeze-dried O/N according to the manufacturer’s program. The dry vaccine was capped under vacuum and stored at −80 °C.
For vaccine formulation in hydrogel, first a 1.2% chitosan solution in 0.1 M HCl was prepared using low molecular weight chitosan (Sigma-Aldrich, St. Louis, MO, USA). Two vials of dry vaccine ERA-2GnRH (~ 8.0 × 10⁸ ffu before inactivation) were loaded gradually into 8 mL of chitosan solution under magnetic stirring, making a vaccine at 1.0 × 10⁸ ffu/mL of hydrogel. The gel vaccine was continuously stirred at 4 °C until it was administered to mice.

SGID was carried out on the hempseed protein samples according to Walsh et al., [36 (link)]. The samples (100 mL, 2% (w/v) protein) were incubated in a water bath (37 °C, 30 min). The pH was adjusted to pH 2 using 1 M HCl. Following addition of pepsin (2.5% (w/v) protein), gastric digestion took place for 90 min before the sample was heated to 90 °C for 20 min, deactivating the enzyme. The pH of the sample was then adjusted to pH 7.5 using 1 M NaOH. Intestinal digestion occurred with the addition of Corolase PP (1% (w/v) protein) and incubation for 150 min at 37 °C before deactivation. The pH values were kept constant throughout each digestion phase using a pH stat (Metrohm 902 Titrando pH-STAT, Herisau, Switzerland). The samples containing digested protein were then freeze-dried (Labconco benchtop freeze-dryer, Kansas City, MO, USA). Therefore, three samples were obtained from each hempseed protein extract: a sample prior to digestion, a sample that had undergone simulated gastric digestion and a sample that had undergone simulated gastric and intestinal digestion. These were subsequently characterized for their degree of hydrolysis (DH%), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and gel-permeation high-performance liquid chromatography (GP-HPLC) profiles, in vitro antioxidant activity, and lipase-inhibitory activity.

Isolation of occidiofungin
was performed as previously reported with some modifications.^{8 (link)} The producing strain, B. contaminans MS14, was grown in modified minimal M9 media with the glucose carbon
source substituted for asparagine (1 g/L). The final pH of the medium
was around 6.9. A 10% inoculum with an O.D.₆₀₀ between
0.6 and 0.8 was made before incubating the culture at 28 °C for
3 to 4 days. Cell free extracts of the culture were passed through
a 0.2 μm filter before preparative HPLC purification. A 5–7
mL sample containing occidiofungin (∼30 mg) in 35:65 (acetonitrile/water),
was loaded onto a SinoChrom ODS-BP 5 μm 20 × 150 mm² column using a CHEETAH Preparative HPLC system (Bonna-Agela,
QBH P100). The mobile phase consisted of acetonitrile (A) and double-distilled
water (B), both with 0.1% formic acid. The gradient was set up as
follows: held at 5% A for 5 min, from 5% to 50% A over 11 min, held
at 50% A for 10 min, from 50% to 90% A over 10 min, and re-equilibrated
by running at 5% A for 5 min. The occidiofungin fraction eluted out
during the 50% A isocratic hold. The preparative HPLC fractions were
checked by mass spectrometry analyses before the samples were freeze-dried
on a benchtop freeze-dryer (Labconco; Kansas City MO). A stock of
occidiofungin at 1 mg/mL in 35:65 (acetonitrile:water) was prepared
for additional chromatography and mass spectrometry analyses.

Maceration was used to extract active components from dried and ground Arthrocnemum macrostachyum samples. Three grams of biomass were added to 100 ml solvent. Two solvents were used for extraction: water and aqueous 50% ethanol (v/v). The biomass and solvent were macerated for 24 h with a speed of 230 rpm using STIK rotary shaker at room temperature. After extraction, the solvent was separated from the solid biomass via vacuum filtration using Supertek 47 mm filtering glass apparatus, Grade 4. Three batches of filtrates were prepared from each crude extract. The filtrates were either dried in the oven at 40 °C (72 h for water – 60 h for aq. EtOH) or 60 °C (48 h for water – 40 h for aq. EtOH) or lyophilized using LABCONCO FreeZone 4.5 Liter (− 100 °C) Benchtop Freeze Dryer for (36 h for water and aq. EtOH). All extraction and drying steps were performed in triplicate.

The plant leaves were dried at room temperature and powdered using a hammer mill (Ika Scientific, Germany) to yield approximately 2 mm mesh size powder. The leaves of each plant were soaked (1:10 w/v) in methanol, ethyl acetate, dichloromethane and distilled water respectively in 200 ml Schott Duran bottles. The bottles were placed in in mechanical shaker (IKA Scientific, Model MF 10 B, Germany) at 100 rpm for 48 h to extract the phytochemicals. The extracts were filtered through a Whatman No.1 filter paper, and then concentrated and dried using a Buchi rotoary evaporator. The water extracts were dried using a bench top freeze dryer (Labconco Corporation, Kansas City, Moussouri, USA) after being frozen at 20 °C. All the solvents used were of AR grade and obtained from Merck, South Africa.