2.9. Ferric reducing antioxidant power (FRAP)
The FRAP reagent was prepared minutes prior to Rapamycin of the assay by mixing 10 mM TPTZ (1 mL, dissolved in 40 mM HCl), 20 mM FeCl3 (1 mL) and 300 mM acetate buffer, pH 3.6 (10 mL) (Benzie & Strain, 1996). The sample (200 μL) was mixed with FRAP reagent (1.5 mL). The reaction was incubated at 37 °C for 5 min and measured at 593 nm. The results are expressed as mg of trolox equivalents per gram of extract (dw).
2.10. High-performance liquid chromatography coupled with electrospray ionization and mass detection (HPLC-ESI-TOF)
2.11. Antimicrobial activity
2.12. Simplex lattice mixture design for optimization of antimicrobial and antioxidant response
A simplex lattice augment mixture design was utilized to determine the effect of interactions among nisin, seed extract, and peel extract on antimicrobial and antioxidant properties. Component proportions were expressed as a fraction of the mixture with a sum of one. Response variables comprised antioxidant (ORAC method) and antimicrobial (turbidimetric assay, measured as lag time) activities. Mixture design experiments were designed and analyzed using Statgraphics Centurion XV, version 15.2.6 (StatPoint Technologies, USA). A total of 10 combinations are presented in Table 1. The following polynomial equation of function Xi was fitted for each factor assessed at each experimental point, where Y is the predicted response and β1, β2, β3, β12, β13, and β23 are constant coefficients for each linear and non-linear interaction terms:equation(1)Y=β1X1+β2X2+β3X3+β12X1X2+β13X1X3+β23X2X3Y=β1X1+β2X2+β3X3+β12X1X2+β13X1X3+β23X2X3
The FRAP reagent was prepared minutes prior to Rapamycin of the assay by mixing 10 mM TPTZ (1 mL, dissolved in 40 mM HCl), 20 mM FeCl3 (1 mL) and 300 mM acetate buffer, pH 3.6 (10 mL) (Benzie & Strain, 1996). The sample (200 μL) was mixed with FRAP reagent (1.5 mL). The reaction was incubated at 37 °C for 5 min and measured at 593 nm. The results are expressed as mg of trolox equivalents per gram of extract (dw).
2.10. High-performance liquid chromatography coupled with electrospray ionization and mass detection (HPLC-ESI-TOF)
2.11. Antimicrobial activity
2.12. Simplex lattice mixture design for optimization of antimicrobial and antioxidant response
A simplex lattice augment mixture design was utilized to determine the effect of interactions among nisin, seed extract, and peel extract on antimicrobial and antioxidant properties. Component proportions were expressed as a fraction of the mixture with a sum of one. Response variables comprised antioxidant (ORAC method) and antimicrobial (turbidimetric assay, measured as lag time) activities. Mixture design experiments were designed and analyzed using Statgraphics Centurion XV, version 15.2.6 (StatPoint Technologies, USA). A total of 10 combinations are presented in Table 1. The following polynomial equation of function Xi was fitted for each factor assessed at each experimental point, where Y is the predicted response and β1, β2, β3, β12, β13, and β23 are constant coefficients for each linear and non-linear interaction terms:equation(1)Y=β1X1+β2X2+β3X3+β12X1X2+β13X1X3+β23X2X3Y=β1X1+β2X2+β3X3+β12X1X2+β13X1X3+β23X2X3