Comparative Lipidomics Profiling of Acylglycerol from Tuna Oil Selectively Hydrolyzed by Thermomyces Lanuginosus Lipase and Candida Antarctica Lipase A

Lipase hydrolysis is an effective method to develop different functional types of lipids. In this study, tuna oil was partially hydrolyzed at 30% and 60% by Thermomyces lanuginosus lipase (TL 100 L) and Candida Antarctica lipase A (ADL), respectively, to obtain lipid-modified acylglycerols. The lipidomic profiling of the acylglycerols was investigated by UPLC-Q-TOF-MS and GC–MS to clarify the lipid modification effect of these two lipases on tuna oil. The results showed that 247 kinds of acylglycerols and 23 kinds of fatty acids were identified in the five samples. In the ADL group, the content of triacylglycerols (TAG) and diacylglycerols (DAG) increased by 4.93% and 114.38%, respectively, with an increase in the hydrolysis degree (HD), while there was a decreasing trend in the TL 100 L group. TL 100 L had a better enrichment effect on DHA, while ADL was more inclined to enrich EPA and hydrolyze saturated fatty acids. Cluster analysis showed that the lipids obtained by the hydrolysis of TL 100 L and ADL were significantly different in the cluster analysis of TAG, DAG, and monoacylglycerols (MAG). TL 100 L has strong TAG selectivity and a strong ability to hydrolyze acylglycerols, while ADL has the potential to synthesize functional lipids containing omega-3 PUFAs, especially DAG.


Introduction
Fish oil is rich in omega-3 PUFAs, such as Eicosapentaenoic acid (EPA) and Docosahexaenoic acid (DHA), which have a wide range of health benefits, including regulating lipid metabolism and cell membrane fluidity, boosting the immune system [1,2], promoting body development, enhancing brain function, as well as preventing and treating chronic diseases (cardiovascular diseases, inflammation, etc.) [3][4][5]. Recent studies have found that ω-3 PUFAs in fish oil have good therapeutic effects on the inflammatory responses induced by coronavirus [6] and that the risk of positive COVID-19 nucleic acid tests in people who consumed ω-3 PUFA supplements has slightly decreased [7].
The EPA and DHA are mixed in natural fish oil, and their total content is generally less than 30%. EPA and DHA have different effects on health. Specifically, EPA has positive effects on preventing and treating hyperlipidemia and neurodegenerative diseases due to its capability of inhibiting oxidative stress and apoptosis [8], while DHA has positive effects on neurological development, human lymphocyte function, and neutrophil function [9]. Therefore, lipases with different selectivity are applied by many in the literature to where C is the concentration of NaOH solution (mol/L); V is the amount of NaOH solution (mL); m is the mass of hydrolysates (g); 0.35 is the proportion of oil phase in the hydrolysates; 187 is the saponification value of tuna oil.

Extraction of Hydrolysates
The HD of the hydrolysates was regularly tested, and the hydrolysates were extracted when they were close to the low HD (30%) and the high HD (60%) by adding 2 mL of anhydrous ethanol. Firstly, the theoretical value of lye required the neutralizing of free fatty acids in the hydrolysates and was calculated by the degree of hydrolysis. After being placed in a magnetic stirrer, 0.5 mol/L KOH 30% ethanol solution was added dropwise to neutralize the free fatty acid. The titration was stopped when the pH of the hydrolyzed solution exceeded 9. The hydrolyzed products were extracted with ether and n-hexane successively. Rotary evaporation was carried out at 50 • C. The residual organic solvent was blown dry with N 2, and the sample was sealed and kept at 4 • C.

Fourier Transform Infrared Spectroscopy (FTIR) Analysis
Based on the method of Feng et al. with slight modifications [19], infrared spectroscopic analysis of the tuna oil and its lipid-modified hydrolysates was performed using a Fourier transform infrared spectrometer (TENSOR 27, Bruker Corporation, Germany).
The analysis conditions were as follows: the resolution was 8 cm −1 , the number of scans was 32, and the wavenumber range was 4000~400 cm −1 . Using the KBr tablet method, the dry KBr powder and the sample were mixed and ground at a ratio of 150:1. This mixture was pressed into a transparent film by using a tablet press and then placed in an instrument for observation.

Fatty Acid Composition Analysis
The methyl esterification of the sample was performed by the method of Xia et al. [20] with slight modifications. An amount of 10 mg of the sample was dissolved in 1 mL of toluene, followed by the addition of 200 µL of 5 mg/mL internal standard (50 mg methyl dodecanoate dissolved in 10 mL of toluene), 200 µL of 1 mg/mL antioxidant (10 mg BHT dissolved in 10 mL of toluene) and 2 mL of 10% acetyl chloride-methanol solution, which was then sealed and kept at 50 • C overnight. A total of 5 mL of NaCl solution (5%, m/v) was added after the mixture cooled to an ambient temperature. An amount of 5 mL of n-hexane was then added to the mixture. The supernatant was taken after shaking the mixture and allowing it to stand. The supernatant was washed with an addition of 5 mL KHCO 3 solution (2%, m/v). A proper amount of anhydrous sodium sulfate was added to the mixture, which was then shaken vigorously and filtered through a 0.22 µm membrane and stored at 4 • C for gas chromatographic analysis.
The fatty acid composition was measured using a TQ8040NX gas chromatographymass spectrometer (Shimadzu Co., Ltd., Japan) equipped with an Inert Cap ® Pure-WAX silica capillary column (30 m × 0.25 mm, 0.25 µm). GC analytical conditions: the carrier gas was helium gas, the pressure was 54.2 kPa, the control mode was line speed, 31.5 cm/s, the total flow was 41.7 mL/min, and the column flow was 0.70 mL/min. The sample was injected in a split mode, and the split ratio was 50:1. FID: injection port temperature 250 • C, detector temperature 250 • C. Column temperature procedure: 130 • C for 5 min, then increased to 240 • C at 4 • C/min and held for 30 min.
The fatty acids in the samples were qualitatively identified by a GC-MS mass spectrometry library combined with the mixed standard of fatty acid methyl esters and quantified by the area normalization method.

Non-Targeted Qualitative and Quantitative Analysis of Acylglycerols
The UPLC-Q-TOF-MS analysis conditions were based on Wu's method [21]. The pretreatment of the samples was carried out using the IPA method. Briefly, 10 mg of a sample was dissolved in 1 mL of isopropanol and vortexed for 30 s. Then, 200 µL of the mixture was taken out and dissolved in 800 µL isopropanol and vortexed for 30 s, then filtered through an organic filter membrane of 0.22 µm. After filtration, 10 µL was taken into an interpolator tube with 80 µL isopropanol and 10 µL of 10 µg/mL internal standard, which was added into the tube. After being sonicated for 10 s, the solution was injected into the instrument.

Statistical Analysis
All experiments were performed in three replicates, with results expressed as the "mean ± standard deviation". Differences with a confidence level of 95% were determined by one-way analysis of variance (ANOVA) and Tukey HSD multiple comparisons using JMP ® Pro 13.0.0 software (SAS, Cary, NC, USA). Cluster heatmaps for the factors controlling the five samples and the acylglycerol species were created using Origin Pro 2022 software (OriginLab Co., Northampton, MA, USA). Clustered heat maps were produced using the group average clustering method. Additionally, the distance measurement algorithm was the Pearson correlation.

FTIR Analysis of Acylglycerols from Tuna Oil Hydrolyzed by Two Lipases
The infrared spectrum of tuna oil and its hydrolyzed acylglycerols are shown in Figure 1. Additionally, eight characteristic peaks on the infrared spectrum map are listed in Table 2. Peak 1 represents the hydroxyl (-OH) stretching vibration; Peak 2 represents the cis carbon-carbon double bond (C=C) stretching vibration; Peak 3 and Peak 4 represent the asymmetric methylene (-CH 2 -) stretching vibration and symmetric methylene (-CH 2 -) stretching vibration, respectively; Peak 5 represents the axial vibration of carbonyl (C=O), which was a unique structure in the acylglycerols [22]. Peak 6 represents the in-plane deformation vibration of methylene (-CH 2 -); Peak 7 is the absorption peak of the ester bond (-COOR) due to the stretching vibration of the ether bond (-C-O-C-); Peak 8 is the long chain methylene (-CH 2 -) absorption peak [23].   The tuna oil ( Figure 1a) had no obvious absorption peaks at Peak 1, while all four hydrolyzed acylglycerols (Figure 1b-e) showed broad and strong absorption peaks on Peak 1, indicating the presence of hydroxyl groups (-OH) in their group structures. This may be because tuna oil existed mainly as TAGs before hydrolysis, while DAG and MAG were generated after hydrolysis. The signal intensity of the tuna oil ( Figure 1a) on Peak 2-7 was significantly greater than that of the four hydrolyzed acylglycerols (Figure 1b-e). This may be because the lipase cleaved the fatty acid esterified on the glycerol backbone during the hydrolysis of the fish oil, destroying the ether bond (-C-O-C-) so that one or two of the triacylglycerols containing a carbon-carbon double bond (C=C), methylene (-CH 2 -), and carbonyl (C=O) functional groups in the fatty acids were replaced by the hydroxyl groups. As a result, the content of the functional groups on Peak 2-7 in the four hydrolyzed acylglycerols (Figure 1b-e) decreased, and the signal intensity dropped.

Analysis of Fatty Acid Composition of Acylglycerols Obtained by Hydrolysis of Tuna Oil by Two Lipases
Tuna oil and its acylglycerols were detected by GC analysis to contain 23 types of fatty acids, whose compositions and contents are shown in Table 3. As can be seen from Table 3, under the catalysis of lipase TL 100 L and ADL, the carbon chain length and saturation of the two hydrolyzed acylglycerols significantly changed; the content of the saturated fatty acids, dominated by C16:0 and C18:0 in the tuna oil, was significantly reduced, and the higher the degree of hydrolysis, the lower the content. This trend is more significant after ADL hydrolysis. ADL-60% has the lowest saturated fatty acid content (reduced to 8.9% for C16:0 and 2.3% for C18:0). Yang's study also showed that most of the free fatty acids produced during the hydrolysis of fish oil were saturated fatty acids [24]. After the hydrolysis of the fish oil, the content of polyunsaturated fatty acids, especially DHA, in the hydrolyzed acylglycerols was significantly increased, and the total content of EPA and DHA was increased to more than 1.5 times at a high degree of hydrolysis. This may be because the molecular conformation of the cis carbon-carbon double bond in EPA and DHA made the fatty acid chain bend and fold so that the methyl group at the end of the chain was very close to the ester bond, forming a steric hindrance effect, which increased the difficulty of the lipase active site to reach the ester bond of the fatty acid and its glycerol backbone [22]. As a result, the EPA and DHA were protected during the hydrolysis process. This indicates that the selective hydrolysis rate of the saturated fatty acids by these two lipases was higher than that of ω-3 PUFAs [25]. Compared with the lipase TL 100 L, ADL showed a higher selectivity and preference for the hydrolysis of the dominated saturated fatty acids, and the content of saturated fatty acid in ADL-30% and ADL-60% decreased from the initial 34.55% to 22.09% and 15.43%, respectively. The trends of the content of monounsaturated fatty acid were different in the two lipase treatment groups. In the TL 100 L group, the content of monounsaturated fatty acids decreased with an increase in HD. Conversely, ADL-30% had the highest content of monounsaturated fatty acids at 26.17% in the tuna oil and its hydrolysates. Akanbi et al. used lipase ADL to partially hydrolyze anchovy oil, and the content of monounsaturated fatty acid in the acylglycerols first increased and then decreased because ADL had a great enrichment of both monounsaturated fatty acids and polyunsaturated fatty acids at 30% HD and began to hydrolyze all the fatty acids when HD was over 40%, which is consistent with the results of this study [17]. Kazuaki et al. found that long-chain monounsaturated fatty acids combined with DHA or EPA could reduce lipid accumulation in the HepG2 cells, and C20:1 n7 when combined with DHA treatment and could significantly reduce cholesterol levels in HepG2 cells [26]. The DHA content in the TL 100 L modified acylglycerols was higher than that of the ADL-modified lipids at both a low and high HD, indicating that lipase TL 100 L had a stronger enrichment effect on DHA. Contrastingly, Lipase ADL had a stronger EPA enrichment effect. Gao et al. found that the lipase OUC-Lipase 6 derived from Streptomyces violascens exhibited a selectivity to enrich EPA over DHA during the hydrolysis of cod fish oil [27].

Lipid Composition Analysis of Acylglycerols Obtained from Hydrolysis of Tuna Oil by Two Lipases
Differences in the structure and composition of EPA and DHA in different lipid or acyl groups can affect the function of Omega-3 lipids. Polyunsaturated fatty acids in the form of acylglycerols are more easily absorbed by the body and have higher antioxidant capacity than free fatty acid and ethyl ester polyunsaturated fatty acids [28,29]. Ding et al. found that the bioavailability of DHA in the form of TAG was higher than that in other forms, which is more likely to be digested and absorbed [30]. The total content of EPA and DHA is closely related to the quality and economic value of fish oil [31]. Therefore, in order to better evaluate the modification effect of different lipases on tuna oil, the composition of acylglycerols in the tuna oil and its hydrolysates was analyzed in this study.
Through the non-targeted qualitative and quantitative analysis of lipids, it was found that 247 kinds of acylglycerols were detected in both the tuna oil and its hydrolysates, including 153 kinds of TAG, 85 kinds of DAG, 7 kinds of MAG, and 2 kinds of diacylglycerol glucuronides. TAG (16:0-16:0-22:6) and TAG (16:0-18:1-22:6) are the two most abundant acylglycerols in tuna oil, accounting for 9.15% and 8.24% of the total TAG in tuna oil, respectively. Zhang et al. [32] found that TAG (16:0-16:0-22:6) and TAG (16:0-18:1-22:6) were the representative TAG used to distinguish deep-sea fish species, which were present in much higher amounts in the tuna oil than other fish oil types. DAG (22:6-22:6) had the highest content in each hydrolysate, which is one of the reasons for the significant increase in the content of DHA after hydrolysis. Figure 2 shows the acylglycerol composition in tuna oil and its hydrolysates hydrolyzed by TL100 and ADL, respectively (TL 100 L-30%, TL 100 L-60%, ADL-30%, and ADL-60%). It can be seen from the figure that the type of lipase and the degree of hydrolysis significantly affected the percentage of each acylglycerol in the fish oil. Before the hydrolysis of the tuna oil, the proportion of TAG in the acylglycerols reached more than 90%. After hydrolysis by the lipase, the content of TAG in the tuna oil decreased substantially, while DAG dominated. This may be because fish oil is generally hydrolyzed at the sn-1,3 position, and the catalytic triplet will preferentially hydrolyze the acyl group at the sn-1,3 position so that the diacylglycerol content increases. Morales-Medina et al. speculated that the reason the content of MAG in the fish oil hydrolysates was much less than the content of DAG was that the fatty acids on TAG were more easily cleaved by enzymatic hydrolysis than those on DAG [33]. Compared with the three other hydrolysates, TL 100 L-60%, ADL-30%, and ADL-60%, TL 100 L-30% had the highest proportion of MAG. This may be because lipase TL 100 L was a specific lipase with selectivity for sn-1,3 positions [34], which selectively hydrolyzed the fatty acids attached at sn-1 and sn-3 positions in the TAG, resulting in the formation of MAG and the enrichment of fatty acids at the sn-2 position. However, the higher the degree of hydrolysis of the tuna oil, the lower the proportion of MAG. This may be because the formation and continuous hydrolysis of MAG was carried out at the same time, but the hydrolysis rate is faster than the formation rate [35]. (Appendix A).
ysis of the tuna oil, the proportion of TAG in the acylglycerols reached more than 90%. After hydrolysis by the lipase, the content of TAG in the tuna oil decreased substantially, while DAG dominated. This may be because fish oil is generally hydrolyzed at the sn-1,3 position, and the catalytic triplet will preferentially hydrolyze the acyl group at the sn-1,3 position so that the diester content increases. Morales-Medina et al. speculated that the reason the content of MAG in the fish oil hydrolysates was much less than the content of DAG was that the fatty acids on TAG were more easily cleaved by enzymatic hydrolysis than those on DAG [33]. Compared with the three other hydrolysates, TL 100 L-60%, ADL-30%, and ADL-60%, TL 100 L-30 had the highest proportion of MAG. This may be because lipase TL 100 L was a specific lipase with selectivity for sn-1,3 positions [34], which selectively hydrolyzed the fatty acids attached at sn-1 and sn-3 positions in the TAG, resulting in the formation of MAG and the enrichment of fatty acids at the sn-2 position. However, the higher the degree of hydrolysis of the tuna oil, the lower the proportion of MAG. This may be because the formation and continuous hydrolysis of MAG was carried out at the same time, but the hydrolysis rate is faster than the formation rate [35]. (Appendix A). Cluster Heatmap Analysis of TAG, DAG and MAG In order to better elucidate the differences in the types and composition of lipids obtained by lipase TL 100 L and ADL at low and high HD, cluster heat map analysis was used to statistically analyze the lipid composition information of the tuna oil and its hydrolyzed acylglycerols. A cluster heat map is a statistical method for classifying the combination of samples and variables. Taking the index variables as the abscissa and the sample information as the ordinate, the method of vertical comparison was adopted to Cluster Heatmap Analysis of TAG, DAG and MAG In order to better elucidate the differences in the types and composition of lipids obtained by lipase TL 100 L and ADL at low and high HD, cluster heat map analysis was used to statistically analyze the lipid composition information of the tuna oil and its hydrolyzed acylglycerols. A cluster heat map is a statistical method for classifying the combination of samples and variables. Taking the index variables as the abscissa and the sample information as the ordinate, the method of vertical comparison was adopted to visually present the global changes and clustering relationships of multi-samples and multi-variables. The cluster heat map of the three acylglycerols is shown in Figure 3.
The , and these TAG were higher in ADL-60% compared to the other four samples (Figure 3a). According to the color scale of the cluster heat map, for lipase TL 100 L, the content of TAG cluster I and TAG cluster II decreased sharply at a low HD, showing the hydrolysis preference for these two types of TAG; conversely, at a high HD, it continued to hydrolyze almost every triacylglycerol. For lipase ADL, its ability to hydrolyze TAG at a low HD was not significantly different from that of TL 100 L, but it significantly increased the content of 48 kinds of TAG, including TAG cluster III. A total of 46 out of these 48 kinds of TAG contained EPA or DHA at a high HD. This may be due to the esterification or transesterification of free fatty acids in the hydrolysis system, especially free forms of EPA and DHA, with TAG at a high HD generating TAG, including TAG (14:1-20:5-22:6), TAG (20:2-22:6-22:6), and TAG (18:4-18:4-20:5). The fatty acid distribution of the TAG cluster III might have been rearranged and improved due to the regiospecificity of the lipase ADL [36]. Wang et al. used immobilized lipase MAS1 derived from the marine Streptomyces sp. strain W007 to catalyze the esterification of glycerol with ω-3 PUFAs and successfully prepared TAG, which was high purity (92.26%) and rich in ω-3 PUFAs. The fatty acid composition of the products was similar to that of ω-3 PUFAs, which were used as reaction substrates [37]. The location distribution of EPA and DHA on TAG also plays a key role in their digestion and absorption in the human body [38]. Combined with a lower saturated fatty acid content (15.43%) ( Table 3) and its lipid composition, ADL-60% has the ability to be used as a structured lipid for omega-3 food fortification. Therefore, compared to TL 100 L, lipase ADL has a higher potential for the lipid modification of fish oil to produce lipids with specific functionalities. in HD (30% and 60%), suggesting that TL 100 L had a stronger ability to hydrolyze than ADL. This result indicated that moderate hydrolysis contributed to the enrichment of acylglycerols containing DHA. DHA in fish oil was usually distributed at the sn-2 position [39], which might also indicate that TL 100 L was more inclined to hydrolyze the sn-1 and sn-3 positions. This result was consistent with the analysis of the percentage of glycerol ester composition (Figure 2).
(a) , and the content of these DAG in TL 100 L-60% was higher compared to the other four samples (Figure 3b). According to the color scale of the cluster heat map, compared with the other four samples, TL 100 L-30% contained a higher content of DAG cluster I and a lower content of DAG cluster II and cluster III, and TL 100 L-60% had a highest content of DAG cluster III. This shows that the degree of hydrolysis had a significant effect on the lipid composition. The effects of lipase ADL and TL 100 L were different. The content of the three categories of DAG in ADL-30% was generally not high, but the content of DAG cluster II increased in ADL-60%. There are 21 DAGs in DAG cluster II, 20 of which contain EPA or DHA, indicating that ADL has the potential to prepare omega-3-rich DAGs.
The seven kinds of MAG include MAG 14:1, MAG 16:0, MAG 18:0, MAG 20:4, MAG 20:5, MAG 22:5, and MAG 22:6, which had the highest content in TL 100 L-30% (Appendix B). Among the seven kinds of MAG, the content of MAG 22:6 accounted for 78.84% of the total content of MAG, which indicated that TL 100 L had better enrichment for containing DHA at a low HD. ADL had less effect on the monoacylglycerols content with a change in HD (30% and 60%), suggesting that TL 100 L had a stronger ability to hydrolyze than ADL. This result indicated that moderate hydrolysis contributed to the enrichment of acylglycerols containing DHA. DHA in fish oil was usually distributed at the sn-2 position [39], which might also indicate that TL 100 L was more inclined to hydrolyze the sn-1 and sn-3 positions. This result was consistent with the analysis of the percentage of acylglycerol composition (Figure 2).

Discussion
As can be seen from Figure 3a, in TAG cluster III, except for TAG 38:0 and TAG 56:12, the content of the other 23 TAGs did not change significantly under the hydrolysis of lipase. The reason for this may be that these TAGs are linked to at least one long-chain polyunsaturated fatty acid dominated by EPA or DHA, and the steric hindrance formed by these fatty acids is more resistant to lipase hydrolysis, while saturated fatty acids and monounsaturated fatty acids have linear and nearly linear structures, respectively, which do not form any obstacle to lipase and are easy to be hydrolyzed [39]. The types of lipases and HD have significant differences in selectivity to TAG. As can be seen from the changes in the color scale, the content of 17 kinds of saturated TAG decreased significantly at a low HD, and the effect of lipase ADL in reducing their content was stronger than that of TL 100 L, which is consistent with the results in Table 3. In 100 L-30%, the content of TAG clusters I and II decreased greatly, and the content of DAG cluster I increased greatly, and it had the highest content of MAG. It is speculated that lipase TL 100 L mainly acts on TAG clusters I and II, and the hydrolysis products are mainly DAG cluster I and all of the MAG. The distribution of DAG in ADL-30% was relatively uniform, which may be due to the fact that lipase ADL was between the selectivity of the sn-2 position and the nonselectivity position, and the production of DAG abounded in randomness [40]. In addition, the lipase preferentially hydrolyzed saturated fatty acids and began to hydrolyze all the fatty acids indiscriminately when a certain degree of hydrolysis was reached, showing fatty acid selectivity, not regioselectivity [17]. The selective hydrolysis of fish oil may be accompanied by esterification and transesterification. In the process of increasing the degree of hydrolysis from 30% to 60%, the content of 48 kinds of TAG increased in the hydrolysis reaction catalyzed by lipase ADL, while the content of only seven kinds of TAG increased in the hydrolysis reaction catalyzed by TL 100 L, and this phenomenon was concentrated on TAG with carbon numbers of 55 to 66 (also unsaturated TAG). This may be due to the presence of esterification or transesterification. TAG with higher carbon numbers was more prone to these reactions, and ADL was more capable of causing these possible reactions than TL 100 L. Therefore, the mechanism of lipase-catalyzed esterification or transesterification may be determined by a combination of the nature of the lipase itself, the type of substrate, the degree of hydrolysis of TAG, the regional distribution of fatty acids in the glycerol backbone, and the unsaturation number or chain length of fatty acids.
Hydrolysis with TL 100 L and ADL improves the tuna oil's polyunsaturated fatty acid profile due to the modification of lipid (DHA and EPA) composition and distribution, which provides the potential to produce healthier lipids with modified properties; these new lipids may be a useful new ingredient for nutritional supplements in human or animal feeding. However, more research is needed to illustrate the functional characteristics, especially the physicochemical properties, metabolic characteristics, and nutritional value of modified tuna oil.

Conclusions
In this study, the lipid modification effect of two lipases on tuna oil at a low and high HD was evaluated by UPLC-Q-TOF-MS and GC-MS. Lipase TL 100 L showed a better enrichment of DHA, while ADL was more inclined to hydrolyze saturated fatty acids and enrich EPA. The hydrolysis reaction catalyzed by lipase ADL might cause the esterification reaction of glycerides or the acid hydrolysis reaction in transesterification. In addition, the selectivity of the two lipases in the tuna oil fatty acids varied significantly at different HDs. TL 100 L had strong specificity for sn-1,3 fatty acid and had a strong ability to continuously hydrolyze acylglycerols, while ADL exhibited the potential to produce omega-3 functional lipids due to its selective hydrolysis of saturated fatty acids and higher yield level of omega-3-containing DAGs. The specific sites of action of the two lipases and the fatty acid composition at the sn-2 position can then be further explored for in-depth analysis. This study provides a scientific basis for designing specific acylglycerol compositions and fatty acid ratios through lipid modification.

Data Availability Statement:
The datasets generated for this study are available on request to the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The datasets generated for this study are available on request to the corresponding author.

Conflicts of Interest: The authors declare no conflicts of interest.
Appendix A Figure A1. UPLC-Q-TOF-MS negative ion mode of five samples. Figure A1. UPLC-Q-TOF-MS negative ion mode of five samples.