Peng Hu1, Xuebing Xu2,Liangli (Lucy) Yu*,3

Supplementary Data for

Analysis of Acer truncatum seed oil rich in nervonic acid, and nutraceutical constituents and physicochemical properties

Peng Hu1, Xuebing Xu2, Liangli (Lucy) Yu*,3

1Institute of Food and Nutraceutical Science, School of Agriculture and Biology, Shanghai Jiao

Tong University, Shanghai, 200240, China

2Wilmar (Shanghai) Biotechnology Research & Development Center Co., Ltd, Shanghai, 200137,

3Department of Nutrition and Food Science, University of Maryland, College Park, MD, 20742,

United States

 Corresponding authors. Tel.: +1 301 405 0761; Fax: +1 301 314 3313.

E-mail addresses: [email protected] 1. Materials and Methods

1.1 Supercritical carbon dioxide extraction

The desired extraction temperature was programmed following extraction vessel sealing. The pressure within the extraction vessel was increased with a constant CO 2 flow rate of 20 mL/min.

The extraction time was calculated once the pressure and temperature reached the predefined values.

The effects of SC-CO2 extraction parameters including temperature, pressure, and time of extraction on oil yield (w/w) were studied using a Box-Behnken design (BBD). Three levels are included for each parameter: Pressure (P): 25, 35, and 45 MPa; Temperature (T): 35, 45, and 55

°C; and Time (t): 6, 8, and 10 h. The extracted oils were stored at 4 °C until further analyses.

A second-order polynomial equation (Eq. 1) was used to express the oil yield (Y1):

2 2 2 Y1 = β0 + β1X1 + β2X2 + β3X3 + β12X1X2 + β13X1X3 + β23X2X3 + β11X1 + β22X2 + β33X3 (1)

Here, X1, X2, and X3 are the independent variables of pressure, temperature, and time, respectively;

β0 is a constant and β1, β2, β3, β11, β22, β33, β12, β23, and β13 are linear, quadratic, and interactive coefficients, respectively. The significance level of the equation parameters for each response variable was assessed by an F ratio at a probability (P) of 0.05. The adequacy of the models was determined using model analysis, a lack-of-fit test and a coefficient of determination (R2) analysis.

For a good fit, the R2 of the model should be at least 0.80. The experimental design matrix, data analysis, and process optimization were performed using a Design-Expert Software (Stat-Ease,

Inc., Minneapolis, USA).

1.2 Extraction kinetics

Extraction kinetics were obtained for optimal operating condition of temperature and pressure and expressed as a function of oil yield with 0.5 h sampling intervals. The extraction can be considered complete when the slope of yield vs. time plot was near zero.

2 Optimization of SC-CO2 extraction parameters

The results for the extraction of ATO by SC-CO2 at different pressures, temperatures, and extraction times are shown in Table S1. The highest oil yield was 42.9 % (w/w) with extraction conditions of 45 MPa, 45 °C, and 10 h.

The following second-order polynomial equation (Eq. 2) was found to represent the experimental data (R2 = 0.9052):

2 2 Y1 = -39.88 + 5.48X1 – 0.28X2 + 3.41X3 + 0.55X1X2 – 1.35X1X3 – 0.55X2X3 – 4.57X1 – 2.07X2 –

2 1.32X3 . (2)

Where X1, X2, and X3 are the independent variables pressure, temperature, and time, respectively.

An analysis of variance (ANOVA) of the regression model is shown in Table S2. The oil yield (Y1) was significantly (P < 0.01) influenced by extraction pressure, time, and the quadratic term of extraction pressure, respectively. The regression model for the response variable was significant via an F test at the 1% confidence level (P < 0.01). The R2 value (0.9052) was larger than 0.80 and the “lack-of-fit” was not significant (P > 0.05) indicating that a satisfactory regression model was developed via this experimental data. The R2 value of 0.9052 implied that 90.5% of the variations related with the ATO extraction yield were attributable to the selected independent variables

(temperature, pressure, and time). Table S1. The Box-Behnken design and experimental results of the response functions for the A. truncatum seed oil yield using SC-CO2 extraction.

Y1 Y0 X1 X2 X3 Experimental Exp. No. Predicted oil yield (%, Residue P/MPa T/°C t/h oil yield (%, w/w) w/w) 1 25 55 8 29.8 26.9 2.9 2 45 35 8 35.6 38.4 2.9 3 35 45 8 39.3 39.9 0.6 4 35 45 8 38.1 39.9 1.8 5 35 45 8 42.9 39.9 3.0 6 35 55 10 37.0 39.1 2.1 7 35 55 6 31.9 33.4 1.5 8 25 45 10 32.5 33.3 0.8 9 25 35 8 27.9 28.6 0.7 10 35 35 6 34.9 32.8 2.1 11 35 45 8 39.2 39.9 0.7 12 45 45 10 42.9 40.7 2.2 13 25 45 6 22.4 23.8 1.4 14 35 35 10 42.2 40.7 1.5 15 45 45 6 38.2 37.4 0.8 16 45 55 8 39.7 39.0 0.7 17 35 45 8 39.9 39.9 0.0

4 Table S2. ANOVA results of the second-order polynomial regression model.

Source P value F ratio Model 0.0075 7.43 Lack of fit 0.1240 3.60 X1 0.0006 34.49 X2 0.7758 0.088 X3 0.0082 13.33 X1X2 0.6880 0.18 X1X3 0.3412 1.04 X2X3 0.6920 0.17 2 X1 0.0093 12.64 2 X2 0.1515 2.59 2 X3 0.3388 1.05 3 Response surface analysis

Fig. S1. Response surface plots for the effect of extraction pressure, temperature, and time on oil yields. (A) The influence of extraction pressure and temperature on oil yield at a fixed extraction time of 10 h. (B) The influence of extraction pressure and time on oil yield at a fixed extraction temperature of 45 oC. (C) The influence of extraction temperature and time on oil yield at a constant extraction pressure of 35 MPa.

4 Extraction kinetics

The SC-CO2 extraction kinetics for ATO were studied under an optimal SC-CO2 operating condition (44 °C and 39 MPa) predicted by RSM. As a result, a 43.1% (w/w) oil yield was obtained at 10 h—this is close to the predicted value (42.9% (w/w)) calculated by Eq. 1. Nearly

33.4% (w/w) of the oil (accounting to 77.6% (w/w) of total extracted oil) was recovered in the first 5 h of extraction. The remaining 9.7% (w/w) (accounting to 22.4% (w/w) of total extracted oil) occurred in the second half of the experiment.

5 Fig. S2. Extraction kinetics of A. truncatum seed oil recovered by SC-CO2 at 39 MPa and 44 °C.