Q2. What was the method for determining the optimal levels of FTase activity?
Since the three factors, named inoculum, packing density and temperature, were found to have a significant effect on the response, their optimal levels were established through a response surface methodology (RSM).
Q3. What was the design used for the FTase synthesis?
A design randomized complete block was also used in the experiments corresponding to application of FTase on aguamiel for the FOS synthesis.
Q4. How much sucrose was used in the FTase synthesis?
Only 43.8% of the initial sucrose was transformed in FOS with yield of product/total substrate equal to0.30 g/g and productivity of 0.097 g/L min.
Q5. How many g/L of sucrose was used in the present study?
In the present study were obtained 1347 U/L at 24 h of fermentation with initial concentration of sucrose of 35 g/L while Sangeetha et al. (2004) used 600 g/L of sucrose as substrate.
Q6. What was the optimal time for the expression of the enzymes?
The first kinetic study showed that 24 h of fermentation was the optimal time for the expression of fructosyltransferase and hydrolase enzymes.
Q7. How long did the enzymatic synthesis take?
In this part of the study, the FOS enzymatic synthesis was carried out for 1 h using aguamiel as substrate and lyophilized enzymatic extract.
Q8. What is the alternative for aguamiel?
Maximum yield, 22.6 g/L, was obtained at 24 h of fermentation with a productivity of 0.94 g/L h. FOS production using aguamiel of maguey as culture medium is a good alternative because aguamiel is a economic by-product, besides, Solid State Fermentation is a bioprocess inexpensive and easy to operate.
Q9. What was the condition for FTase activity assay?
For FTase activity assay, 100 lL crude enzymatic extract were mixed with 900 lL sucrose (4% in acetate buffer, pH 5.0, 50 mM) and incubated at 30 C for 20 min in a water bath.
Q10. What was the maximum activity of the fsf enzyme?
The maximum fructosyltransferase activity was at 42 h; however, hydrolytic activity was also present during fermentation,increasing significantly between 24 and 48 h.
Q11. How much sucrose is used in the enzymatic synthesis?
Several studies have focused in the FOS production obtaining yields of 50–60% from initial sucrose, but they particularly using high levels of sucrose between 15 and 60%, while in this work only is used the sucrose present in the aguamiel (35 g/L) that corresponds to 3.5% (Sangeetha et al., 2004, 2005; Mussatto et al., 2009).
Q12. How long did the fermentation process take?
Fermentation process was carried out for 48 h and samples were withdrawn at regular time intervals (0, 18, 24, 30, 42 and 48 h of fermentation).
Q13. What was the method used for the analysis of aguamiel?
Analysis of aguamiel consisted in the determination of total sugars (TS) by method Anthrone (Dreywood, 1946; Pearson et al., 1984 and Campana et al., 2006), reducing sugars (RS) by the method previously reported by Miller (1959) using 3-amino-5-dinitrosalicylic acid reagent with some modifications (Gonçalves et al., 2010), protein by Bradford’s method (Bradford, 1976), lipids, ashes (i.e. mineral content), total solids, moisture and pH using the AOAC methods (AOAC, 1990) and degrees brix and density with a densitometer (Anton Paar, DMA 35N, Austria).
Q14. What was the optimal time for the FTase activity?
24 h of fermentation was selected because hydrolytic activity after this time was major than fructosyltransferase activity.
Q15. What is the significance of the results obtained?
The results obtained indicate that the aguamiel represents economical alternative for the FOS and enzyme FTase industrial production.
Q16. What was the FTase activity of the crude extract?
The regression coefficients for linear, quadratic and interactions for each variable were determined and adjusted to a polynomial second order equation (Eq. (1))Y ¼ b0 þ b1X1 þ b2X2 þ b1X21 þ b2X22 þ b1X1X2 þ b2X1X2 ð1Þ where Y = fructosyltransferase activity (FTase in U/L of crude extract), X1 and X2 are the significant variables (temperature and packing density, respectively) and bi is the regression coefficient for each factor.
Q17. What is the effect of the pH on the synthesis of FOS?
Sangeetha et al. (2004) studied the FOS production by FTase from A. oryzae CFR 202 and obtained maximum FOS of 52% after 8 h of reaction, but high levels of sucrose were used as substrate (600 g/L).
Q18. What was the effect of the temperature and packing density on the FTase activity?
An analysis of variance (ANOVA) (Table 5) revealed that incubation temperature (X2) and packing density (X3) had a linear effect statistically significant at a 95% confidence level on the FTase production.