No 1 filter

Thiobarbituric acid reactive substances (TBARS) values were measured using the method of Mielnik et al. (2006 (link)) with some modification. Five gram sausage was homogenized with 30 mL of 7.5% aqueous solution of trichloroacetic acid using a Ultra‐Turrax (T‐18 digital Ultra‐Turrax, IKA Works GmbH & Co. KG, Staufen, Germany) homogenizer (9500 rpm, 60 s). Then the homogenate was centrifuged (Hettich Zentrifugen Mikro 220R, Andreas Hettich GmbH & Co., Tuttlingen, Germany) at 6000 rpm for 20 min at 20°C. After filtration (Whatman No.1 filter) using vacuum filter, 5.0 mL extract was transferred in a stopper test tube and 5.0 mL of 0.02 M aqueous thiobarbituric acid (TBA) was added. The test tubes were incubated in a water bath at 100°C for 35 min. Then the tubes were cooled under running water. Absorbance measurements were taken at 532 nm (Lambda 25 UV/Vis Spectrometer, Perkin Elmer, CT) against a blank sample prepared with 5 mL distilled water and 5 mL TBA solution. A standard curve was prepared with 1,1,3,3 tetraethoxypropane standards and TBARS values were expressed as mg malondialdehyde/kg of sausage.

One hundred grams of Acacia senegal leaves powder was macerated with petroleum ether (500 ml) at room temperature for 24 h as a first step. Afterwards, the mixture was filtered through the Whatman No.1 filter; the marc was dried and macerated again in 70% ethanol (V/V) overnight. The supernatant was collected, concentrated and frozen before being lyophilized to obtain the powder of A. senegal extract (HEASG).

The sericin and purple waxy corn cob extractions were performed, as previously described [13 (link)]. The sericin from silkworm cocoons (J108 strain) was obtained from Queen Sirikit Sericulture Center (Khon Kaen, Thailand) in January 2020. Sericin was extracted by autoclave. The cocoons were aged 42 days at collection. The silkworm cocoon was cut into small square pieces of 1 × 1 cm. Twenty-four grams of the cocoon material was added to 700 mL of purified water and autoclaved (Tomy-SX-700, Tokyo, Japan) at 120 °C, 2.5 bar for 60 min. Then, the mixture was filtered through a Whatman No. 1 filter. The filtrate was then freeze-dried at −85 °C, 0.595 bar (Labconco, Kansas, MO, USA), to obtain the sericin extract.
The purple waxy corn cobs (Thai purple waxy corn) were obtained from the Plant Breeding Research Center for Sustainable Agriculture (Khon Kaen University, Thailand) and extracted by maceration in 50% ethanol (ratio of powder to solvent 1:25), with stirring at 25 °C for 24 h. Then, the filtrate was collected. The residue was then reextracted twice following the same procedure. The filtrates were pooled, evaporated, and then freeze-dried at −85 °C, 0.595 bar (Labconco), to obtain the crude extract (purple waxy corn cob extract, PWCC) [13 (link)]. Both sericin and PWCC were stored and protected from the light at −20 °C.

To extract antifungal compounds, strain SCA3-4 was inoculated in a 5-L Erlenmeyer flask containing 1 L of fermentation broth (15 g of corn flour, 10 g of glucose, 0.5 g of K₂HPO₄, 0.5 g of NaCl, 0.5 g of MgSO₄, 3 g of beef extract, 10 g of yeast extract, 10 g of soluble starch, 2 g of CaCO₃, pH 7.2–7.4). The flask was cultured in a rotary shaker (150 rpm) at 28°C for 7 days. The fermentation broth was extracted with an equal volume of ethyl acetate. The mixture was filtered through a Whatman No. 1 filter and shaken vigorously in a separating funnel. Then, the collected organic solvent extract was evaporated using a rotary vacuum evaporator (EYELA, N-1300, Japan). The crude extract was dissolved in 10% dimethyl sulfoxide (DMSO) with a final concentration of 20.0 mg/ml. After filtering through a 0.22-μm sterile filter (Millipore, Bedford, MA, United States), the crude extract solution was kept in a refrigerator at 4°C for the antifungal bioassay and the GC-MS analysis.

Aliquots of 5 g of ground samples were weighed into a suitable container and 25 mL of distilled water was added and shaken for three min. Whatman No. 1 filter was used to filter the extract. Then 50 µL of standard or sample were added to the wells in duplicate. Then each well received 50 µL of the conjugate. Subsequently, each well received 50 µL of the anti-DONantibody, which was carefully mixed by hand shaking the plate and incubated for 30 min at room temperature (20 to 25 ℃) in the dark. After that, the well contents were discarded, and the assay was continued as mentioned previously for AFs.

For each experimental run, 40.0 g of FA was used with the urea previously dissolved in 95% ethanol (1:3.7 ratio based on the weight of the urea). The urea/FA content ratio was established according to each essay of the experimental design (Table 2). The FA/ethanolic urea solution mixture was heated to 60 °C in a low reflux system with constant stirring until completely dissolved. The solution was cooled to room temperature and the subsequent crystallization stage. At the end of this stage, the urea-FA adducts were also separated from the uncomplexed fraction by vacuum filtration with a Whatman No. 1 filter. The uncomplexed fraction was diluted with 400 mL distilled water and acidified to pH 4.5 with 6N HCl. The uncomplexed fraction was washed twice with 200 mL hexane each time in a decantation funnel and the hexane phases obtained were filtered in the presence of anhydrous Na₂SO₄. The solvent was evaporated in vacuum at 47 °C on a rotary evaporator. The EPA + DHA concentrate product was stored at −80 °C without adding antioxidant.

Ground samples (5 g) and 25 mL of 70% methanol were mixed for 10 min at room temperature by vortexing and filtered through a Whatman No. 1 filter or centrifuged (10 min / 3500 g / room temperature). Then, 100 µL of the filtrate/supernatant was diluted with 600 µL distilled water. The wells were then filled with 50 µL of standard or sample in duplicate together with 50 µL of the conjugate. Then, 50 µL of the antibody was added to each well, and the plate was gently shaken for mixing and then allowed to sit at room temperature (20 to 25 ℃) in the dark for 30 min. After incubation, the well contents were discarded and the microwell holder was tapped upside down strongly (three times) on absorbent paper. Then wells were washed with 250 µL wash buffer 3 times after which 100 µL of substrate/chromogen was added to each well, gently mixed by hand shaking the plate, and incubated for 15 min at room temperature (between 20 and 25 ℃) in the dark. The stop solution (100 µL) was then pipetted into each well followed by manual shaking of the plate. After 30 min, the extinction was determined at 450 nm.