Origin 2019b

Lymecycline and fucosterol with GRB2 were rationally docked in our previous work. This complex was analyzed by all atom molecular dynamics (MD) simulations for 50 ns. All the simulations were carried out with Gromacs 2020.2 software package and gromos54a7_atb.ff force field. The force field parameters of fucosterol and Lymecycline was generated by ATB (https://atb.uq.edu.au/register.py). To ensure the total charge neutrality of the simulated system, the corresponding amount of sodium ions were added to the system to replace water molecules to produce solvent boxes of the appropriate size. Initial the energy of 50,000 steps of the whole system was minimized by (EM) at 300 K. Subsequently, the systems were equilibrated by position restraint simulations of NVT and NPT ensembles. Equilibrated systems were used to simulate a 50 ns no restraint production run. In the end MD analyses were performed, this includes root mean square deviation (RMSD), root mean square fluctuations (RMSF) and Hydrogen bond analysis. The data of RMSD, RMSF and Hydrogen bond analysis is generated by Origin2019b (https://www.originlab.com/).

Regarding centrifugation optimization procedures, using response surface methodology, STATISTICA v. 10 software was used to obtain the response surfaces. Also, an ANOVA analysis was performed, to determine significant factors (α = 0.05). To adjust the obtained rheological data to the Cross model, in viscosity determinations, Origin 2019b (OriginLab) software was used. All the remaining data were analyzed using Prism 5 (GraphPad) software, calculating the means and standard errors of the replicates. ANOVA was performed to determine significant differences between the means, using the Tukey test to compare more than two samples, and the T-test to compare only two samples, setting for both a significance level at p < 0.05.

Viscosity curves were carried out using a rotational rheometer Haake Mars Modular Advanced Rheometer System (Thermo Fisher Scientific, Massachusets, USA) associated with the refrigeration system (Peltier) Thermo Scientific Haake MarsIII Controller and to the air compression system Eheim professional 3. The PP60 probe (60 mm of diameter, rough surface) was used for the thick samples—puree, pomace, and snack/formulations—and the CC25 DIN Ti probe (concentric cylinders) was used for the other liquid samples. Viscosity curves were obtained in triplicate, with a 1.5 mm gap, at 20 ± 2°C, with shear rates ranging between 1 × 10⁻⁸ and 100 s⁻¹, stepping up every 35 s to ensure the steady-state on each shear rate. Data treatment was carried out using Origin 2019b (OriginLab) software, adjusting the obtained curves to the Cross model (Equation 4):
where η_∞ (Pa.s) is the infinite shear viscosity, η₀ (Pa.s) the zero-shear viscosity, k the consistency coefficient (Pa.s), m the Cross rate (dimensionless) constant, and ${\stackrel{\bullet }{\gamma }}^{.}$ The shear rate (s⁻¹).

The figures were prepared using Origin 2019b (OriginLab Corporation, USA). The contents of N and Mg in fruits and leaves were determined for 30 biological replicates and three technical repetitions; other indexes were determined for six biological replicates and three technical repetitions. Data were analyzed with SPSS 17.0 (Statistics software, version 20.0, IBM, USA) using one-way analysis of variance (ANOVA). The significant differences were considered at a probability level of P ≤ 0.05.

All data are expressed as the mean difference ±standard deviation. Differences between experimental groups were analyzed using Student’s t-test. The Pearson correlation coefficient was calculated to estimate the linear correlation between two variables. Statistical analysis was performed using Origin 2019b (OriginLab, America). All tests were 2-tailed, and P < 0 .05 was considered statistically significant.

Kinetic parameters were determined measuring NADH formation at 340 nm. Reaction mixes were prepared in 96 well plates for UV measurement by triplicate. For (+)‐endo‐1 a kinetic study: 0.5 μM of SrBDH1 (tetramer), 1 mM NAD⁺, (+)‐endo‐1 a in a range between 0.5 and 6 mM, 5 % DMSO, buffer Tris‐HCl 100 mM pH 9. For NAD⁺ kinetic study: 0.5 μM of SrBDH1, NAD⁺ in a range between 0.05 and 2 mM, 5 mM of (+)‐endo‐1 a, 5 % DMSO, buffer Tris‐HCl 100 mM pH 9. All the reactions were started adding NAD⁺ to the reaction mix. Absorbance at 340 nm was measured every 15 s for 30 min. The linear range of the curves was used to calculate the initial rates. Origin 2019b (OriginLab Corporation, USA) was used for the nonlinear fitting using Michaelis‐Menten model to obtain the kinetic parameters.
Specific activities were obtained in a similar way under the following conditions: 20 μM of the alcohol dehydrogenase (monomer) (AaADH2, PsBDH, SoBDH2, SoBDH3, SrBDH1 or SrBDH2), 2 mM NAD⁺, 2 mM of substrate, 1 % DMSO in Tris‐HCl 100 mM pH 9.