Two-stage Enzymatic Hydrolysis of Soybean Concentrated Phospholipid to Prepare Glycerylphosphorylcholine: Optimized by Response Surface Methodology

A two-stage enzymatic hydrolysis method, in which phospholipase A 1 (PLA 1 ) was added after phospholipase A 2 (PLA 2 ) was added for a certain time, was successfully carried out to prepare glycerylphosphorylcholine (GPC) from soybean concentrated phospholipid. Effects of reaction variables on hydrolysis reaction were optimized using response surface methodology, and the optimal conditions were as follows: PLA 2 load of 1.25%, PLA 1 load of 0.70%, substrate concentration of 13%, reaction temperature of 41°C, and stirring rate of 680 rpm. Under the optimal conditions, the GPC yield reached 83.07%, which is close to the predicted value by the fitted model. This paper not only provides an efficient and low-cost method to prepare GPC, but also improves the high-value utilization of soybean concentrated phospholipid.

cient in theory due to the occurrence of acyl migration 20 . Therefore, some studies have attempted to prepare GPC under the concerted catalysis of phospholipase and lipase, but the reaction time was quite long 11 . In theory, PLA 1 and phospholipase A 2 PLA 2 are sn-1 and sn-2 specific phospholipases, the combinational catalysis of which is effective for GPC preparation 21,22 . According to our previous study, compared with the concerted catalysis of PLA 1 and PLA 2 , the catalytic efficiency of the method, in which PLA 1 was added after PLA 2 was added for a certain time PLA 2 A 1 , was more beneficial to improve the GPC yield and shorten the reaction time 23 .
In terms of GPC preparation, high-purity soybean powder phospholipid or lecithin phospholipid is generally used as raw material 24 , which leads to high cost. In comparison, soybean concentrated phospholipid SCP , which is a primary by-product of soybean oil processing, has high yield and low price. Moreover, phospholipid is quite unstable when exposed to air or sunlight, and is easily oxidized and rancid. The oil in SCP can prevent phospholipid from oxidation and rancidity, which is beneficial to the storage 25 . Therefore, the preparation of GPC from SCP can not only reduce the production cost and broaden the comprehensive utilization of SCP, but also improve the oxidative stability and thermal stability of phospholipid.
In this paper, the processing conditions of GPC preparation with the PLA 2 A 1 method were optimized with SCP as the raw material and GPC yield as the result index. The effects of reaction time, reaction temperature, stirring speed, substrate concentration, PLA 1 load, and PLA 2 load on the preparation of GPC were investigated and evaluated using response surface methodology RSM .

Materials
SCP was supplied by COFCO Jiayue Co., Ltd. Tianjin, China . PLA 1 was purchased from Novozyme Biotechnology Co., Ltd., and PLA 2 was purchased from DuPont Danisco Co., Ltd. GPC standard purity ≥ 98 was purchased from Sigma-Aldrich Chemical Co., Ltd. Chloroform and methanol were chromatographic grade Kermio Chemical Reagent Co., Ltd., Tianjin, China . All other solvents were of analytical grade Tianjin Chemical Reagent Co., Ltd., Tianjin, China . Ultrapure water was made in laboratory.

Enzymatic hydrolysis reaction
SCP and water were mixed in a 250 mL round bottom flask with stirring for 30 min, then the mixture was homogenized at 10000 rpm for 5 min with a high-speed shear dispersion emulsifier. After adjusting the temperature and pH, PLA 2 was added to start the reaction. When the reaction was performed for a certain time, PLA 1 was added to con-tinue the hydrolysis reaction for some times.

GPC analysis
Once the reaction was finished, the reaction product was taken to dehydrate in vacuum at 80 . Then, it was washed 3 times using acetone. After the oil in the product was removed, the solvent was evaporated in vacuum at 40 . The mixed solvents chloroform/methanol, v/v, 2:1 were added with shaking, then it was centrifuged at 10000 rpm for 10 min. The upper layer was filtered by a 0.22 μm polypropylene filter and analyzed by high-performance liquid chromatography HPLC .
GPC was analyzed by HPLC Agilent 1260 equipped with an evaporative light scattering detection ELSD . A Sun-Fire TM Prep silica column 5 μm, 4.6 250 mm, Agilent, USA was employed to analyze samples, and the column temperature was set at 35 . The evaporation and atomization temperatures of ELSD were respectively set at 40 and 65 with a nitrogen gas flow rate of 1.6 L/min. The mobile phase consisted of methanol A and water B at a flow rate of 1.0 mL/min. A gradient elution was used starting with 85 phase A, then reached to 75 in 7 min and decreased to 70 in 6 min, returned to the initial 85 in 0.1 min and held for 6.9 min.
The concentration of GPC was measured by the external standard method. The yield of GPC was calculated as follows: Where, C GPC is the concentration of GPC in the sample mg/mL , V is the sample volume mL , and m GPC is the theoretical yield of GPC mg .

Experimental design for RSM
A three-level-five-factor Box-Behnken design was employed to evaluate the interaction effects of reaction variables on the GPC yield. The factors and levels were as follows: reaction temperature 30, 40, and 50 , substrate concentration 1, 10, and 15 , PLA 1 load 0.45, 0.60, and 0.75 , relative to the weight of total substrates , PLA 2 load 0.75, 1.00, and 1.25 , relative to the weight of total substrates , and stirring rate 300, 500, and 700 rpm Table S1 .

Statistical analysis
The experimental data were analyzed by Design-Expert 8.0. The mathematical models between the reaction variables with the responses can be obtained by the following quadratic polynomial equation: Where Y is the predicted response GPC yield , and X i and X j represent the independent variables. β 0 , β i , β ii , and β ij are the intercept, linear, quadratic, and interaction terms, respectively.
To ensure the validity of the results, all experiments were performed at least in triplicate. The results were expressed as averages. Significant difference p 0.05 was estimated using a one-way analysis of variance ANOVA .

Results and Discussion
The SCP product mainly contained PC 17.02 , phosphatidylethanolamine PE , phosphatidylinositol PI , phosphatidic acid PA , and a small amount of sn-2-lysophosphatidylcholine sn-2-LPC, Fig. S1 . After enzymatic hydrolysis, the generated GPC was quantified by external standard method.

Effect of reaction time
According to our previous study, the total reaction time of at least 120 min was required for PC to be adequately hydrolyzed by PLA 2 and PLA 1 for obtaining a high yield of GPC. The influence of the respective time of PLA 2 and PLA 1 on the GPC yield was shown in Fig. 1. As PLA 2 time increased, GPC yield increased, followed by flattening. In theory, when PLA 2 is initially added to act on PC, a large amount of sn-1-LPC are generated, which is relatively difficult to undergo acyl migration 26,27 . When the reaction time of PLA 2 is insufficient, less sn-1-LPC is generated, which leads to reduce the catalytic efficiency of subsequent PLA 1 . When the reaction time of PLA 2 was 60 min, the GPC yield reached equilibrium 68.50 . This was consistent with the results reported by Vikbjerg et al. 20 , who pointed out that although increased reaction time promoted the hydrolysis rate, acyl migration was also difficult to be avoided. Therefore, the total reaction time was preferably 120 min, and the times of PLA 2 and PLA 1 were both 60 min.

Effect of reaction temperature
The influence of reaction temperature on the GPC yield was shown in Fig. 2. The GPC yield increased from 32.04 to 58.68 when the reaction temperature increased from 30 to 40 . However, when the temperature further increased from 40 to 70 , the GPC yield remained constant, followed by a significant decrease. These results indicated that the activity of PLA 2 is relatively high at 40-50 , thus increasing PC conversion and providing sufficient reactants for subsequent PLA 1 ; when temperature continues to rise, enzyme activity decreases and acyl migration is promoted 27 . Similar results were also observed in some studies 24,28,29 . Hence, considering the production cost, the optimal temperature was chosen at 40 .

Effect of substrate concentration
As shown in Fig. 3, when the substrate concentration increased from 5 to 10 , the GPC yield increased by 14.58 . However, the GPC yield sharply decreased from 67.20 to 33.16 with the increase of the substrate concentration from 10 to 25 , which might be due to the fact that when substrate concentration is relatively low, the viscosity of reaction system is low, where phospholipid exists in the form of single molecule or small aggregates to obtain a large reaction interface area. Although excessive phospholipids can theoretically accelerate the equilibrium of enzymatic hydrolysis reaction, the number of phospholipase active sites is limited. Therefore, when substrate concentration exceeds a certain range, single and dispersed phospholipids gather and accumulate, resulting in the decrease of reaction system dispersion and reaction interface and subsequent decrease of GPC yield 30 . Liu et al. also found a similar trend, which the inhibitory effect of microenvironment and products on enzymatic hydrolysis increased with the increase of substrate concentration 31 . Considering  Fig. 2 these results, the optimum substrate concentration was 5 .
3.1.4 Effect of stirring rate As the reaction system is heterogeneous, the mixing strength of SCP-water might affect the rate of enzymatic reaction. Effect of stirring rate on the GPC yield in enzymatic hydrolysis reaction was shown in Fig. 4. With increasing stirring rate, GPC yield increased initially and then flattened with the highest level of 65.07 at 500 rpm. The results could be explained that high stirring rate increases the interface area of SCP at the aqueous phase and the probability of phospholipase active sites on SCP, resulting in the increase of the GPC yield. However, when stirring rate is too high, the centrifugal force on the SCP-water mixture and phospholipase is so large that they are thrown onto the inner wall of the reactor, which reduces the amounts of SCP-water mixture and phospholipase in the reaction, and slightly reduces the GPC yield 32 . Thus, the stirring rate was preferably 500 rpm. 3.1.5 Effect of phospholipase A 2 load PLA 2 load directly determines the amount of primary hydrolysate sn-2-LPC , which indirectly affects the catalytic efficiency of PLA 1 in the second stage 33 . The influence of PLA 2 load on the GPC yield was shown in Fig. 5. When the PLA 2 load increased from 0.25 to 1.00 , the GPC yield increased from 30.78 to 75.07 . The phenomenon indicated that the addition of more PLA 2 in the early stage benefited generation of more sn-1-LPC, which led to a high GPC yield. It is due to the fact that the catalytic rate increases with the increase of enzyme load per unit volume 34 . Nevertheless, excessive PLA 2 had no positive effect on improving GPC yield, and it might even have a negative effect on the reaction interface of substrate and enzyme. Some studies have reported that the rate of enzymatic reaction sometimes does not increase with the increase of enzyme concentration 35 . Overall, the PLA 2 load was selected to be 1.00 .

Effect of phospholipase A 1 load
The amount of phospholipase directly affects GPC yield. As shown in Fig. 6, the trend of GPC yield with the increase of PLA 1 load was similar to that of PLA 2 . The GPC yield increased from 42.02 to 64.97 with the PLA 1 load from 0.15 to 0.60 , whereas there was no significant difference in the GPC yield with the PLA 1 load from 0.60 to 0.90 . In the primary stage of hydrolysis reaction, a large number of sn-1-LPC are generated. In the second stage, the addition of more PLA 1 generates more active sites per unit volume, which accelerates the enzymatic reaction. However, sn-1-LPC generated is limited, excessive PLA 1 has no an obvious effect on enzymatic reaction when the active sites and substrates of PLA 1 reach saturation 36 . Therefore, the PLA 1 load was chosen at 0.60 .

Model tting
In this work, RSM was employed to examine the effects  Preparation of Glycerylphosphorylcholine by Two-stage Enzymatic Hydrolysis Reaction of reaction temperature, substrate concentration, stirring rate, PLA 1 load, and PLA 2 load on the GPC yield Table  S1 . According to the statistical method, the data of 46 experimental runs were analyzed to simulate multiple response values of regression analysis. As shown in Table 1, the quadratic regression model was significant p 0.0001 , which indicated that the model was predominant and adequate to predict the actual relationship between these reaction parameters and GPC yield. The multinomial regression equation of GPC yield was obtained as follows: The interaction of reaction variables can be better un-   Preparation of Glycerylphosphorylcholine by Two-stage Enzymatic Hydrolysis Reaction derstood by 3D surface plots from the predicted models Fig. 7 . In the selected range of substrate concentration and reaction temperature, reaction temperature was the main affecting factor for GPC yield. Moreover, the interaction had no significant effect Fig. 7A . When PLA 1 load was constant, GPC yield initially increased and then slightly decreased with the increase of temperature. Specially, when reaction temperature was 35 , the GPC yield reached the maximum value Fig. 7B . When reaction temperature was 30-45 and PLA 2 load was 0.85-1.25 , GPC yield was relatively high Fig. 7C . As seen from Fig. 7D, when temperature was below 40 , GPC yield had an obvious increasing trend with the increase of stirring rate, indicating that stirring rate was the main affecting factor. However, high temperature 40 had no significant effect on GPC yield. When PLA 1 load was 0.45-0.52 and substrate concentration was 5-15 , the effect of PLA 1 load on GPC yield was greater than that of substrate concentration Fig. 7E .
With the increase of substrate concentration and PLA 2 load, the yield of GPC initially increased and then decreased. The interactive effect of substrate concentration and PLA 2 load was highly significant Fig. 7F . As seen from Fig. 7G, when stirring rate was 300-460 rpm and substrate concentration was 5-15 , the influence of stirring rate on GPC yield was greater than that of substrate concentration, whereas the interaction had no significant effect. When PLA 2 load was less than 1.00 and PLA 1 load was 0.45-0.75 , PLA 2 load was the main affecting factor. Nevertheless, when PLA 1 load was less than 0.60 and PLA 2 load was 0.75-1.25 , PLA 1 load was the main factor Fig. 7H . Figure 7I showed that the interaction between stirring rate and PLA 1 load had no significant effect on GPC yield. When PLA 2 load was 0.75-1.05 and stirring rate was 300-540 rpm, the interaction was greatly significant Fig. 7J .

Optimal conditions and model veri cation
The optimal process conditions were obtained by RSM as follows: reaction temperature of 40.73 , substrate concentration of 12.98 , PLA 2 load of 1.24 , PLA 1 load of 0.71 , and stirring rate of 676.97 rpm. Under the conditions, the predicted yield of GPC was 85.16 . Considering the practical application of industry, the processing parameters were adjusted as follows: reaction temperature of 41 , substrate concentration of 13 , PLA 2 load of 1.25 , PLA 1 load of 0.70 , and stirring rate of 680 rpm. The average GPC yield of triplicate under these conditions was 83.07 Fig. S2 , which was in accordance with the predicted value.

Conclusions
In this work, a two-stage enzymatic hydrolysis of SCP catalyzed by PLA 2 A 1 was successfully carried out with RSM for optimizing processing parameters. The reaction conditions were optimized as follows: PLA 2 load of 1.25 , PLA 1 load of 0.70 , substrate concentration of 13 , reaction temperature of 41 , and stirring rate of 680 rpm. Under these conditions, the GPC yield reached 83.07 , which was in agreement with the predicted value. This paper not only provides an efficient and low-cost method for GPC preparation, but also improves the high-value utilization of SCP. However, further purification of the obtained crude GPC is required in further study.