3.3. Biological Assay

In order to determine the antioxidant activity, 0.1 g of each inclusion complex was placed in a centrifuge tube containing 1.5 mL of a solution of NaCl (10%)–methanol (1:1). The sample was then vortexed for 10 min, and 8 mL of a hexane–acetone (1:1) solution was added and centrifuged at 10,000 rpm for 15 min. The antioxidant activity was measured using the 2,2′-azinobis-3 ethylbenzthiazoline-6-sulphonate (ABTS) radical. Trolox was used as a standard reference. The ABTS radical scavenging assay was determined following the method of Re et al. [43], with some modifications; the blue–green ABTS radical cation chromophore (ABTS ●⁺) was prepared by reacting ABTS stock solution (7 mmol) with potassium persulphate (2.45 mmol) and allowing the mixture to stand at room temperature in the dark during 16 h. The solution was diluted with ethanol to obtain an absorbance of 0.700 ± 0.02 at 734 nm in a Genesys 5 (spectronic) spectrophotometer.

The antioxidant activity was calculated as a percentage of inhibition according to the following Equation (2):

Ab represents the absorbance of the control (without test oil), and as represents the absorbance of the test oil. A calibration curve was determined for Trolox standard for the radical (ABTS) at a concentration range of 10–160 μmol. The antioxidant activity values were expressed as μmol Trolox equivalent (TE)/mg oil.

The antifungal assay was performed according to the semi-quantitative method reported by Rosas-Taraco et al. [44] and Sánchez et al. [45] with some modifications. The strains of Aspergillus flavus and A. niger kindly provided by Dr. Eduardo Sánchez from Chemistry Laboratory of UANL, were used to determine the Fungal Inhibitory Concentration (FIC) and were maintained on potato dextrose agar (PDA) slants (Difco). Inoculum was prepared from PDA cultures incubated for seven days at 28 °C until they sporulated. Fungal conidia were collected with a loop and suspended in saline solution containing 0.05% of Tween 20 and adjusted to (1 × 10⁴) by microscopy in a Neubauer chamber. An amount of 200 μL of this suspension was added into tubes containing 1.8 mL of PDA broth plus inclusion complexes in different concentrations, ranging from 1.25 to 10 mg/mL. The tubes were incubated at 28 °C for 5 days. The antifungal activity was assigned according to a symbolic scale (+) and arbitrary from 0 to 3, where (-) represents total absence of growth, (+) represents little growth, (++) represents medium growth and (+++) represents high growth compared to the control growth of the pure strain without inclusion complexes. The FIC was defined as the minimum concentration that did not allow the visual mycelial growth of the fungus. Analyses with not encapsulated COEO were carried out to compare the activity before and after encapsulation. β–CD without COEO was used as negative control.

Antibacterial activity of inclusion complexes was tested against Salmonella typhi (ATCC 19430) and Listeria monocytogenes (ATCC 13061), which were kindly provided by the Laboratory of Sanitary Microbiology, FCB, UANL. These strains were selected for their importance to the food industry and are representatives of pathogenic bacteria, commonly occurring in various food products. The strains were stored at −80 °C in brain heart infusion (BHI) with 20% v/v glycerol (Difco Laboratories, Sparks, MD, USA). Fresh cultures (18 h) were obtained in Mueller–Hinton broth or agar (MH, Difco Laboratories). Listeria monocytogenes was cultivated on trypticase soy broth or agar (TS, Difco Laboratories). The antimicrobial activity was determined through a dilution method according to Castillo et al. [46]. The minimum inhibitory concentration (MIC) was defined as the lowest concentration of inclusion complex that decreased growth by about 90% of the bacterial population after 24 h of incubation [34,47]. Aliquots (10 μL) of fresh and adjusted cultures (0.1 OD 600nm) (1 × 10⁶) of each bacterium were added separately in tubes containing 2 mL of MH broth plus different concentrations (1.25, 2.5, 5, and 10 mg/mL) of the inclusion complexes (5x, 10x, and 20x); after that, they were incubated at 37 °C for 24 h. Subsequently, plate counts were made using the Miles and Misra method. For Listeria monocytogenes, the same procedure was used, but TS medium instead of MH was used. The control was the same preparation without inclusion complexes. β–CD was used as a negative control while previous results of MIC of free COEO were used as a positive control [11].