A method for pretreating camellia seeds using low-temperature plasma

Abstract

This invention discloses a method for pretreating camellia seeds using low-temperature plasma, relating to the field of bioengineering technology. The method involves discharging the entire surface of the camellia seed using DBD, JET, DF, or RF at atmospheric pressure, preferably DBD 120kV×1min or RF 3m / min at a distance of 0-3mm for 20 passes. This achieves microporousization of the seed coat and targeted disruption of the cell wall to promote mass transfer. The treatment reduces lipase and lipoxygenase activities by approximately 31.72% and 35.69% (or 29.54%), respectively, and significantly enhances the antioxidant capacity of total phenols and DPPH / ABTS / FRAP in the methanol extract (up to 1.58 times the total phenols). This method simultaneously promotes the release of active ingredients, inhibits lipid oxidation, and improves oil yield and quality under low-temperature, non-thermal conditions, making it suitable for continuous industrial applications.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a method for pretreating camellia seeds using low-temperature plasma. Background Technology

[0002] Camellia oleifera is an important woody oilseed crop unique to my country. Camellia seeds, as a raw material for oil extraction, possess both nutritional and functional properties. However, the naturally dense seed coat and complex cell wall structure limit mass transfer, resulting in suppressed oil yield and solvent extraction efficiency. Production practices typically improve the tissue structure and rheology through pretreatment methods such as drying, crushing, and steaming. However, high-temperature processes easily induce oxidation and loss of some active ingredients, making it difficult to achieve a balance between "high oil yield—low oxidation—active ingredient enrichment." With increasing demands for quality and functionality, developing novel pretreatment technologies that achieve structural remodeling and synergistic quality improvement under mild conditions has become a trend.

[0003] Heat treatments such as steaming, frying, and baking can soften cell walls and improve oil fluidity, but they also promote lipid oxidation and cause thermal degradation of natural antioxidants such as polyphenols, affecting oil stability and flavor. Enzymatic pretreatment can specifically degrade cell wall components, but the reaction cycle is long, the cost is high, and residual enzymes or degradation products may affect subsequent processes, limiting its suitability for continuous industrial application. While physical field treatments such as ultrasound and microwaves can generate pores and cracks, the energy coupling and hot spot effect management are complex, and there are still drawbacks such as narrow process windows and large repeatability fluctuations in protecting heat-sensitive components and inhibiting deterioration reactions.

[0004] Cold plasma (CP) is a non-thermal plasma, typically generated under ambient pressure by a high electric field, resulting in the coupling of multiple factors including active particles, free radicals, ozone, and ultraviolet radiation. It can etch, activate, and modify the surface layer of biomass at near-room temperature. Unlike traditional heat treatment, cold plasma is suitable for selective structural remodeling that "affects only the surface without damaging the bulk phase," balancing tissue opening and protection of active components. Common discharge modes include dielectric barrier discharge (DBD), jet plasma discharge (JET), differential dielectric barrier discharge (DF), and radio frequency (RF), allowing for flexible switching between laboratory and continuous industrial scenarios.

[0005] Studies on camellia seeds show that CP treatment can effectively disrupt cell structure and form micropores on the seed coat surface, thereby improving the accessibility of water and solute migration channels and facilitating mass transfer in subsequent drying, pressing, and extraction. DBD and RF treatments showed outstanding results, reducing lipase activity by 31.72% and 34.19%, and lipoxygenase activity by 35.69% and 29.54%, respectively, inhibiting the initiation of the oil hydrolysis and oxidation chain reaction at the enzymatic level. RF treatment also significantly increased the total phenolic content and antioxidant capacity of the camellia seed methanol extract, suggesting that CP can promote the release of phenols and other active substances through cell wall microperforation and non-covalent bond breaking, achieving a synergistic effect of "structure opening - activity release - quality enhancement".

[0006] Studies have found that low-power or short-duration treatment is more conducive to enzyme activity inhibition and active ingredient protection, while excessive discharge may trigger secondary reactions or degradation of active ingredients, exhibiting a "rise then fall" response characteristic. Therefore, it is necessary to match the process window in terms of voltage, time, distance, and number of scan passes. Specifically, DBD 120kV treatment for 1 min can significantly inactivate lipase / lipoxygenase while maintaining or improving antioxidant capacity indicators, such as the enhanced free radical scavenging and reducing abilities measured by DPPH, ABTS, and FRAP methods. The RF single-electrode solution achieves surface roughening and inner seed coat voiding at a transmission speed of 3 m / min, a treatment distance of 3 mm, and 20 reciprocations, adapting to continuous production lines while considering modification depth and thermosensitive safety.

[0007] Unlike high-temperature steaming and frying, which focuses on softening and flavor development, CP emphasizes the reconstruction of surface energy and chemical activity at low temperatures, reducing adverse effects on heat-sensitive active ingredients and inhibiting the initiation of oil oxidation. Compared to enzymatic methods, CP requires no exogenous reagents and has a shorter residence time, reducing the risk of cleaning and residues, making it more suitable for pre-treatment at the raw material pretreatment stage and seamlessly integrating with drying and pressing processes. Compared to physical field technologies such as ultrasound and microwave, CP achieves etching and activation under normal pressure and low heat load conditions, and its process window is more conducive to large-scale, repeatable control. Because CP can be carried out at normal pressure and its device form is diverse, it can be easily integrated into existing cleaning-drying-pressing production lines to form online pretreatment units, reducing modification costs and facilitating scale-up validation. Currently, it is still necessary to construct process window maps around raw material moisture content, particle size, surface cleanliness, and discharge parameters to clarify the relationship between "target indicators - parameter combinations - quality response" and avoid over-processing that could lead to loss of active ingredients or structural embrittlement. Taking camellia seed with a moisture content of about 8.92% as an example, short-time discharge is beneficial to achieving a balance between increasing total phenols and protecting flavonoids, reflecting the optimal process range under the joint regulation of moisture state and plasma chemistry.

[0008] Existing research has confirmed the effects of CP on the microstructure modification and enzyme activity inhibition of camellia seeds. However, the process adaptation model for different varieties, origins, and batches remains incomplete, affecting the stability assessment for large-scale promotion. Although experimental indicators support the causal chain between improved antioxidant system and enhanced virgin oil quality, the mechanisms regarding free radical types, surface functionalization pathways, and selective degradation of cell wall polymers require further in-situ characterization and multi-scale modeling. Furthermore, closed-loop control for online process monitoring (such as discharge uniformity, surface energy, and hydrophobicity / hydrophobicity changes) and end-quality prediction needs to be established to meet the comprehensive requirements of continuous, standardized, and low-carbon industrial processes.

[0009] In summary, low-temperature plasma, with its non-thermal, atmospheric pressure, rapid, and modular integration characteristics, offers a new technological approach to address the mass transfer bottleneck and quality deterioration risks caused by the dense structure of camellia seeds. By rationally selecting DBD, RF, JET, or DF methods and optimizing voltage, time, and path parameters, it is expected to achieve the synergistic goal of "structure activation - enzyme activity inhibition - activity enrichment - quality improvement," laying a pretreatment foundation for improving oil yield and antioxidant stability. This invention addresses a clear real-world pain point and has significant industrialization potential, making the development of parameterized, continuous, and quality-traceable technical solutions both necessary and urgent. Summary of the Invention

[0010] In view of this, the purpose of this invention is to propose a method for pretreating camellia seeds using low-temperature plasma to solve the problems of limited mass transfer caused by the dense outer shell and inner seed coat of camellia seeds, thermal degradation of active ingredients and oil oxidation caused by traditional high-temperature pretreatment, and hydrolysis and oxidative deterioration caused by high lipase and lipoxygenase activity. This method achieves targeted modification of the microstructure, inhibition of key enzyme activity, and efficient release of active ingredients under normal pressure and low temperature conditions, synergistically improving oil yield and oil antioxidant quality. This invention uses dielectric barrier discharge (DBD), jet plasma (JET), differential dielectric barrier discharge (DF), or radio frequency (RF) methods, and optimizes the voltage, treatment time, treatment distance, and number of repetitions to roughen the seed coat surface and form micropores. This allows the plasma to penetrate to the non-covalent bonds of the inner seed coat and kernel cell walls, establishing channels conducive to the migration of water and solutes, while simultaneously opening tissue and protecting heat-sensitive components. Under optimal conditions, DBD treatment at 120kV for 1 min combined with RF parameters significantly inhibited lipase and lipoxygenase activities. DBD reduced lipase and lipoxygenase activities by approximately 31.72% and 35.69%, respectively, while RF reduced lipoxygenase by approximately 29.54%. Furthermore, it significantly increased the total phenol content and antioxidant indicators such as DPPH, ABTS, and FRAP in the methanol extract (total phenol content increased by up to 1.58 times, DPPH by up to 2.61 times, ABTS by up to 1.51 times, and FRAP by up to 2.42 times), thereby reducing the oxidation risk during oil processing and storage. This invention also proposes a parameter boundary control strategy for DBD and RF modes to avoid adverse effects such as an initial increase followed by a decrease in total phenol content and flavonoid degradation caused by excessive treatment time and discharge intensity, thus improving the stability and repeatability of the active ingredients and antioxidant capacity after pretreatment. By embedding the above process into the pre-drying, pressing, or solvent extraction stage, this invention achieves rapid, non-thermal, and continuous surface modification of camellia seed raw materials, providing a reliable industrial solution for increasing oil yield, improving oil quality, and extending shelf life.

[0011] The adopted technical solution is as follows: a method for pretreating camellia seeds using low-temperature plasma, comprising the following steps: S1, selecting camellia seeds with a moisture content of 8.0-60.0% as the treatment object; S2, performing surface discharge pretreatment on the camellia seeds obtained in step S1 using low-temperature plasma technology under normal pressure conditions. The surface discharge pretreatment methods include dielectric barrier discharge, plasma flow, differential dielectric barrier discharge, or radio frequency single electrode plasma. The voltage range of dielectric barrier discharge is 90-150kV, and the treatment time is 1-15min; the plasma flow method uses direct plasma jet; the differential dielectric barrier discharge method uses differential output dielectric barrier discharge; the transmission speed of radio frequency single electrode plasma is 2-4m / min, the treatment distance is 0-3mm, and the number of reciprocating treatments is 15-25 times; S3, after surface discharge pretreatment in step S2, the camellia seeds are placed at room temperature and left to stand for 10-30min before subsequent drying, pressing, or solvent extraction processes.

[0012] Preferably, the discharge gas in the dielectric barrier discharge method described in step S2 is air, the voltage is 90-150kV under normal pressure, and the processing time is 1-15min; at the same time, the parameters for low voltage short-time processing are as follows: 90kV, 1-5min; the parameters for medium voltage processing are as follows: 120kV, 1-10min; and the parameters for high voltage long-time processing are as follows: 150kV, 10-15min.

[0013] Preferably, the dielectric barrier discharge method described in step S2 has a voltage of 120kV under normal pressure and a processing time of 1min.

[0014] Preferably, the discharge parameters of the radio frequency single-electrode plasma method in step S2 are a transmission speed of 3 m / min, a processing distance of 3 mm, 20 reciprocations, and the gas medium is air and / or argon. Preferably, the plasma flow method described in step S2 uses a plasma flow jet power of 50-200W, a jet distance of 5-20cm, a processing time of 30s-2min, and the gas is air and / or nitrogen. Preferably, the differential dielectric barrier discharge method described in step S2 adopts differential output dielectric barrier discharge, with a voltage of 80-120kV, a frequency of 10-50kHz, a processing time of 1-5min, and air as the gas. Preferably, the moisture content of the camellia seeds in step S1 is 8.92%.

[0015] Preferably, the processing power and time in step S2 need to be controlled so that the total energy input is ≤500kJ / kg.

[0016] In summary, the beneficial effects of this invention are as follows: In summary, the beneficial effects of this invention are as follows: It achieves microporousization and roughening of the camellia seed coat surface under non-thermal conditions of normal pressure and low temperature, significantly improving the mass transfer channels for water and solutes and reducing adverse effects on heat-sensitive active ingredients. By directionally disrupting the non-covalent bonds of the outer shell, inner seed coat, and kernel cell walls and inducing microperforation, it promotes the release of cell contents, mechanistically supporting the improvement of subsequent drying, pressing, and extraction efficiency. Under optimized parameters, it achieves key enzymatic inhibition, with DBD (120kV-1min) and RF treatments reducing lipase by approximately 31.72% and 34.19%, and lipoxygenase by approximately 35.69% and 29.54%, respectively, effectively reducing the risk of oil hydrolysis and oxidation. It significantly enhances the antioxidant system; RF treatment can increase the total phenol content to 1.58 times that of the control, and antioxidant indicators such as DPPH, ABTS, and FRAP can be increased by up to 2.61 times, 1.51 times, and 2.42 times, respectively, contributing to improved oil quality stability. A clear parameter window and response pattern were established, demonstrating that low-power or short-time treatment is superior to high-power, long-time treatment, avoiding the initial increase followed by a decrease in total phenols and excessive degradation of flavonoids, thus improving process controllability and repeatability. Optimal modes and conditions suitable for direct scale-up are provided, with DBD (120kV-1min) and RF (3 m / min, 3 mm treatment distance, 20 passes) showing the best overall performance. JET and DF modes, when applied within specific boundaries, can avoid a decrease in antioxidant capacity. The process is easily integrated into existing production lines and can serve as an online pretreatment unit before drying, pressing, or solvent extraction, enabling rapid, continuous, and standardized modification. Sufficient microscopic evidence is provided; SEM reveals features such as seed shell cavitation, inner seed coat damage, and kernel curling, directly verifying the physical basis of structural opening and the release of active ingredients. Quality indicators are synergistically improved, with enhancements in total phenols and overall antioxidant capacity combined with inhibition of key enzyme activity, providing a reliable guarantee for improving the quality and stability of virgin oil. A parameterized control framework adaptable to different raw materials and operating conditions can be used to construct standard process windows based on factors such as voltage, time, distance, and number of passes, facilitating consistent quality and large-scale application. Attached Figure Description

[0017] Figure 1 This is a diagram of CP processing methods (A: dielectric barrier discharge, B: plasma flow, C: radio frequency, D: differential).

[0018] Figure 2 This is a diagram showing the effect of CP treatment on the microstructure of camellia seeds (A: seed coat, B: inner seed coat, C: kernel, CK: control group, DBD: S3, JET: S10, DF: S11, RF: S12).

[0019] Figure 3 These are comparison images showing the effects of cold plasma treatment on the appearance of camellia seeds. Figure 3 A represents the control group (untreated) camellia seeds. Figure 3B represents camellia seeds treated with cold plasma.

[0020] Figure 4 This is a comparative graph showing the effects of cold plasma treatment on the total phenolic and total flavonoid content of camellia seed methanol extract. Figure 4 A is a graph showing the change in total phenol content. Figure 4 B is a graph showing the change in total flavonoid content.

[0021] Figure 5 This is a graph showing the effect of cold plasma treatment on the antioxidant capacity of camellia seed methanol extract. Figure 5 A represents the result of the DPPH free radical scavenging capacity assay. Figure 5 B represents the result of the ABTS free radical scavenging ability test. Figure 5 C represents the result of the FRAP iron ion reducing power determination. Detailed Implementation

[0022] The present invention will now be described in detail through specific embodiments. However, these exemplary embodiments are for illustrative purposes only and are not intended to limit the actual scope of protection of the present invention in any way, nor are they intended to restrict the scope of protection of the present invention to these embodiments. For parameter ranges not mentioned, intermediate values ​​are selected.

[0023] Materials and Methods The experimental materials are as follows: camellia seeds with a moisture content of 8.92%.

[0024] The reagents are as follows: methanol, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, ferric chloride, sodium acetate, acetic acid, sodium nitrite, aluminum nitrate, anhydrous sodium carbonate, sodium hydroxide (analytical grade, Sinopharm Chemical Reagent Co., Ltd.), 99% linoleic acid, Tween 20, 4-nitrophenyl laurate, p-nitrophenol, gallic acid, rutin, quinoline dimethacrylate, ABTS working stock solution, 2,2-biphenyl-1-picrylhydrazine, 2,4,6-tripyridyltriazine (Shanghai Maclean Biochemical Technology Co., Ltd.), borate buffer, and Folin-Ciocalteu (Hefei Bomei Biotechnology Co., Ltd.).

[0025] Experimental equipment: CPCS-I type high voltage electric field low temperature cold plasma sterilization experimental equipment, CTP-2000S differential output low temperature plasma equipment, radio frequency single electrode low temperature plasma treatment equipment (Nanjing Suman Plasma Technology Co., Ltd.), GZX-9240 digital display forced-air drying oven (Shanghai Boxun Industrial Co., Ltd.), Biotek microplate reader (Berten Instruments Co., Ltd., USA), UV-1200 ultraviolet spectrophotometer (Shanghai Aoyi Instrument Co., Ltd.), LC-LX-HR185C benchtop high-speed refrigerated centrifuge (Shanghai Lichen Bangxi Instrument Technology Co., Ltd.), SHY-2 constant temperature water bath shaker (Changzhou Guoyu Instrument Manufacturing Co., Ltd.), SU8600 scanning electron microscope (Hitachi High-Tech International Trading Co., Ltd.).

[0026] The experimental method is as follows: CP pretreatment of camellia seeds: Naturally sun-dried camellia seeds were used as the control group (CK). Camellia seeds were treated with four methods: DBD, JET, DF (differential-feed dielectric barrier discharge), and RF, respectively. Figure 1 As shown. The sample processing methods are: S1 (90kV-1min), S2 (90kV-5min), S3 (90kV-10min), S4 (120kV-1min), S5 (120kV-5min), S6 (120kV-10min), S7 (120kV-15min), S8 (150kV-10min), S9 (150kV-15min), S10 (JET), S11 (DF-DBD), and S12 (RF, 3 m / min, distance 0-3 mm, 20 passes).

[0027] Effects of CP pretreatment on lipase and lipoxygenase activities in Camellia oleifera seeds: The effects of CP pretreatment on lipase and lipoxygenase activities in Camellia oleifera seeds were investigated according to the Tolouie method (TOLOUIE H, MOHAMMADIFAR MA, GHOMI H, et al. The impact of atmospheric cold plasma treatment on inactivation of lipase and lipoxygenase of wheat germs[J]. Innovative Food Science and Emerging Technologies, 2018, 47: 346-352). The lipase activity was determined as follows: 1 g of crushed Camellia oleifera seeds were weighed into a 50 mL centrifuge tube, 5 mL of phosphate buffer (100 mmol / L, pH=7.0) was added, and the mixture was extracted by shaking at 200 rpm at a constant temperature (30℃) for 20 min. The enzyme extract was then centrifuged at 4℃ for 15 min and filtered for later use. Enzyme activity of the enzyme extract was measured using 4-nitrophenyl laurate as a substrate. 1 mL of enzyme extract was added to 2.5 mL of 420 µmol / L 4-nitrophenyl laurate solution, and the reaction was allowed to proceed for 30 min. The absorbance at 410 nm was then measured. One unit of lipase activity (U) was defined as the amount of enzyme required to release 1 µmol / L of p-nitrophenol per minute under the assay conditions. Enzyme activity results were expressed as U / mg. The method for determining lipoxygenase was as follows: The conjugated diene product formed by linoleic acid under the catalysis of lipoxygenase was determined using a spectrophotometer. The linoleic acid substrate solution was prepared according to the Tolouie method (TOLOUIE H, MOHAMMADIFAR MA, GHOMI H, et al. The impact of atmospheric coldplasma treatment on inactivation of lipase and lipoxygenase of wheat germs[J]. Innovative Food Science and Emerging Technologies, 2018, 47: 346-352). 30 µL of the substrate solution was added to 2.97 mL of borate buffer (pH 6.0) and 5 µL of enzyme extraction solution. The absorbance at 234 nm was immediately measured. The lipoxygenase activity results were referenced in the literature (Wang Jing, Tang Xuxiao, Zhang Yingzhong, et al. Effects of lipoxygenase on the quality and flavor of tea oil[J]. Modern Agricultural Science and Technology, 2023(06):). Calculate (193-196+201), and the result is expressed as U / g.

[0028] Effects of CP pretreatment on the microstructure of camellia seeds: The microstructure of the outer shell, inner seed coat, and kernel of camellia seeds was observed using SEM (scanning electron microscopy), following the method of Zhang (ZHANG RY, LIU AB, LIU C, et al. Effects of different extraction methods on the physicochemical properties and storage stability of tiger nut). Cyperus esculentus The effect of CP pretreatment on the cell structure of camellia seed was observed. Effect of CP pretreatment on the antioxidant capacity of camellia seed: The method of methanol extract of camellia seed was carried out according to the reference (OBIED HK, SONG Y, FOLEY S, et al. Biophenols and antioxidant properties of Australian canola meal[J]. Journal of Agricultural and Food Chemistry, 2013, 61(38): 9176-9184). The total phenol content, total flavonoids and antioxidant capacity (DPPH free radical scavenging capacity, ABTS free radical scavenging capacity, FRAP reducing capacity) of methanol extract were determined according to Zeng Xueying's method (Zeng Xueying. Preliminary study on antioxidant components of camellia meal based on solvent extraction and online detection[D]. Changsha: Central South University of Forestry and Technology, 2022). Total phenol content was determined using the Folin-Ciocalteu method. Gallic acid was used as the standard, and gallic acid standard solutions of different concentration gradients were prepared. A standard curve was plotted with known concentrations of gallic acid on the x-axis and absorbance values ​​on the y-axis: y1 = 1.4943x1 - 0.0019, R0. 2 =0.9997 The result is expressed as gallic acid equivalent (mgGAE / g). Total flavonoids were determined using the sodium nitrite-aluminum nitrate colorimetric method. A standard curve equation was established with known concentrations of rutin as the x-axis and absorbance values ​​as the y-axis: y² = 0.0007x² + 0.0759, R0 2=0.9939, the result is expressed as mgRE / g. DPPH free radical scavenging capacity determination: Accurately weigh 0.0039 g DPPH powder, dilute to 100 mL with methanol to prepare a 0.1 mM DPPH solution, prepared fresh for use; take 10 µL of Camellia oleifera seed methanol extract, add 190 µL of DPPH methanol solution, react in the dark for 30 min, and use methanol as a blank, perform three parallel measurements, measuring the absorbance at 517 nm. Prepare Trolox methanol solutions of different concentrations and react with DPPH methanol solution to establish a standard curve equation. Antioxidant capacity is expressed as µmol TE / L. ABTS+ free radical assay: Take 1 mL of ABTS stock solution, dilute with methanol to make the absorbance of the working solution at 734 nm 0.7 ± 0.02, and prepare fresh before use. Take 10 µL of camellia seed methanol extract, add to 190 µL of the working solution, react in the dark for 30 min, and use methanol as a blank. Perform three parallel measurements, and measure the absorbance at 734 nm. The Trolox standard curve is established in the same way as above. The antioxidant capacity is expressed as µmol TE / L. FRAP reducing power assay: Prepare acetate buffer, TPTZ solution, and 20 mmol / L ferric chloride solution separately. Mix them in a 10:1:1 ratio to prepare the FRAP working solution before use. Take 10 µL of camellia seed methanol extract, add to 190 µL of the working solution, react in the dark for 30 min, and use methanol as a blank. Perform three parallel measurements, and measure the absorbance at 593 nm. The Trolox standard curve is established in the same way as above. The antioxidant capacity is expressed as µmol TE / L.

[0029] Results and Analysis Effects of CP pretreatment on the microstructure of Camellia oleifera seeds: Scanning electron microscopy can reveal changes in the microstructure of Camellia oleifera seeds. CP treatment resulted in varying degrees of alteration in the microstructure. Taking DBD-treated S3, JET-treated S10, DF-treated S11, and RF-treated S12 samples as examples, the effects of CP treatment on the seed coat, inner seed coat, and kernel structure of Camellia oleifera seeds are as follows: Figure 2 As shown. The surface structure of untreated camellia seed shells is dense, smooth, and intact. Figure 2After CP treatment, the seed coat of the camellia seed (-CK) underwent significant changes, generally exhibiting roughness and etching marks, as well as obvious cavities (e.g., DBD, RF) and varying degrees of structural damage (e.g., DBD, DF, RF). Furthermore, the effects of CP treatment penetrated the outer shell and extended to the inner seed coat, causing structural damage and cavities in the inner seed coat of some samples (e.g., DBD, DF, RF). In the control group, the internal structure of the camellia seed kernel remained intact, with wavy wrinkles in the cell walls due to drying and dehydration. In contrast, the CP-treated experimental group showed varying degrees of curling in the kernels, indicating damage to the cell structure. This damage to the cell walls and other structures may contribute to oil release, explaining why CP treatment can increase the oil yield of camellia seeds. The high-energy particles generated by CP mainly act on the outer shell of camellia seeds. By breaking the non-covalent bonds (such as hydrophobic bonds and hydrogen bonds) between cell wall polymers (such as cellulose, hemicellulose and pectin), the mechanical strength of the cell wall skeleton is weakened and micropores are induced. These micropores provide channels for water to enter, which is conducive to the release of active ingredients such as phenols. This proves that CP pretreatment has the application prospect of improving oil yield and active ingredients.

[0030] Effects of CP treatment on lipase and lipoxygenase activities in Camellia oleifera seeds: CP treatment reduced lipase activity in Camellia oleifera seeds, with S2 (90kV-5min), S4 (120kV-1min), and S10 (JET) showing reductions of 37.56%, 31.72%, and 34.19% in lipase activity compared to the control group, respectively. Treatment power and treatment time significantly affected lipase activity; low-power, short-duration CP treatment was more effective in reducing lipase activity. Lipoxygenase catalyzes the oxidation of unsaturated fatty acids to produce hydroperoxides, and CP treatment reduced lipoxygenase activity in Camellia oleifera seeds. Compared to the control group, S4 (120kV-1min) and S12 (RF) showed significant reductions in lipoxygenase activity of 35.69% and 29.54%, respectively. The reduction in enzyme activity is generally related to changes in the secondary and tertiary structures of the enzymes caused by CP treatment. In summary, the lipase and lipoxygenase activities of S4 (120kV-1min) and S12 (RF) were significantly lower than those of the control group.

[0031] Effects of CP treatment on the total phenolic and total flavonoid content of camellia seed methanol extract: After CP treatment, the total phenolic content of camellia seed methanol extract was 4.84-7.86 mg GAE / g ( Figure 4 A). Compared with untreated samples, the total phenol content of samples S1-S4, S6, and S8-S11 increased by 1.82%-26.41%, but the differences were not significant. P >0.05), the total phenol content of S12 was 1.58 times higher than that of the control group, and significantly higher than that of all other experimental group samples ( P<0.05). Under the same power treatment conditions, the total phenol content usually increases first and then decreases with prolonged treatment time. This study is consistent with previous results; an appropriate treatment time can increase the content of active ingredients, while excessive treatment time leads to the degradation of total phenols and other active ingredients. The reactive oxygen species generated by plasma discharge act on the aromatic rings of phenolic substances, causing hydroxylation or the formation of quinone compounds, initiating aromatic ring cleavage and phenolic degradation. The total flavonoid content of the methanol extract of Camellia oleifera seeds after CP treatment was 3.45-7.09 mgRE / g ( Figure 4 B). Compared with untreated samples, the total flavonoid content of CP-treated samples (S1-S3, S5, S7-S11) decreased by 6.71%-46.18%, which may be related to the increased treatment power. Some flavonoids can be degraded into small molecule compounds such as phenols after CP treatment. The total flavonoid content of samples S4, S6, and S12 increased by 1.25, 1.00, and 1.11 times respectively compared with untreated samples, but the differences were not significant. P >0.05). Therefore, selecting appropriate CP treatment parameters can increase the flavonoid content in the sample.

[0032] Effect of CP treatment on the antioxidant capacity of Camellia oleifera seed methanol extract: The DPPH free radical scavenging capacity of Camellia oleifera seed methanol extract after CP treatment was 124.76-373.09 µMTE / L ( Figure 5 A) CP treatment reduced the DPPH radical scavenging ability of S3 (90kV-10min), S7 (120kV-15min), and S11 (DF), but the effect was not significant. P >0.05). CP treatment was significant ( P <0.05) increased the DPPH radical scavenging ability of S12 (RF) and S4 (120kV-1min) methanol extracts by 2.61 times and 2.23 times, respectively, compared with the control group. The ABTS radical scavenging ability of the camellia seed methanol extract after CP treatment was 220.16-373.84µMTE / L ( Figure 5 B), CP treatment significantly reduced the ABTS radical scavenging ability of S7 (120KV-15min), S10 (JET), and S11 (DF). P <0.05) enhanced the free radical scavenging ability of S2 (90kV-5min), S4 (120kV-1min), S5 (120kV-5min), S8 (150kV-10min), and S12 (RF) ABTS by 1.40, 1.24, 1.28, 1.40, and 1.51 times, respectively, compared with the control group. The FRAP reducing power of the methanol extract of Camellia oleifera seeds after CP treatment was 333.92-848.92 µMTE / L (…). Figure 5(C) Among them, S4 (120kV-1min), S5 (120kV-5min), S8 (150kV-10min), S9 (150kV-15min), S11 (DF), and S12 (RF) showed increases of 1.57, 1.18, 1.45, 1.40, 1.22, and 2.42 times compared to the control group, respectively, indicating a significant improvement in antioxidant capacity. In summary, compared to the control group, the methanol extracts of S4 (120kV-1min) and S12 (RF) showed significantly enhanced antioxidant capacity.

[0033] In summary, CP treatment, as a non-thermal processing technique, can significantly alter the surface structure of Camellia oleifera seed coats. CP treatment disrupts the cellular structure of the seed coat and inner seed coat, facilitating the dissolution of intracellular active ingredients, thus resulting in significantly higher total phenolic content and antioxidant capacity in the methanol extract of Camellia oleifera seeds compared to the control group. Different CP treatment modes showed differences. Under the DBD mode, high-power, short-time treatment (120kV-1min) effectively reduced lipase activity while improving antioxidant capacity; JET treatment may have reduced total flavonoid content and antioxidant capacity due to the introduction of moisture; DF treatment reduced lipase activity but negatively impacted antioxidant capacity; RF treatment showed good effects in both reducing enzyme activity and enhancing antioxidant capacity. Overall, RF treatment and DBD (120kV-1min) treatment were the most effective in altering the structure of the seed coat and inner seed coat of Camellia oleifera seeds and promoting the release of active ingredients. The results of this study provide a theoretical basis for the application of CP technology in the pretreatment and subsequent processing of Camellia oleifera seeds.

[0034] The detailed descriptions listed above are merely specific illustrations of feasible embodiments of the present invention and are not intended to limit the scope of protection of the present invention. All equivalent embodiments or modifications made without departing from the spirit of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for pretreating camellia seeds using low-temperature plasma, characterized in that, Includes the following steps: S1. Select fresh or dried camellia seeds with a moisture content of 8.0-60.0% as the treatment target; S2. Under normal pressure, the camellia seeds obtained in step S1 are subjected to surface discharge pretreatment using low-temperature plasma technology. The surface discharge pretreatment methods include dielectric barrier discharge, plasma flow, differential dielectric barrier discharge, or radio frequency single-electrode plasma. For dielectric barrier discharge, the discharge gas is air, the voltage is 90-150kV at normal pressure, and the treatment time is 1-15min. The parameters for low-voltage short-time treatment are as follows: 90kV, 1-5min; for medium-voltage treatment, 120kV, 1-15min; and for high-voltage long-time treatment, 150kV, 1-15min. For plasma flow, direct plasma jetting is used. For differential dielectric barrier discharge, differential output dielectric barrier discharge is used. For radio frequency single-electrode plasma, the transmission speed is 2-4m / min, the treatment distance is 0-3mm, and the number of reciprocating treatments is 15-25 times. S3. After surface discharge pretreatment in step S2, the camellia seeds are left to stand at room temperature for 10-30min before subsequent drying, pressing, or solvent extraction processes.

2. The method for pretreating camellia seeds using low-temperature plasma according to claim 1, characterized in that, The dielectric barrier discharge method described in step S2 has a voltage of 120kV under normal pressure and a processing time of 1min.

3. The method for pretreating camellia seeds using low-temperature plasma according to claim 1, characterized in that, The discharge parameters for the radio frequency single-electrode plasma method described in step S2 are: transmission speed of 3 m / min, processing distance of 3 mm, reciprocating 20 times, and the gas medium is air and / or argon.

4. The method for pretreating camellia seeds using low-temperature plasma according to claim 1, characterized in that, The plasma flow method described in step S2 uses a plasma flow jet power of 50-200W, a jet distance of 5-20cm, a processing time of 30s-2min, and the gas is air and / or nitrogen.

5. The method for pretreating camellia seeds using low-temperature plasma according to claim 1, characterized in that, The differential dielectric barrier discharge method described in step S2 uses differential output dielectric barrier discharge with a voltage of 80-120kV, a frequency of 10-50kHz, a processing time of 1-5min, and air as the gas.

6. The method for pretreating camellia seeds using low-temperature plasma according to claim 1, characterized in that, The moisture content of the camellia seeds mentioned in step S1 is 8.92%.

7. The method for pretreating camellia seeds using low-temperature plasma according to claim 1, characterized in that, The processing power and time in step S2 need to be controlled so that the total energy input is ≤500kJ / kg.

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