Preparation for shaddock skin polysaccharide derivatives by response surface method

The derivation of polysaccharide has an important impact on its properties. The preparation process of phosphorylated-shaddock skin polysaccharides (SSP) and acetylated-SSP was optimized by the response surface method. The constructed model was accurate and reliable in predicting the substitution of acetylated-SSP and the phosphate content of phosphorylated-SSP. This method was simple and easy to operate, which provided a basis for the preparation of a large number of derivatives.


Single-factor experimental analysis
From Fig. 1a, it could be learned that the degree of acetylation substitution showed an increasing trend when the reaction time was increased from 1.5 h to 3.0 h.However, when the reaction time reached 3 h, the degree of acetylation substitution showed a significant decreasing trend.It might be that with the increase of reaction time, the collision probability between the reactants increases, leading to the degradation of some polysaccharides, and thus affecting the degree of acetyl substitution of acetylated-SSP 8 .From Fig. 1b, it could be seen that the increase in the amount of acetic anhydride made the SSP dissolve more fully, and the constant contact between SSP and acetic anhydride eventually led to an increase in the degree of substitution, but when the percentage of acetic anhydride exceeded a certain range, the degree of acetyl substitution started to decrease, which was most likely due to the increase in the side reaction of acetic anhydride 9 .As shown in Fig. 1c, the degree of substitution tended to increase and then decrease as the reaction temperature increases.This trend might be because an appropriate temperature increase accelerated the raw material's swelling, allowing the polysaccharides and acetic anhydride to react more fully with each other.Therefore, when the temperature increased, the degree of acetyl substitution also increased.However, when the temperature was too high, the acetic anhydride in the reaction would be hydrolyzed and SSP could only react with part of the acetic anhydride, so the acetyl substitution decreased 10 .
From Fig. 1d, it can be learned that the phosphate content of phosphorylated-SSP did not change much when the ratio of sodium tripolyphosphate and sodium trimetaphosphate was 1:6 to 4:3.This is most likely because sodium tripolyphosphate and sodium trimetaphosphate have a mutually inhibiting effect.When the mass ratio of phosphorylation reagents was 4:3 to 6:1, the phosphate content of phosphorylated-SSP gradually increased with the increase of the proportion of sodium tripolyphosphate, which indicated that an appropriate increase of the proportion of sodium tripolyphosphate could promote phosphorylation.As shown in Fig. 1e, when the temperature is between 30℃ and 80℃, the content of the phosphate group increases gradually with the increase in temperature, which may be because the increase in temperature enhances the polymer bond activity of SSP 11 .From Fig. 1f, it is known that the phosphate content increased with increasing reaction time when the reaction time was from 1 to 5 h.This may be because the contact between the phosphorylation reagent and the SSP molecules was more adequate when the reaction time was properly extended, so it made the phosphate content increase.When the reaction time was between 5 and 6 h, the phosphate content showed a decreasing trend.It may be because the reaction time was too long, which leads to phenomena such as breakage or curling of the sugar chain of the polysaccharide 12,13 .

Model fitting and statistical analysis
Based on the preliminary single-factor experimental results, the three parameters of reaction time, material ratio and reaction temperature were further investigated using response surface analysis to obtain the optimum acetylation conditions.The design matrix and corresponding experimental results are shown in Table 3, and the Design-Expert software (version 8.0.6) is used for data analysis.The response variable of the degree of acetylation substitution can be described by the following second-order polynomial equation: Degree As can be seen from Table 4, F = 475.25 with a P-value < 0.0001, which indicates that the linear relationship between the dependent variable and the three independent variables is good and that the model reaches an extremely significant level.The misfit term of the model was used as a measure of the measurement data that it cannot represent within the experimental range, and the P-value for the misfit term in this experiment was 0.1325 > 0.05, which is insignificant compared to the pure error.This indicates that other factors have little effect on the acetylation substitution.The coefficient of variation of the equation CV = 1.18%, which indicates that   5).All three independent variables examined in the model affected the degree of substitution of the SSP acetylation modification.The order of influence was A (reaction time) > B (material-liquid ratio) > C (reaction temperature).The effect of BC on the degree of substitution reached an extremely significant level in the interaction term, while the effect of AC and AB on the degree of substitution was not significant.In the quadratic term, the effect of the quadratic term on the degree of substitution for all three factors was at an extremely significant level.Model fitting and statistical analysis were carried out using Design-Expert 8.0.6software to regress the phosphate content of the samples from the 17 specimen points obtained in Table 6 and the relationships between the response values and the factors were obtained as follows: The F-value and P-value can be used to test the significance of each coefficient and to assess the strength of the interaction between each factor.As can be seen from Tables 7 and 8, the F-value of the model is 274.83 with a P-value < 0.0001, which indicates that the regression model reaches an extremely significant level and that the    linear relationship between the dependent variable and the three independent variables is good.In addition, the P-value for the misfit term was 0.7164 > 0.05, indicating that the difference between the model and the test values was small.The equation coefficient of variation CV = 1.96%, the coefficient of determination R 2 = 0.9972 and the correction coefficient Adj R-Squared = 0.9935 indicate that the fit between the values predicted by the model and those of the actual test is excellent and adequate to represent the true relationship between the independent and response variables.The order of influence of the three independent variables examined in the model on the phosphate content of phosphorylated-SSP was D (phosphorylated reagent mass ratio) > E (temperature) > F (time).In the interaction term, the effect of DE on phosphate content reached an extremely significant level, while the effects of DF and EF on phosphate were not significant.Otherwise, the secondary terms of all three factors had an extremely significant level of effect on phosphate content.

Analysis of the interaction of the model factors
The shape of the surface plots can be used to examine the important interaction of each element, and the slope degree of the surface has a positive correlation with significance 14 .If the response surface has a steeper slope and the contours are tight, it indicates that the interaction between the two factors has a significant effect on the response value 15 .If the surface is gentler and the contour lines are loose, it indicates that the interaction between the two factors has no significant effect on the response value.As can be seen from Fig. 2, the interaction between B and C, D and E forms the steepest slope of the surface, indicating that the interaction between the two groups of factors has the most significant effect on the response values.This is consistent with the ANOVA results.

Determination and validation of optimum process conditions
The predicted optimal preparation solution based on the software and the actual measured results are shown in Tables 9 and 10.After analysis, the difference between the predicted and actual results of acetylation substitution is only 1.4%, and the difference between the predicted and actual values of phosphate content is 0.187%, which proves that the constructed model is accurate and reliable in predicting the substitution of acetylated-SSP and the phosphate content of phosphorylated-SSP.

Conclusion
The derivation of polysaccharides has an important impact on their properties.Using SSP as the starting raw material, the preparation method of SSP derivatives was systematically studied.At the same time, the response surface method was used to optimize the above preparation process and the expected effect was achieved.This preparation method provided a basis for the application of SSP.
www.nature.com/scientificreports/ the proportion of unexplained total variation is small, and the experimental value is reliable.The coefficient of determination R 2 = 0.9984 and the correction coefficient Adj R-Squared = 0.9963 indicate a very good fit between the values predicted by the model and the actual experimental values (Table

Figure 1 .
Figure 1.Effect of each single factor experiment on acetylated-SSP substitution and phosphorylated-SSP phosphate content.

Figure 2 .
Figure 2. Response surface and contour plots of the interaction of the experimental factors.

Table 1 .
Box-Behnken test factors and levels of acetylated-SSP.

Table 2 .
Box-Behnken test factors and levels of phosphorylated-SSP.

Table 3 .
Three-variable encoding level and response value of SSP acetylation modification based on BBD.

Table 6 .
Three-variable encoding level and response value of SSP phosphorylation modification based on BBD.

Table 8 .
Reliability analysis of regression model.

Table 9 .
Validation of response surface results for acetylation modification.

Table 10 .
Validation of response surface results for phosphorylation modification.