Enzymatic Degumming of Arachidonic Acid Oil Using Immobilized Phospholipase A1 on Hollow Double-Layer Mesoporous Silica Nanoparticles

Abstract

This study explores the application of immobilized phospholipase A1 (PLA1) on hollow double-layer mesoporous silica nanoparticles (PLA1@NH₂/C₈-HdlMS) for the degumming of crude arachidonic acid (ARA) oil for the first time. The immobilized enzyme was comprehensively characterized, and the reaction conditions were optimized via single-factor experiments. Under the optimized conditions (enzyme dosage 0.3% w/w, 35 °C, water addition 3%, and reaction time 90 min), PLA1@NH₂/C₈-HdlMS achieved a remarkable phosphorus removal rate of 97.9%, reducing the phosphorus content from 441.21 mg/kg to 9.29 mg/kg in 90 min (well below the food-grade standard of <10 mg/kg). The fatty acid composition of the oil remained almost unchanged, while the oxidative induction time of the degummed oil significantly improved by 42%. Notably, PLA1@NH₂/C₈-HdlMS demonstrated broad applicability across crude oils, with initial phosphorus contents ranging from 294.98 mg/kg to 537.44 mg/kg, and it maintained ~93% of its initial activity after 11 reuse cycles. Compared to traditional hydration degumming (with a phosphorus removal rate of 56.3%), this enzymatic method offers superior efficiency at lower temperatures, minimizing energy consumption and the thermal degradation of ARA. This green, efficient, and sustainable method for degumming heat-sensitive oils offers significant potential for the industrial application of high-quality functional oils by preserving PUFA integrity and reducing environmental impact.

1. Introduction

Arachidonic acid (ARA, C20:4, ω-6) is an essential polyunsaturated fatty acid (PUFA) in the human diet, playing a critical role in health and nutrition [1,2]. As a key component of membrane phospholipids, particularly in the brain and retina, ARA is involved in modulating cell signaling [3], facilitating normal inflammatory responses [4,5], and supporting the normal development and function of the nervous system [6,7,8]. Due to its nutritional importance, ARA has broad applications in the food, pharmaceutical, and nutritional industries, particularly in infant formula [9,10], functional foods [11], high-end edible oils [12], and anti-inflammatory and neuroprotective agents [13,14]. The growing market demand for ARA has driven research into sustainable production and efficient refining technologies, particularly in the degumming process.

Degumming is the initial and critical step in edible oil refining, aiming to remove phospholipids, proteins, and mucilaginous gums that affect oil quality, stability, and downstream processing [15,16]. Traditional degumming methods (hydration, acidification, and physical adsorption) often involve high-temperature heating and vigorous mixing, which exacerbate the oxidation of thermosensitive oils like ARA, leading to the formation of harmful byproducts (e.g., 3-chloropropanol esters and glycidyl fatty acid esters) and loss of nutritional value. These methods also fail to effectively remove non-hydratable phospholipids, leading to off-flavors and complications in downstream processing. Additionally, they suffer from drawbacks such as high wastewater generation, oil losses, and the removal of valuable nutritional components [17,18]. Despite the growing demand for high-quality ARA oil, degumming technologies tailored to thermosensitive PUFAs remain limited, highlighting an urgent need for green, safe, and efficient alternatives.

Enzymatic degumming has emerged as a promising solution due to its high dephosphorization efficiency (<10 mg/kg phosphorus content), broad applicability, minimal wastewater generation, and low energy consumption [19]. Phospholipase A1 (PLA1) and phospholipase C (PLC) are widely used for hydrolyzing phospholipids at specific ester bonds [20]. For example, the commercial PLA1 Lecitase^® Ultra has been shown to reduce the phosphorus content while preserving oil quality and nutritional value [21,22]. Similarly, Purifine^® PLC not only effectively reduces the phosphorus content but also enhances oil yield by generating diglycerides [23]. Ultrasound-assisted enzymatic degumming has also been explored, achieving high dephosphorization rates of up to 98.82%, thereby providing a deep degumming effect [24]. However, despite these advantages, the industrial application of free phospholipases is constrained by high costs, poor reusability, and difficult separation from the oil matrix [25].

To address these challenges, enzyme immobilization has been developed to enhance enzyme stability, reusability, and overall cost effectiveness [26,27]. Various carrier materials have been developed for phospholipase immobilization (e.g., polydopamine nanoparticles [28], polycarboxylated magnetic nanoparticles [29], and protein–inorganic hybrids [30]), but their performance in ARA oil degumming remains underexplored. Notably, these traditional carriers exhibit drawbacks that are particularly incompatible for the degumming of thermosensitive ARA oil: (1) inadequate catalytic activity under mild reaction conditions (30–40 °C), requiring excessive enzyme dosage to meet dephosphorization standards; (2) poor interfacial affinity between the carrier and the oil–water system, as most carriers are either overly hydrophilic or hydrophobic, failing to concentrate phospholipids at the reaction interface and thus limiting enzyme–substrate interactions; and (3) inadequate stability, as the immobilized enzyme tends to leach or denature during reuse, compromising reusability. In essence, the lack of precise surface modification in traditional carriers results in a trade-off between enzyme activity, stability, and compatibility with thermosensitive oils, which severely restricts their application in high-quality ARA oil refining. Our previous work demonstrated that bifunctional modification of silica-based carriers with alkyl and nitrogen-containing alkyl amines enhances the activity and stability of immobilized enzymes by strengthening hydrogen-bonding interactions between the carrier and enzyme and maintaining a hydrophobic microenvironment at the oil–water interface that facilitates substrate enrichment [31,32,33]. We hypothesized that this strategy could be extended to phospholipase immobilization for the degumming of thermosensitive oils, thereby filling the gap in tailored carriers for thermosensitive PUFA-rich oil refining.

In this study, hollow double-layer mesoporous silica nanoparticles surface-modified with aminoalkyl and alkyl groups were prepared for PLA1 immobilization (PLA1@NH₂/C₈-HdlMS). The objectives were to: (1) optimize the enzymatic degumming conditions for crude ARA oil; (2) characterize the immobilized enzyme and clarify the structure–activity relationship; (3) evaluate the degumming efficiency, fatty acid retention, oxidative stability, and reusability of PLA1@NH₂/C₈-HdlMS; (4) compare the performance with traditional hydration degumming; and (5) discuss the potential applications in functional food production. This work provides a comprehensive reference for the industrial application of immobilized phospholipases in the degumming of thermosensitive oils.

2. Results and Analysis

2.1. Screening of PLA1

Upon assaying, the activities of four phospholipases were determined as follows: S-PLA₁ = 165 U/mL, PLB = 180 U/mL, V-PLA₁ = 178 U/mL, and Lecitase Ultra = 200 U/mL, with values relatively close to each other. In contrast, PLC exhibits a distinct hydrolysis site, resulting in extremely low activity and negligible fatty acid release under this assay system (Figure 1). Then the degumming efficiencies of five phospholipases—PLC, S-PLA1, PLB, V-PLA1, and Lecitase Ultra (PLA1) were compared under optimal conditions (phospholipase solution 24 mg/mL, 0.5 mL, pH 5.8, 45 °C, 3 h) [34], with a crude ARA oil containing 441.2 mg/kg of phospholipids. As shown in Figure 1, after degumming, the phosphorus content and degumming rate of the treated oils were 335.1 mg/kg, 58.6 mg/kg, 14.6 mg/kg, 11.0 mg/kg, and 8.6 mg/kg and 19.5%, 86.7%, 96.6%, 97.5%, and 98.0%, respectively. The results indicated that Lecitase Ultra achieved the best degumming performance. Thus, Lecitase Ultra was selected for the subsequent experiments.

2.2. Characterization of HdlMS and PLA1@NH₂/C₈-HdlMS

The morphology of HdlMS was thoroughly characterized using scanning electron microscopy (SEM, SU8010, Hitachi, Tokyo, Japan) and transmission electron microscopy (TEM, FEI Tecnai G2 F30, Hillsboro, America). TEM images revealed that HdlMS exhibited a uniform double-layered hollow spherical structure with an average diameter of 500 nm. The outer shell thickness of HdlMS was approximately 56 nm, while the inner shell thickness was about 26 nm (Figure 2a,b). In the Fourier transform infrared (FTIR, TENSOR 27 FTIR, Bruker, Karlsruhe, Germany) spectrum, the peak at 1089 cm⁻¹ corresponded to the symmetric bending vibration of Si-O-Si bonds, confirming the presence of silica in the HdlMS structure. The peaks at 2927 cm⁻¹ and 2856 cm⁻¹ were attributed to the stretching vibrations of -CH and -CH₂ groups, respectively, indicating the successful introduction of alkyl groups. Additionally, the broad peak at 3363 cm⁻¹ was indicative of the NH₂ group, confirming that both the octyl and amino groups have been successfully grafted onto the HdlMS surface. The FTIR spectrum of PLA1@NH₂/C₈-HdlMS showed unique peaks at 1463 cm⁻¹ (C-H bending vibration of protein) and 1630 cm⁻¹ (amide I band, C=O stretching vibration of PLA1), confirming the successful immobilization (Figure 2c). The nitrogen adsorption-desorption isotherms, analyzed via the Brunauer–Emmett–Teller (BET) method, revealed the specific surface areas were 1158.3 m²/g for HdlMS, 129.7 m²/g for NH₂/C₈-HdlMS, and 1.8 m²/g for PLA₁@NH₂/C₈-HdlMS. The progressive reduction in specific surface area following surface modification and enzyme immobilization indicated effective immobilization of PLA1 onto the modified carrier (Figure 2d, Figure S1, Table S1). In addition, X-ray photoelectron spectroscopy (XPS, FEI Tecnai G2 F30, Hillsboro, America) was employed to further characterize HdlMS, NH₂/C₈-HdlMS, and PLA1@NH₂/C₈-HdlMS. As shown in Figure 2e, XPS analysis revealed characteristic peaks for silicon (Si2p and Si2s), carbon (C1s), nitrogen (N1s), and oxygen (O1s), with binding energies of 103.08 eV, 154.08 eV, 285.08 eV, 400.08 eV, and 532.08 eV, respectively. The intensities of the C1s and N1s peaks gradually increased from HdlMS to NH₂/C₈-HdlMS and PLA1@NH₂/C₈-HdlMS (Table S2), further corroborating the successful modification with amino and alkyl groups and the immobilization of PLA1 on the HdlMS carrier. Thermogravimetric analysis (TGA, Netzsch Thermogravimetric Analyzer, Selb, Germany) was conducted to assess the thermal stability of HdlMS, NH₂/C₈-HdlMS, and PLA1@NH₂/C₈-HdlMS (Figure 2f). All samples exhibited an initial weight loss of approximately 6% when heated to 100 °C, likely due to the removal of adsorbed moisture. HdlMS demonstrated robust thermal stability, with minimal mass loss up to 700 °C. In contrast, NH₂/C₈-HdlMS showed a mass loss of 11% between 250 °C and 600 °C, attributed to the degradation of the surface-modifying groups. PLA1@NH₂/C₈-HdlMS exhibited two distinct mass loss stages: 34% between 100 °C and 250 °C and 20% between 250 °C and 600 °C. The first stage corresponded to the thermal decomposition of the immobilized PLA1, while the second stage was associated with the degradation of the surface groups of the modified carrier. These results highlight the successful immobilization of PLA1 and the enhanced thermal stability of the modified HdlMS carrier. The enzyme loading and specific activity for PLA1@NH₂/C₈-HdlMS were determined to be 18.6 mg/g and 175 U/g, respectively.

2.3. Optimal Conditions for Degumming of Crude ARA Oil

The activity of immobilized enzyme is significantly influenced by reaction conditions, which, in turn, affect degumming efficiency. This section investigated four key factors—reaction time, enzyme dosage, reaction temperature, and water addition—on degumming efficiency. Optimizing immobilized enzyme dosage and reaction time is crucial for controlling reaction costs while maximizing efficiency. For both immobilized and free enzymes, degumming efficiency increased rapidly within the first 30 min and then plateaued with prolonged reaction time. This trend was likely attributed to the decreasing concentration of phospholipids as the reaction progressed, which reduced the likelihood of enzyme–substrate interactions and thereby affected the reaction rate (Figure 3a). Moreover, the degumming efficiency increased significantly with the addition of immobilized enzyme. When the amount of immobilized enzyme was 0.67 g/kg, the phosphorus removal rate reached approximately 90.7% after 180 min of reaction, but equilibrium was not achieved. However, when the dosage of PLA1@NH₂/C₈-HdlMS increased to or exceeded 5.0 g/kg, the reaction reached equilibrium within only 30 min, with a degumming efficiency exceeding 97.0%. In contrast, free enzyme (16.67 mL/kg) achieved a maximum degumming efficiency of 94.5% after 140 min of reaction, which was slightly inferior to the performance of immobilized enzyme at a dosage of 1.67 g/kg. The increase in the number of reaction active centers enhanced the likelihood of phospholipids adsorbing onto the immobilized enzyme active sites, thereby improving the efficiency of the reaction rate. When the amount of added enzyme approaches saturation, further increases in enzyme quantity have minimal impact on the reaction rate. This phenomenon is primarily due to the fact that phospholipase-catalyzed phospholipid hydrolysis occurs at the oil–water interface [26]. When factors such as enzyme quantity, interfacial area, and phospholipid concentration reach saturation levels, the microscale mass transfer at the interface becomes the predominant limiting factor for degumming efficiency. Considering industrial feasibility, excessive reaction time may lead to oil oxidation and enzyme inactivation. Therefore, a balanced approach was adopted: a ratio of 100 mg of immobilized enzyme to 30 g of oil was selected for further experiments. Reaction temperature is a critical factor, influencing both energy consumption and product quality. Figure 3b showed that as the reaction temperature increased from 30 °C to 50 °C, the phosphorus removal rate dropped from 98% to 75%. This trend is inconsistent with previous reports [35], which reported maximal phospholipase activity at higher temperatures. The discrepancy may be attributed to the different effects of temperature on the lipase and phospholipase activities of PLA1. The authors hypothesized that the immobilization process altered the temperature sensitivity of PLA1. Initially, PLA1@NH₂/C₈-HdlMS rapidly hydrolyzed phospholipids at 50 °C. However, as the incubation time was extended in this higher temperature environment, the immobilized enzyme exhibited increased esterification activity. This activity re-esterified the hydrolyzed fatty acids back into the molecular skeleton of the phospholipids, thereby reducing the overall degumming efficiency. After comprehensive evaluation, a reaction temperature of 35 °C was chosen as the optimal, balancing efficiency and practicality. This observation can be ascribed to two key underlying mechanisms: (1) PLA1@NH₂/C₈-HdlMS adopts a constrained conformation via its interactions with the support matrix, and temperatures ≥40 °C induce subtle alterations in the three-dimensional structure of the enzyme’s active site—including the displacement of critical serine residues—thereby diminishing its substrate-binding affinity. (2) Elevated temperatures augment the inherent esterification activity of PLA1, which promotes the re-esterification of hydrolyzed fatty acids and lysophospholipids back into intact phospholipid species; this consequently lowers the overall phosphorus removal efficiency [23]. Collectively, this dual-mechanism framework underscores the critical role of low-temperature conditions in the enzymatic degumming of thermosensitive oils while also furnishing a theoretical foundation for the precise temperature regulation of industrial enzymatic degumming operations. Concurrently, it is noteworthy that enzymatic degumming can be effectively achieved at lower temperatures, thereby operating under more energy-efficient and environmentally friendly conditions. This aligns with the broader goals of green chemistry and sustainable industrial processes. Water dosage plays a dual role in enzymatic degumming: stimulating the “interface activation effect” of phospholipases and aiding the separation of hydrolysis products. Figure 3c within 90 min demonstrated that the phosphorus removal rate initially increased from 96.6% to 97.9% and then decreased to 97.5% as water addition rose from 1.5% to 3.5%. Optimal water content enhanced enzyme–substrate interaction, but excessive water potentially exacerbating oil emulsification, which hinders separation. Considering these factors, a water addition of 3% was determined to be optimal. Through systematic optimization, the ideal enzymatic degumming process for crude ARA oil using PLA1@NH₂/C₈-HdlMS was established: a reaction temperature of 35 °C, an enzyme dosage of 0.3% (w/w), a water addition of 3%, and a reaction time of 90 min. Under these conditions, the phosphorus removal rate reached 97.9%, reducing the phosphorus content of ARA crude oil from 441.21 mg/kg to 9.29 mg/kg, well within the safety standards for edible oils.

2.4. Analysis of Fatty Acid Composition and Oxidative Stability

Table 1. Fatty acid profile of ARA oil: pre- and post-degumming (mean ± SD, n = 3).

	Crude ARA Oil	Degummed ARA Oil
C16:0	6.55 ± 0.23	6.25 ± 0.16
C18:0	5.14 ± 0.18	4.97 ± 0.13
C18:1	4.40 ± 0.15	4.18 ± 0.11
C18:2	3.70 ± 0.13	3.81 ± 0.04
C18:3	2.44 ± 0.10	2.28 ± 0.03
ARA	57.62 ± 1.14	56.50 ± 1.10
C22:1	3.57 ± 0.11	3.79 ± 0.09
C22:6	12.73 ± 0.41	13.99 ± 0.36
other fatty acids	3.87 ± 0.17	4.26 ± 0.19

and Figure S2 provide a detailed overview of the fatty acid composition of crude and degummed ARA oil. In the pre-degumming sample, ARA was the predominant fatty acid, comprising 57.62% of the total fatty acids, followed by docosahexaenoic acid (DHA) at 12.73%. After degumming, the ARA content slightly decreased to 56.50%, while DHA content increased to 13.99%. The minor changes in the fatty acid profile indicated that the degumming process has a negligible impact on the overall fatty acid composition of ARA oil. These minimal changes in fatty acid composition were attributed to the low reaction temperature (35 °C), which avoided the oxidative degradation of ARA that often occurs in high-temperature traditional degumming.

The oxidative stability of ARA oil was evaluated via the Rancimat method, with oxidative induction time serving as a key indicator of its resistance to oxidation. The degumming process significantly improved the oxidative stability of ARA oil, increasing the Rancimat induction time from 36 to 51 min, a 42% enhancement (Figure S3). This improvement can be attributed to two primary mechanisms: (1) effective removal of phospholipids, which act as prooxidants under thermal stress through decomposing into reactive species that accelerate lipid peroxidation; and (2) preservation of endogenous antioxidants enabled by the mild conditions of enzymatic degumming. Such enhanced oxidative stability is crucial for preserving the functional quality and nutritional value of ARA oil during storage and processing.

2.5. Wide Applicability and Reusability

To evaluate the universality of the optimized reaction conditions, degumming experiments were conducted on crude ARA oils with varying initial phosphorus contents. As illustrated in Figure 4a, the phosphorus contents of crude ARA oils, initially at 537.44 mg/kg, 441.21 mg/kg, 322.88 mg/kg, and 294.98 mg/kg, were reduced to 8.86 mg/kg, 9.76 mg/kg, 8.22 mg/kg, and 9.42 mg/kg, respectively, after the degumming process. The phosphorus removal rate decreased slightly from 98.3% to 96.7% as the initial phosphorus content in the crude oil decreased. However, regardless of the initial phosphorus content of the crude oil, the degumming process consistently reduced the phosphorus content to below 10 mg/kg for all tested oils. This finding demonstrated the robustness and broad applicability of PLA1@NH₂/C₈-HdlMS. Enzymatic degumming was subsequently compared with the hydration degumming method. In the hydration degumming process, the ARA oil was treated at 75 °C with the addition of 5% water, and the reaction was stirred at 300 r/min for 90 min [36]. Under these conditions, the phosphorus content of the degummed ARA oil was 192.81 mg/kg, corresponding to a phosphorus removal rate of only 56.3% (Figure 4b). This indicated that hydration degumming can only remove the hydratable phospholipids from the crude oil and has limited ability to remove non-hydratable phospholipids. Additionally, the repeatability of the immobilized enzyme was assessed through multiple cycles of degumming reactions. After each reaction, the immobilized enzyme was separated by centrifugation, recovered, and reintroduced into a new reaction system to continue the degumming process. The relationship between the relative activity of the immobilized enzyme and the number of reuse cycles is depicted in Figure 4c. Although the relative activity of the immobilized enzyme decreases gradually with increasing reuse cycles, it is noteworthy that after 11 cycles, the enzyme still retains approximately 93% of its initial activity. This indicated that the immobilized enzyme maintains high efficiency and stability over multiple uses, which is advantageous for industrial applications where enzyme reusability is crucial for cost effectiveness and sustainability.

It should be noted that scaling up from laboratory scale to industrial ton-scale batches presents distinct challenges, particularly for non-magnetic nanoparticle carriers. Drawing on practical experience in physical refining and moderate processing conditions, we anticipate that the technical maturity and scalability of the proposed approach can be further improved. This strategy is expected to achieve a favorable balance among catalytic efficiency, environmental sustainability, and nutrient retention, making it promising for industrial applications in high-quality functional oil refining.

3. Materials and Methods

3.1. Materials

The ARA crude oil was generously provided by CABIO Biotech Co., Ltd. (Wuhan, China) Lecitase Ultra and PLC were purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). S-PLA1 was provided by Ningxia Xiasheng Biotechnology Co., Ltd. (Yinchuan, China), V-PLA1 by Qingdao Weilan Biotechnology Co., Ltd. (Qingdao, China), and PLB by Angel Enzyme Preparation (Yichang) Co., Ltd. (Yichang, China). n-Octyltrichlorosilane (OTCS), 3-[2-(2-aminoethylamino)ethylamino]propyl-trimethoxysilane (AEPTES), tetraethyl orthosilicate (TEOS), cetyltrimethylammonium bromide (CTAB), sodium molybdate, and hydrazine sulfate were purchased from Aladdin (Shanghai) Co., Ltd. (Shanghai, China). Polyvinyl alcohol, concentrated sulfuric acid, ammonia water, ethanol, sodium carbonate, hydrochloric acid, and toluene were all purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Deionized water was produced using a Milli-Q Direct 8 pure water system (Carlsbad, CA, USA).

3.2. Preparation of HdlMS

Hollow double-layer mesoporous silica spheres (HdlMS) were synthesized via a modified literature method [37]. Briefly, ammonia water (24 mL), water (32 mL), and ethanol (400 mL) were thoroughly mixed in a three-necked flask under stirring. Subsequently, TEOS (12 mL) was added, and the reaction was continued at room temperature for 6 h. Meanwhile, CTAB (4.8 g) was dissolved in a mixture of water (880 mL) and ethanol (40 mL) by stirring for 6 h. The CTAB solution was poured into the three-necked flask described above, and stirring was continued for 30 min. TEOS (4 mL) was added, and the reaction was carried out for 12 h. After the reaction was completed, the product was collected by suction filtration, washed three times with absolute ethanol, and then dried. The dried sample was dispersed in a sodium carbonate solution (0.2 M, 500 mL), and an etching reaction was performed at 55 °C for 6 h. After the reaction was finished, the sample was collected again by suction filtration, washed three times with absolute ethanol in the same manner, and then dried. The sample was then dispersed in an absolute ethanol solution of hydrochloric acid (2.4 mol/L) and stirred at 55 °C for 12 h. This operation was repeated twice to thoroughly wash away the CTAB. Finally, the white particles were collected by suction filtration, washed with absolute ethanol, and then dried to obtain the HdlMS.

3.3. Modification of HdlMS

The modification procedure was carried out according to previous methods with slight adjustments [31]. OTCS and AEPTES were used to carry out the hydrophilic–hydrophobic modification of the carrier. The specific operation steps are as follows: First, take HdlMS (0.5 g) and disperse it in anhydrous toluene (15 mL). Next, add AEPTES (20 μL) to the dispersion, followed by the addition of OTCS (120 μL). After the addition is completed, subject the mixed solution to ultrasonic treatment for 5 min. Then, place the mixture on a shaker at room temperature and oscillate at a rotation speed of 220 rpm for 2 h. After the modification reaction is completed, collect the sample by filtration, wash it three times with anhydrous ethanol, and finally dry it to obtain the modified carrier.

3.4. Preparation of PLA1@NH₂/C₈-HdlMS

The method for immobilizing PLA1 refers to reference [38] and has been slightly modified. The steps are as follows: First, dilute the phospholipase A1 solution by a factor of two with a phosphate buffer solution (pH = 6.0, 50 mmol/L). Then, take anhydrous ethanol (100 μL) to moisten the modified carrier (0.1 g), and subsequently add it to the diluted PLA1 solution (10 mL). Carry out ultrasonic dispersion treatment on the mixed solution for 5 min to ensure thorough mixing of the carrier and the enzyme solution. Subsequently, remove air bubbles between the carriers by vacuum filtration to ensure the uniformity of the subsequent immobilization reaction. After completing the above operations, place the mixed system in a constant temperature shaker at 30 °C and oscillate at a rotation speed of 220 rpm for 1 h for immobilization. After the immobilization is complete, separate the immobilized enzyme by centrifugation. To remove excess impurities, wash the immobilized enzyme three times with a phosphate buffer solution. Finally, after freeze-drying treatment, the final product of the immobilized enzyme is obtained. The determination methods of enzyme loading capacity and enzyme activity can be found in the Supporting Information [39]. The enzyme activity was determined according to the method described by Wang et al. [22].

3.5. Degumming Process of PLA1@NH₂/C₈-HdlMS

The degumming process is based on the reported method [24] with specific modifications. Initially, crude ARA oil (30 g) was weighed and placed in a 50 mL conical flask, which was then heated to 70 °C. Subsequently, a citric acid solution (0.2 mL, 40% concentration) was added, and the mixture was homogenized at high speed for 1 min followed by continuous stirring in a water bath for 20 min. After cooling the oil to 30–50 °C, sodium hydroxide was added to adjust the pH of the oil–water mixture to 5.8, and the mixture was stirred for an additional 5 min. The immobilized enzyme (0.06–0.6% of the substrate mass) and water (1.5–3.5% of the substrate mass) were then added. The mixture was homogenized again at 10,000 r/min for 1 min to ensure uniform distribution. The degumming reaction was initiated and maintained for 180 min, with samples collected every 30 min to monitor the process.

3.6. Determination of Phosphorus Content

The determination of the phosphorus content was conducted using the molybdenum blue colorimetry method, according to a previous method [20,40], and the specific procedure can be found in the Supplementary Information.

3.7. Reusability of PLA1@NH₂/C₈-HdlMS

After the degumming reaction was completed, the reaction mixture was subjected to centrifugation to separate the immobilized enzyme and the colloid. The resulting mixture was washed three times with n-hexane to isolate the immobilized enzyme, which was then freeze-dried for the next round of degumming reaction. The recovered immobilized enzyme was reintroduced into the newly pretreated crude ARA oil to continue the degumming reaction, with each reaction cycle lasting 90 min.

3.8. Determination of Fatty Acid Composition of ARA Oil

The fatty acid composition was detected by high-performance gas chromatography [41], and the specific detection conditions can be found in the Supplementary Information.

3.9. Analysis of Oxidative Stability

Referring to the reported method [42], the oxidative stability of ARA oil was evaluated based on the oxidation induction time using a Rancimat device (Model 743, manufactured by Metrohm KEBO Lab AB, Herisau, Switzerland). The specific operation process is as follows: weigh the sample (3.0 g) into a Rancimat glass reaction tube, add ultrapure water (50 mL) to each conductivity cell, set the air flow rate at 20 L/h, and place the reaction tube after heating the heating cell to 110 °C using the dotted line control for temperature. During the oxidation of the oil, small molecules generated will be carried into the conductivity chamber. Among them, polar small molecules will dissolve in the deionized water, causing a change in electrical conductivity. By calculating this change in electrical conductivity, the oxidation induction time (OIT) is recorded and finally reported in minutes (min).

3.10. Statistical Analysis

All the data are expressed as the mean ± SD (standard deviation). The quantitative results were calculated using Microsoft Office Excel. Origin 2022b software from OriginLab (North Andover, MA, USA) was used to plot all the graphs.

4. Conclusions

In summary, this study developed an efficient enzymatic degumming method for crude ARA oil using immobilized PLA1 on hollow double-layer mesoporous silica nanoparticles (PLA1@NH₂/C₈-HdlMS). The optimized process achieved a phosphorus removal rate of 97.9%, reducing phosphorus content from 441.21 mg/kg to 9.29 mg/kg while maintaining the fatty acid composition and significantly enhancing oxidative stability by 42%. The proposed scheme exhibited remarkable efficacy in degumming ARA crude oils, which had phospholipid contents varying from 537.44 mg/kg to 294.98 mg/kg. After the degumming process, the phosphorus content was effectively reduced to below 10 mg/kg. Compared to hydration degumming, which achieved only a 56.3% removal rate, the enzymatic method demonstrated superior efficiency at a lower temperature, minimizing energy consumption and reducing thermal degradation. The total content of ARA and DHA was 70.35% in crude oil and 70.49% in degummed oil, with no significant difference. The immobilized enzyme also retained high activity over 11 reuse cycles, highlighting its potential for industrial application. This green and sustainable approach offers a promising alternative for degumming heat-sensitive oils.