Optimization of the ultrasound-assisted aqueous enzymatic extraction of Pinus koraiensis nuts oil by response surface methodology

Abstract Response surfaces methodology was established in order to optimize ultrasound-assisted aqueous alkaline protease extraction parameters of Pinus koraiensis nuts oil (PNO) in this short communication. On the oil yield, the impacts of single factors were studied. The solid–liquid ratio, enzyme concentration, enzyme hydrolysis temperature, and enzyme hydrolysis duration were chosen for further optimization of the extraction process utilizing a Box–Behnken design based on statistical significance analysis. Under ideal extraction conditions, a maximum oil recovery of 68.35% was achieved: solid–liquid ratio, enzyme concentration, enzyme hydrolysis temperature, and enzyme hydrolysis duration were 1:5 (g/mL), 3.23 mg/g, 44 °C, and 2.84 h, respectively. Furthermore, physicochemical properties testing revealed that the oil was of higher quality than other approaches. Meanwhile, the DPPH radical-scavenging activities increased with increased content compared to olive oil, with an IC50 value of 0.082 mg/mL. The method has a lot of potential when it comes to extracting oils from plants. Graphical Abstract


Introduction
Pinus koraiensis belongs to the pinnate pine family and is an evergreen needle-leaf tree. In East Asia, particularly in Northeast China, it is a significant economic forest (Incegul et al. 2020). Previous research had shown that Pinus koraiensis nuts oil (PNO) can lower blood lipids, have anti-inflammatory and immune-boosting properties (Xie et al. 2016). Since these physiological functions of PNO, the requirements for PNO quality are more environmentally friendly and economical. Then, oil extraction methods are constantly evolving.
In various extraction methods, cold pressing, hot pressing, hexane extraction, subcritical extraction, and supercritical carbon dioxide extraction were commonly investigated (Chen et al. 2011, Li et al. 2011, Lixia et al. 2018. Due to the obvious internal high temperature, the unsaturated fatty acid in the oil is easily decomposed even during hot pressing process. The flavor and color of the fat can be retained by cold pressing, but the extraction rate is low. The oil extraction yield of n-hexane is high and the quality is good, but the environmental pollution and production safety caused by organic solvents have also been widely concerned (Li xia et al. 2018). The supercritical CO 2 extraction and subcritical extraction require a lot of energy (Sparks et al. 2006).
Aqueous enzyme extraction is a safe alternative oil extraction technology that has been widely used in the extraction of various oil seeds and fruits (Najafian et al. 2009, Teixeira et al. 2013. The aqua-enzymatic method works on the principle of using enzymes that hydrolyze plant cell walls or lipoproteins, lipopolysaccharides, and other complexes to enzymatically hydrolyze oils after mechanical crushing, allowing oils to be easily released from the oils, and non-oil components are used to treat oil and water. The difference in affinity between oil and water, as well as the difference in oilwater density, separate the oil from the water (Li et al. 2011). Ultrasound causes portion of the cell wall structure to collapse, resulting in cell swelling. This can hasten the release of lipids, improve oil production, and reduce processing time (Shah et al. 2005, Sharma andGupta 2006). In comparison to other extraction methods, the aqua-enzymatic approach offers softer conditions, safer operation, reduced energy usage, and is more ecologically friendly and cost-effective. Therefore, the aims of this short communication are to assess and compare the best condition for extracting PNO by ultrasound-assisted aqueous enzymatic and its physicochemical properties compared other methods.

Results and discussion
2.1. The analysis of single factor experiment Figure S2A-C showed that the yields increased with the ultrasonic power, time, and temperature, reaching a peak when these variables arrived at 400 W, 40 min and 50 C, respectively. The yield reached its greatest value, which was attributable to the mechanical vibration induced by the ultrasonic speeding up cell wall deterioration (Vilkhu et al. 2008, Zhang et al. 2009).
The solid-liquid ratio had a considerable effect on the PNO, as seen in Figure S2D. The yield of oil reached its maximum at 1:5 as the amount of solvent increased. Despite this, the yield started to decrease below 1:5. The phenomena can be explained by the fact that a lower solvent concentration, a higher substrate concentration in the reaction system, a higher viscosity, and a lower fluidity affect the enzyme's outcome on the substrate (Moradi et al. 2018).
In most cases, the yield increased with the enzyme concentration, enzyme duration and temperature. The optimum condition for the yield obtained above was found to be at 3.0 mg/g, 4 h and 45 C in Figure S2E-G. These differences indicated that as the increased in the amount of enzymes promoted interaction between the enzyme and the substrate more effective. Table S2 showed the yield results calculated by the DX12 software. The model is significant was presented by F-value of 119.41 (Table S3), which explained just a 0.01% chance could occur due to noise. The P values were used to test the significance of each coefficient, which may suggest the pattern of the interactions among the variables. In general, a high coefficient of determination R 2 value of 0.9917 intimated that a model provides a credible fit for the results. Using equation (3) presented by DX12 we can forecast the yield of extracted. Y ¼ 68:35 þ 0:08X 1 À0:35X 2 þ 0:26X 3 À0:36X 4 À0:29X 1 X 2 À0:38X 1 X 4 À0:25X 2 X 3 þ 1:32X 2 X 4 À0:78X 1 2 À2:52X 2 2 À1:96X 3 2 À2:14X 4 2 (3) Figure S3 showed the 3 D models of the effect of factors on the yield of PNO obtained from the extraction process by UAEE. The relationship between various factors and the response value were reflected directly in response surface diagram. The results acquired after optimization were liquid-solid ratio of 1:5 (g/mL), enzyme addictive amount of 3.23 mg/g, enzymatic hydrolysis duration of 2.84 h and enzymatic hydrolysis temperature of 44 C with expected yield of 68.35%. According to the given data, we can assume that the quadratic model properly anticipated the experimental values.

Physicochemical properties of pine nut oils
The following conclusions were drawn from Table S4. The color of the oil obtained by UAEE was pale yellow (Y30 R0.6) and transparent.
The acid values of the pine nut oils were within the range of 0.36-0.99 mg KOH/ g, reaching the National Standard of the People's Republic of China for third-class soybean oil (GB 1535(GB -20172017 2017. While in terms of the peroxide values of 2.47-4.34 mmol/kg, the pine nut oils meet the National Standard of the People's Republic of China for the first-class soybean oil (GB 1535(GB -20172017 2017. The peroxide value of UAEEO was 2.62 ± 0.04 mmol/kg, which was lower than that of other methods. Combining the acid value and peroxide value, we can see that UAEEO with decent quality. The Iodine value of UAEEO was 141.65 ± 0.02 g/100 g, which was higher than that of other methods. The saponification values reflect the C-chain length of the fatty acids. The saponification value of UAEEO was 185.73 ± 0.45 mg KOH/g, indicated that the triglycerides of the PNO had smaller molecular weights than the oils form other extraction methods (Li xia et al. 2018). Above the results indicated that the PNO obtained by UAEE had better quality compared other methods.

Determination of antioxidant activity of PNO
The experimental results of PNO scavenging ability of DPPH free radicals were observed among the various experiments as shown in Figure S4. The free radical scavenging ability of PNO was universally above 30%. Meanwhile, the scavenging ability increased with increased content. A lower value of IC 50 indicated a higher antioxidant activity. Its IC 50 value is 0.082 g/mL. Furthermore, olive oil is recognized as an oil resource with rich antioxidant activity and powerful nutritional value. Its IC 50 value is 0.054 g/mL, which slightly higher than PNO as a positive control. It was indicated that the PNO obtained by the UAEE had good anti-oxidation properties and completely retained its natural unsaturated fatty acid and other nutrients (Blomhoff et al. 2006, Qu et al. 2019).

Conclusions
PNO was extracted from P. koraiensis nuts at 68.35% under optimum conditions, with 0.9917 of R 2 in this communication. The PNO by UAEE had superior quality than those obtained by conventional methods, according to physicochemical properties tests. Furthermore, the PNO antioxidant activity tests revealed that the scavenging ability increases dramatically. Last but not least, PNO obtained by UAEE method had better anti-oxidation properties and completely retained its natural unsaturated fatty acid and other nutrients, which has potential applications in the production and development of functional food raw materials.

Disclosure statement
No potential conflict of interest was reported by the authors.