RSM-Modeling and Optimization of High Titer Functional Xylo-oligosaccharides Production by Edible Gluconic Acid Catalysis

Xylo-oligosaccharides have great value in food, feed fields. Previous studies have shown that organic acids catalyze the hydrolysis of xylan-rich sources for the production of xylo-oligosaccharides. In this study, gluconic acid of aldonic acid generated xylo-oligosaccharides via hydrolysis of xylan from corncob. In order to maximize efficiency of xylo-oligosaccharides production, the optimum conditions was ascertained by Box-Behnken design-based response surface methodology. The developed process resulted in a maximum xylo-oligosaccharides yield of 57.73% using 4.6% gluconic acid at 167 °C for 28 min, which was similar to the predicted value and fitted models of xylo-oligosaccharides production. The results showed that the reaction temperature was crucial to xylo-oligosaccharides production, and by-product yields (xylose and furfural) could be effectively controlled by both reaction temperature and time. In addition, 44.87 g/L XOS was achieved by decreasing the solid-liquid ratio. Overall, the described process may be a preferred option for future high concentration xylo-oligosaccharides production.


Raw Materials
Xylan extracted from corncob was obtained from Jiangsu province in China. The main components were determined according to the National Renewable Energy Laboratory and contained 69.15% xylan, 2.98% glucan, and 10.36% arabinan. D-Gluconic acid solution was purchased from the Shanghai Aladdin Biochemical Technology Co. Ltd (Shanghai, China).

Pre-experimental Design for Gluconic Acid Hydrolysis
The experiments were designed on the basis of the Box-Behnken Center combination test design principle. In order to optimize XOS yield, three factors were used as independent variables: temperature 130-170 °C, retention time 5-75 min, and GA concentration 2.5-20% (w/w). The independent variables and center point values are listed in Table 1. Eighteen experimental runs were conducted according to the Box-Behnken Design matrix.
One gram of xylan and 10 mL GA with different concentrations were mixed in a digestion tube and stirred. Then, the tube was placed into the multifunctional intelligent digestion instrument (GL-16, Greencarey Shandong), which was preheated to a set temperature and set to a specific time. When the reaction was completed, the digestion tube was immediately removed and allowed to cool naturally to room temperature. The solid and liquid were collected and separated by centrifugation. XOS content in the liquid was analyzed.

Methods of Analysis
Xylose and furfural were analyzed using high-performance liquid chromatography (HPLC) (Agilent 1260, USA) which has an Aminex Bio-Rad HPX-87 H column (Bio-Rad Laboratories, USA). The mobile phase of HPLC was 0.005 M sulfuric acid at a flow rate of 0.6 mL/min and kept working at 50 °C. GA, xylobiose (X2), xylotriose (X3), xylotetraose (X4), xylopentaose (X5), and xylohexaose (X6) produced in pretreatment were analyzed by a high-performance anion exchange chromatography (HPAEC) (Dionex ICS-3000, USA) which was equipped with a CarboPacTM PA200 column [27]. The mobile phase of HPAEC-PAD was 0.1 M sodium hydroxide and 0.5 M sodium acetate, and the flow rate was set at constant 0.3 mL/min. The yields of furfural, xylose, and XOS were calculated as following formula:

Statistical Analysis
Statistical software design expert (version 11.0) was used for regression analysis of experimental data and response surface graphs [28]. One-way analysis of variance (ANOVA) and Duncan's multiple range test (P < 0.05) were used to determine the statistical significance of the data. The relationship between independent variable and response variable was calculated by quadratic polynomial equation : where Y is the yield of the predicted XOS (%), A 0 is the constant term, X is the independent variables, and A i , A ii , and A ij are coefficients of linear, quadratic, and interaction parameters, respectively.

Production of XOS from Xylan Hydrolyzed by Gluconic Acid
Reaction temperature, retention time, and acid concentration are important factors affecting the degradation of xylan, as well as the selectivity of XOS production. Therefore, these three factors needed to be optimized by response surface methodology (RSM) to obtain the highest XOS yield. RSM is an effective statistical and analytical method, which only requires minimum experiments to determine the mathematical model and optimal conditions, and can be used to maximize the XOS yield. In this work, 1 g xylan was hydrolyzed with 2.5-20% GA at 130-170 °C for 10-75 min (Table 1). During acid hydrolysis, xylan was first degraded into saccharides with relatively high DP, which were further hydrolyzed into XOS, xylose, and furfural. Thus, after hydrolysis of xylan Xylose yeild(%)= Xylose content in hydrolyates (g) by GA, these products and by-products were simultaneously analyzed in order to evaluate the effects of different conditions (Fig. 1a-c). The highest XOS yield was 52.6% when xylan was hydrolyzed with 2.5% GA at 170 °C for 30 min. As shown in Fig. 1a, only 15.4% yield was obtained when the reaction was conducted at 130 °C with 10% GA for 30 min, with the yield rapidly increasing to 46.9% at 150 °C with 10% acid concentration after 30 min. Therefore, high temperature could accelerate the degradation of xylan to XOS under the same conditions. As known in Fig. 1, XOS with lower DP, such as X2 and X3, have higher XOS content at higher temperature, which may be caused by XOS degradation with higher DP into XOS with low DP at high temperature. Also, Fig. 1 shows that with increasing reaction temperature, retention time, and acid concentration, XOS was further degraded into xylose and furfural. Just as the furfural content was 0.25 g/L and xylose could reach 10.23 g/L under the condition of 10% GA at 170 °C for 20 min, which was much higher than the concentration of furfural and xylose obtained at 150 °C with 10% GA for 15 min. Moreover, under constant temperature, high acid concentration and long retention time promoted high xylose and furfural yield. Figure 1b shows that the concentration of xylose obtained with 20% GA for 20 min is 11.7 g/L, while that with 10% acid concentration was only 1.8 g/L for 15 min. Hence, xylan could be hydrolyzed efficiently to prepare XOS at high temperature, but xylose and furfural were also obtained. Thus, a kinetic study was conducted to gain greater insight into its degradation trend. Figure 2 shows the degradation trend of xylan with 5% GA at 170 °C. In the first 10 min, the distribution of X2 to X6 was relatively average, which may be caused by the random action of GA on β-1,4 glycosidic bond resulting in cleavage. As the time increased, X5 and X6 decreased, while X2 and X3 gradually increased, which was due to the continuous depolymerization of X5 and X6 with high DP to form XOS with lower DP. In addition, X5 and X6 content in all hydrolysates were lower, while the contents of X2 and X3 with low polymerization degree were higher, which was also preferred in food application [29].
The trend of by-products over time is shown in Fig. 2. Prolongation of retention time produces xylose and furfural reaching a concentration of 1.35 g/L and 16.42 g/L, respectively. The growth trend of by-products slowed down after 20 min of retention time, which decreased XOS yield after 20 min. Therefore, the acid hydrolysis conditions could be controlled both XOS and by-product yield. However, the design of optimal conditions was based on the reasonable model, which was verified.

Fitting Model of XOS Produced by Hydrolysis of Xylan
As listed in Table 1, the reaction temperature, retention time, and acid concentration are important factors affecting the production of XOS. When temperature was too high, XOS was further degraded into xylose and furfural, while at too low temperature, the reaction would take an undesirable length of time, which was not economically viable. Therefore, a suitable model was required to determine optimal conditions. On the basis of the fit summary reports generated by the statistical analysis software, the Box-Behnken design-based response surface methodology is an effective method that can effectively establish the mathematical model of acid hydrolysis and optimize the conditions. The regression equation fitted by Design-Expert 11.0 is as follows: where P 1 , P 2 , and P 3 are the reaction temperature, retention time, and GA concentration, respectively. That the model had P-value of 0.0001 was shown by ANOVA analysis, which indicates that the fitting model was in good agreement with the actual experiment. R 2 , the decisive coefficient, of 0.9598 indicates that the regression model was in good agreement with the model as well. Also, the value of adeq precision was 15.7234; hence, the fitting model was reasonable. The P-value of P 1 , P 2 , and P 3 were <0.0001, 0.0084, and 0.2006, respectively. P-value could reflect the order of importance of the factors affecting the hydrolysis of xylan by GA, which should be ranked as follows: reaction temperature > retention time > GA concentration, and the interaction of retention time and reaction temperature had the most important effect on the yield of XOS. Moreover, the 3D response surface generated by Design Expert 11 is shown in Fig. 3. According to Fig. 3a-c, the red area represents better conditions, in which the optimal conditions were 4.6% GA, 28 min, and 167 °C. Under these conditions, the contents of X2, X3, X4, X5, and X6 were 11.59 g/L, 10.57 g/L, 9.52 g/L, 4.61 g/L, and 3.62 g/L, respectively, and the yield of XOS reached 57.7%. The predicted yield of XOS was 54.3%, which was comparable to the actual XOS yield, which also verifies that the model could well predict XOS yield. After fine-tuning the established model, the kinetic profile could be studied.

Improved XOS Content by Increasing Solid-Liquid Ratio
High concentration XOS are easier to be collected and purified in industry, and the highest yield of XOS can be optimized based on response surface methodology. Therefore, the solid-liquid ratio was optimized to obtain higher concentration of XOS. Figure 4 shows the composition distribution of different solid-liquid ratios under optimal Fig. 2 The concentration of furfural, X1-X6 with 5% GA at 170 °C at different times conditions. The solid-liquid ratio of 1:7.5 effectively increased XOS concentration to 44.9 g/L, whereas the ratio of 1:7.5 improved the yield of XOS for subsequent industrial production. However, when the solid-liquid ratio was 1:5, XOS concentration was only 38.42 g/L, which did not increase but decreased. This is due to the gluconic acid solution cannot fully mix with xylan, resulting in incomplete hydrolyzation when coking and adhering to the inner wall of digestion tube at high temperature. Furthermore, when the ratio of solid to liquid was 1:10, the concentration of XOS decreased because XOS were diluted due to excessive liquid added. Therefore, for industrial production, solid-liquid ratio of 1:7.5 is recommended for pretreatment to obtain high concentration of XOS. Additionally, XOS content reached 5%, which could reduce the cost of subsequent purification. Overall, it was expected to produce high concentrations of XOS. A total of 44.9 g/L XOS could be produced by GA under optimized conditions.

Conclusions
This work presents a green, feasible pretreatment method that can effectively convert xylan into XOS, and RSM is employed to define the point wherein maximum production of XOS, which is achieved and proved experimentally. All results indicate that the yield of XOS depends on both acid concentration, time, and temperature, where 44.9 g/L XOS at a yield of 48.67% is produced with 4.6% GA at 167 °C for 28 min and