1. Introduction
Peony (
Paeonia suffruticosa Andr.), which belongs to the family Paeoniaceae, is a deciduous shrub that is widely planted in China, especially in Heze and Luoyang [
1,
2]. Peony seeds are rich in resources, the annual production of which can reach tens of thousands of tons, and the industry shows considerable development potential. Peony seed contains 29–34% oil, 18–22% protein, and a higher unsaturated fatty acid content than other vegetable oils [
3]. The unsaturated fatty acids in peony seed oil are mainly oleic acid, linoleic acid, and linolenic acid [
4,
5], which can delay aging and prevent the occurrence of obesity [
6,
7,
8]. In 2011, the China Ministry of Health listed peony oil as a new food resource [
3,
8].
The traditional technique of oil extraction using organic solvents leads to health, safety, and environmental problems [
9]. Among the modern extraction methods, green solvents such as water, ethanol, and carbon dioxide (CO
2) are receiving increasing attention [
10]. The aqueous enzymatic method has the advantages of simple operation and mild conditions, is a safe and non-polluting oil-extraction method, and can better ensure oil quality [
11]. This is because the cell wall can undergo enzymatic hydrolysis under mild conditions, and thus enzyme-assisted extraction can improve oil yields [
12,
13]. Wei et al. [
14] found that cellulase and hemicellulose help improve the yield of peony seed oil extraction via the aqueous enzymatic method. However, the industrial production scale of this approach is hindered due to enzyme costs. Specifically, enzyme composition and process conditions need to be optimized to obtain higher oil yields and lower production costs. We also previously found that peony seed oil is present in residues after extraction using the aqueous enzymatic method.
In comparison with enzymatic extraction, the supercritical CO
2 extraction technique is considered efficient [
15] and thus, can be used to continue extracting oil from aqueous enzymatic method residue (AER). For example, while supercritical extraction equipment is typically expensive, this can be overcome by applying supercritical CO
2 extraction to low-volume AER, which avoids the need for large and expensive equipment. Currently, no studies have been published on the extraction of peony seed oil using a two-step strategy combining hydroenzymatic and supercritical CO
2 extraction. Nevertheless, this strategy likely requires more control of the process condition parameters. To address this, the response surface methodology can be effectively employed. For example, the extraction conditions of olive oil,
Lawsonia inermis essential oil, and hemp seed oil were optimized using the response surface methodology, enabling significant oil yield increases [
16,
17,
18].
In this study, we adopt a two-step strategy of aqueous enzymatic method combined with supercritical CO
2 extraction to extract peony seed oil. The Plackett–Burman design (PBD) [
19] and mixture design [
20] are used to optimize the composition of enzymes in the extraction process, and central composite design (CCD) [
21] is used to optimize the conditions of supercritical extraction. We then compare the differences in fatty acid compositions of peony seed oil extracted using these two extraction methods, and also compare the physicochemical indices of commercial peony seed oil and the oil extracted using the aqueous enzymatic method. Our results provide a new strategy for the effective development and utilization of peony seeds as a new food resource.
2. Materials and Methods
2.1. Materials
Shelled peony seed kernels were collected from Heze, Shandong, and stored in a cold storage at 4 °C. They were crushed by hammer crusher into particles of less than 40 mesh before use. Enzymes including cellulase, β-glucanase, α-amylase, glucoamylase, pectinase, and xylanase were purchased from SUNSON Industry Group Co., Ltd. (Beijing, China), and papain was purchased from Pangbo Biotechnology Engineering Co., Ltd. (Nanning, China). The enzyme activities were re-tested.
2.2. Enzyme Assays
The required amounts of commercial cellulase, β-glucanase, α-amylase, glucoamylase, pectinase, xylanase, and papain enzyme powder were weighed and dissolved in the corresponding buffers with the respective optimal pH to prepare the enzyme solutions.
Cellulase, β-glucanase, xylanase, and pectinase were measured using filter paper, carboxymethyl cellulose, beech xylan, and pectin as the substrate, respectively [
22,
23,
24,
25]. The enzyme activities of both α-amylase and glucoamylase were measured using starch as a substrate, the only difference being the concentration, as described in the literature [
26,
27]. The dinitrosalicylic acid (DNS) method with glucose as the standard was used to determine the amount of reducing sugars released [
28]. One enzyme activity unit (U) was defined as the amount of enzyme that liberates 1 μmol glucose, xylose, or galacturonic acid equivalent per minute under the assay conditions.
For the measurement of papain activity, 2 mL of enzyme solution and 2 mL of 1% concentration casein solution were mixed. After 10 min of reaction at 40 °C, 4 mL of trichloroacetic acid solution (0.4 M) was added to terminate the reaction. After shaking well, the sample was centrifuged at 300×
g for 5 min. One unit of enzyme activity was defined as the amount of enzyme required to produce 1 μg of tyrosine by hydrolyzed casein in 1 min [
29].
2.3. Determination of Protein Concentration
Protein concentrations were determined using the Bradford Protein Assay Kit (Sangon, Shanghai, China) according to the manufacturer’s instructions [
30].
2.4. Extraction of Peony Seed Oil Using the Aqueous Enzymatic Method
We placed 30 g of crushed peony seeds (<40 mesh) in a jacketed beaker. The corresponding amount of enzyme and five times the volume of distilled water were added. The reaction solution underwent enzymolysis in a water bath at 55 °C for 3 h and was then heated to 90 °C to inactivate the enzyme. The enzymatic hydrolysate was centrifuged at 5000×
g for 20 min, after which the solution was stratified. The upper oil layer was collected to calculate the yield of peony seed oil.
Figure 1 illustrates the workflow relating to the aqueous enzymatic extraction method.
Finally, the oil yield was calculated according to the following formula:
2.4.1. PBD
The PBD can quickly and effectively identify the most important factors from multiple examined variables, thereby enabling subsequent optimization tests. Here, key enzyme components were screened for their major effects using PBD. The variables selected were the respective activities of cellulase, papain, β-glucanase, α-amylase, glucoamylase, pectinase, and xylanase.
Table 1 shows the experimental design of the PDB analysis.
For mathematical modeling, the following first-order polynomial model was used:
where Y is the predicted oil yield, β
0 is the model intercept, β
i is the linear coefficient, and x
i is the independent variable.
2.4.2. Mixture Design
The peony seeds were treated with pectinase, cellulase, and papain synergistically. The effects of formulations with different enzyme mixture ratios on the oil yield were investigated using the simplex centroid design method while considering cost as a prerequisite. The most suitable pre-treatment cocktail enzyme formulation for the extraction was screened with the aim of optimizing the oil yield. With reference to the experimental results of the PBD and considering economic cost (accepting that the more enzyme used, the better the yield), the dosage was limited to 10 mg enzyme protein/g peony seeds. The experimental design is shown in
Figure 2, in which each point represents one formulation out of a total of ten tested). The specific schemes and results of the oil yield measurement are shown in
Table 2.
The mathematical relationship of the oil yield (Y) to the significant independent variables (x
1, x
2 and x
3) is provided by the following quadratic polynomial equation:
where Y is the response (oil yield); x
1, x
2, and x
3 are significant independent variables corresponding to the ratio of pectinase, cellulase, and papain, respectively; β
1, β
2, and β
3 are linear regression coefficients; and β
12, β
13, and β
23 are interactive regression coefficients.
2.5. Optimization of Supercritical CO2 Extraction Using Response Surface Methodology
We weighed 25 g of peony seed AER (dried) after aqueous enzymatic extraction and placed the sample in a 100 mL extraction kettle for supercritical CO2 extraction. We adopted the process conditions of previous direct supercritical CO2 extraction of peony seed oil (unpublished data), with an extraction pressure of 55–65 MPa, extraction temperature of 45–55 °C, and extraction time of 60–120 min. The extracts were collected at regular intervals. The oil was collected, and the yield calculated.
Response surface methodology (RSM) can conveniently determine the best conditions for a process. CCD was employed to obtain information regarding the significant effects and interactions between the selected variables and identify the optimal values of each variable to maximize oil yield. The three factors selected for further optimization were temperature (°C), pressure (MPa), and time (h), which were denoted as x
1, x
2, and x
3, respectively. Each factor was assessed at five different levels combining factorial points (−1, +1), axial points (−α, +α), and the central point (0) (
Table 3).
Oil yield was analyzed using a second-order polynomial equation, and the data were fitted by multiple regression. The mathematical relationship of the response (
) to the significant independent variables (x
1, x
2, and x
3) is provided by the following quadratic equation:
where Y is the response (oil yield); x
1, x
2, and x
3 are temperature, pressure, and time, respectively; β
0 is a constant; β
1, β
2, and β
3 are linear regression coefficients; β
11, β
22, and β
33 are quadratic regression coefficients; and β
12, β
13, and β
23 are interactive regression coefficients.
2.6. Analysis of the Fatty Acid Composition of Peony Seed Oil
Fatty acid analysis was slightly improved with reference to the method of Li et al. [
31]. Agilent 7890 gas chromatography and Agilent 5975 mass spectrometry were performed to analyze the fatty acid composition of the peony seeds. The BF
3-methanol solvent method was used for methyl esterification; 1 g of peony seed oil was placed in a round bottom flask, and 6 mL of 0.5 M potassium hydroxide-methanol solution was added for reflux saponification in a water bath at 60 °C for 30 min. After cooling, 12 mL of BF3-methanol solution with a 40% mass fraction was added for reflux methylation in a water bath at 60 °C for 5 min. After cooling, 4 mL of hexane and 4 mL of saturated sodium chloride were added, mixed thoroughly, centrifuged, and analyzed using gas chromatography-mass spectrometry (GC-MS) according to the following conditions: chromatographic column HP-88 quartz capillary column (100 mm × 0.25 mm, 0.2 μm); heating procedure: held at 80 °C for 5 min, increased to 150 °C at a rate of 10 °C min
−1, held for 2 min, increased to 230 °C at a rate of 5 °C min
−1, and held for 10 min. The injection volume was 1 μL, the inlet temperature was 250 °C, the split ratio was 10:1, the carrier gas was helium, the pre-column pressure was 100 kPa, the ionization voltage was 70 eV, and the mass scan range was 50–550 amu.
2.7. Analysis of Physicochemical Properties of Peony Seed Oil
The International Organization for Standardization (ISO) standards were used for the determination of oil relative density (ISO 3675, 1998), acid value (ISO 1242, 1999), peroxide value (ISO 3960, 2007), and iodine value (ISO 3961, 2013).
2.8. Statistical Analysis
Minitab® 17 (Minitab Inc, State College, PA, USA) software was employed for designing PBD, mixture design and CCD experiments as well as for regression analysis of the experimental data obtained. Statistical analysis of was performed using analysis of variance (ANOVA). All assays were performed in triplicate, and mean values were calculated.
4. Conclusions
A two-step method was used to extract edible oil from peony seeds. First, peony seed oil was extracted using an aqueous enzymatic method. Pectinase, cellulase, and xylanase had positive effects on peony seed oil extraction using the aqueous enzymatic method with optimal proportions of 16.15%, 31.33%, and 52.53%, respectively, at an enzyme dosage of 10 mg/g peony seed. These conditions provided an oil yield of 16.41%. We subsequently extracted a further yield of 6.30% from the AER using supercritical CO2 extraction over 1.58 h at 49.41 °C and 59.75 Mpa. Notably, the fatty acid compositions of the oils extracted using the two methods were comparable. Compared with commercial peony seed oil extracted using organic solvent leaching, the oil prepared in this study also shows better relative density and iodine values.