Use of hydrolyzed glycerides in increasing antibacterial activity

Abstract

The application relates to the technical field of specific therapeutic activity of compounds, and particularly relates to application of hydrolyzed glyceride in improving bacteriostatic activity. The application provides application of hydrolyzed glyceride in improving bacteriostatic activity, and a preparation method of the hydrolyzed glyceride, which comprises the following steps: (1) placing cold-pressed coconut oil in a reaction container, and sequentially adding lipase and a phosphate buffer solution, and hydrolyzing under the conditions of 35-45 DEG C and 500-600 r / min for 1-2 h to obtain a hydrolysis liquid; (2) adding anhydrous ethanol into the hydrolysis liquid to terminate the reaction, and after alkaline neutralization, organic solvent extraction and nitrogen blowing treatment, the hydrolyzed glyceride is obtained; the lipase is selected from at least one of 20000L, TLIM, ADL and 10SD. The high-melting-point natural coconut oil is selectively hydrolyzed by an enzyme method, and is directionally converted into a novel oil product with high glycerol diglyceride content, low melting point and significant bacteriostatic activity.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of specific therapeutic activity of compounds, and particularly relates to application of hydrolyzed glycerides in improving bacteriostatic activity. BACKGROUND

[0002] Coconut oil is a natural vegetable oil rich in medium-chain triglycerides (MCT), with unique flavor and functional properties, and is widely used in food, cosmetics and pharmaceutical fields. However, natural coconut oil has the disadvantages of high saturated fatty acid content, single lipid composition, high solidification point and poor oxidation stability, which limits its application in more fields. In addition, the correlation between high saturated fatty acid intake and cardiovascular disease risk also prompts the industry to seek healthier and more functional coconut oil modification solutions.

[0003] At present, the modification methods of oil and fat mainly include fermentation method, plasma method and the like, but these methods often have problems such as harsh reaction conditions, low specificity, high energy consumption or complex products. Enzymatic modification gradually becomes an important means of oil and fat modification due to its mild reaction conditions, high specificity and environmental friendliness. Studies have shown that enzymatic modification can improve the functional properties of fish oil, milk fat and other oils and fats, but there is still insufficient research on the selective hydrolysis of coconut oil using specific lipases, the influence of which on the lipid composition, physicochemical properties and bacteriostatic activity of the oil is not fully understood.

[0004] Therefore, it is of important research value and application prospect to develop an enzymatic hydrolysis method for modifying coconut oil and elucidate its application potential in bacteriostasis. SUMMARY

[0005] The present application uses four lipases screened to selectively hydrolyze and modify coconut oil, analyzes the changes in lipid composition, lipidomics and physicochemical properties before and after hydrolysis, explores the influence of hydrolysis of different fatty acids on the structural properties and thermal stability of coconut oil, and analyzes the differences in bacteriostatic effects of different hydrolyzed glycerides on Staphylococcus aureus, Escherichia coli and Shewanella. The present application aims to further tap the application value of coconut oil as a special medical food or functional food, and to provide a reference for the development and application of functional products of coconut oil.

[0006] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0007] In one aspect, the present application provides the application of hydrolyzed glycerides in improving bacteriostatic activity.

[0008] Preferably, the preparation method of the hydrolyzed glycerides comprises the following steps:

[0009] (1) Place cold-pressed coconut oil in a reaction vessel, add lipase and phosphate buffer solution in sequence, and hydrolyze for 1-2 h at 35-45℃ and 500-600 r / min to obtain hydrolysate;

[0010] (2) The reaction was terminated by adding anhydrous ethanol to the hydrolysate. After neutralization with alkali, extraction with organic solvent, and nitrogen blowing, hydrolyzed glycerol esters were obtained.

[0011] The lipase is selected from at least one of 20000L, TLIM, ADL and 10SD;

[0012] The amount of lipase added is 5-6% of the mass of the cold-pressed coconut oil;

[0013] The mass-to-volume ratio of the cold-pressed coconut oil to the phosphate buffer solution is 1-1.2:2.

[0014] More preferably, the lipase is 10 SD.

[0015] It should be noted that existing research has shown that enzymatic modification can improve the functional properties of oils such as fish oil and milk fat, but for coconut oil, especially using specific lipases (such as Palatase 20000L and Lipozyme), the effects are not ideal. ® The study on the effects of selective hydrolysis of TLIM, NovoCorADL, and Lipase MHA (Amano 10SD) on lipid composition, physicochemical properties, and antibacterial activity is still insufficient.

[0016] More preferably, the pH of the phosphate buffer solution is 7.0.

[0017] More preferably, the hydrolyzed glycerol ester contains 37% triglycerides and 59.81% diglycerides.

[0018] More preferably, the method of alkali neutralization is as follows: neutralize the free fatty acids produced by hydrolysis with 0.3 mol / L potassium hydroxide containing 5% anhydrous ethanol, and adjust the pH to 9.2.

[0019] More preferably, the organic solvent extraction method is as follows: first, anhydrous diethyl ether of the same volume as the hydrolysate is added for a first extraction at 30°C, and then n-hexane of the same volume as the hydrolysate is added for a second extraction at 45°C.

[0020] In this invention, the hydrolysate refers to the mixture in the reaction vessel containing cold-pressed coconut oil, phosphate buffer solution, lipase, and the generated monoglycerides, diglycerides, free fatty acids, and unreacted substrates after the lipase hydrolysis reaction is completed.

[0021] In another aspect, the present application provides the use of hydrolyzed glycerides in the preparation of an antibacterial preparation having inhibitory effect on Staphylococcus aureus, Escherichia coli and / or Shewanella.

[0022] Preferably, the preparation method of the hydrolyzed glycerides comprises the following steps:

[0023] (1) Put cold-pressed coconut oil into a reaction container, and sequentially add lipase and phosphate buffer solution, and hydrolyze at 35-45℃ and 500-600 r / min for 1-2 h to obtain a hydrolysis solution;

[0024] (2) Add anhydrous ethanol into the hydrolysis solution to terminate the reaction, and after alkaline neutralization, organic solvent extraction and nitrogen blowing treatment, obtain the hydrolyzed glycerides;

[0025] The lipase is selected from at least one of 20000L, TLIM, ADL and 10SD;

[0026] The added amount of the lipase is 5-6 % of the mass of the cold-pressed coconut oil;

[0027] The mass-volume ratio of the cold-pressed coconut oil to the phosphate buffer solution is 1-1.2:2.

[0028] More preferably, the lipase is 10SD.

[0029] More preferably, the pH value of the phosphate buffer solution is 7.0.

[0030] More preferably, the content of triglycerides in the hydrolyzed glycerides is 37 %, and the content of diglycerides in the hydrolyzed glycerides is 59.81 %.

[0031] More preferably, the method of alkaline neutralization is to neutralize the free fatty acids generated by hydrolysis with 0.3 mol / L potassium hydroxide containing 5 % anhydrous ethanol, and adjust the pH to 9.2.

[0032] More preferably, the method of organic solvent extraction is to first add anhydrous diethyl ether with the same volume as the hydrolysis solution for the first extraction at 30℃, and then add n-hexane with the same volume as the hydrolysis solution for the second extraction at 45℃.

[0033] Compared with the prior art, the present application has the following beneficial effects:

[0034] (1) The present application can efficiently and specifically convert triglyceride into diglyceride by selecting specific lipase (preferably 10SD) for selective hydrolysis of coconut oil under mild conditions. The lipase 10SD can make the content of diglyceride in the product as high as 59.81 %, the content of lauric acid as low as 34.69 %, the sliding melting point of CO group as low as 22.3 ℃ from 28.2 ℃, and the reduction as high as 20.92 %, realizing the change from solid state to liquid state at room temperature.

[0035] (2) The modified product shows unexpected excellent antibacterial activity, and has significant inhibition effect on Staphylococcus aureus, Escherichia coli and Shewanella. The antibacterial effect of 10SD is the most obvious, the pure coconut oil has no inhibition effect on Escherichia coli, and the inhibition circle diameter of 10SD hydrolysis product on Escherichia coli is 12.96±0.48 mm. The inhibition effect on Staphylococcus aureus and Shewanella is also very strong, which may be due to the increase of lauric acid glyceride (especially MAG12:0) and the synergistic effect of medium and short chain fatty acid derivatives after hydrolysis.

[0036] (3) The reaction conditions of the method are mild, which avoids the damage of high temperature and strong acid and alkali treatment to the quality of oil and fat, and the immobilized enzyme can be used, which meets the requirements of green chemistry and sustainable production. The method provides a reliable technical scheme for developing coconut oil-based products with specific functional characteristics, and expands the application potential of coconut oil in the fields of high value-added food, medicine and cosmetics.

[0037] (4) The present application explores the structure-activity relationship of coconut oil lipid composition-functionality, which provides a basis for the directional preparation of coconut oil derivative products with high diglyceride and high antibacterial activity. Future research can focus on the accurate identification of functional medium and short carbon chain fatty acids and their derivatives in coconut oil, and balance the functional characteristics and stability of the product by combining with the optimization of hydrolysis conditions; at the same time, the application in the fields of food preservation and functional oil products can be expanded, which provides scientific basis for the functional exploration of coconut oil and the development and application of functional products. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 The figure shows the relative content of lipid composition and the change of sliding melting point of coconut oil and its hydrolyzed glyceride; different sizes and letters represent significant differences in relative content of lipid composition / sliding melting point P <0.05).

[0039] Figure 2a The figure shows the acylglycerol (triglyceride) clustering heat map of coconut oil and its hydrolyzed glyceride.

[0040] Figure 2b The figure shows the acylglycerol (diglyceride) clustering heat map of coconut oil and its hydrolyzed glyceride.

[0041] Figure 2cFigure 1 is a heat map of coconut oil and its hydrolyzed glycerides acylglycerols (monoglycerides).

[0042] Figure 3 Figure 2 is an infrared spectrum of coconut oil and its hydrolyzed glycerides.

[0043] Figure 4a Figure 3 is a differential scanning calorimetry (DSC) analysis of coconut oil and its hydrolyzed glycerides.

[0044] Figure 4b Figure 4 is a thermogravimetric (TG) analysis of coconut oil and its hydrolyzed glycerides.

[0045] Figure 5 Figure 5 is the antibacterial effect of coconut oil and its hydrolyzed glycerides on Staphylococcus aureus, Escherichia coli, and Shewanella.

[0046] Figure 6a Figure 6 is the effect of coconut oil and its hydrolyzed glycerides on the growth curve of Staphylococcus aureus.

[0047] Figure 6b Figure 7 is the effect of coconut oil and its hydrolyzed glycerides on the growth curve of Escherichia coli.

[0048] Figure 6c Figure 8 is the effect of coconut oil and its hydrolyzed glycerides on the growth curve of Shewanella. DETAILED DESCRIPTION

[0049] Hereinafter, the technical solutions of the present application will be described in conjunction with examples, but the present application is not limited to the following examples. The experimental methods and detection methods described in each example are all conventional methods unless otherwise specified; and the reagents and materials described are all commercially available unless otherwise specified.

[0050] Example 1 Materials and Methods

[0051] 1.1 Materials

[0052] Cold-pressed coconut oil was purchased from Hainan Baoting Ezefang Food Co., Ltd. and stored in a dry and cool place. Lipase NovoCo ADL (ADL) derived from Candida antarctica, Candida antarctica Patalase 20000L (20000L) derived from Mucor miehei, Rhizopus oryzae Immobilized lipase Lipozyme®TLIM (TLIM) derived from Thermomyces lanuginosus, Thermomyces lanuginosus were all purchased from Beijing Gao Ru Sen Technology Co., Ltd. Immobilized lipase Lipase MHA "Amano" 10SD (10SD) was purchased from Jiangsu Tianye Enzyme Co., Ltd.

[0053] 1.2 Method

[0054] 1.2.1 Hydrolysis of coconut oil and glyceride extraction

[0055] The method disclosed in the reference "Xuan J, Xia Q, Li Y, et al. Enzymatically produced acylglycerol and glycerin monostearate additives improved the characteristics of gelatin-stabilized omega-3 emulsions and microcapsules [J]. Food Chemistry, 2024, 448: 139135. DOI (https: / / doi.org / 10.1016 / j.foodchem.2024.139135)" is referred to. Specifically, 30 g of cold-pressed coconut oil is placed in a round-bottom flask, 5% of the mass fraction of the coconut oil is added to the lipase (the amount of lipase added is 5% of the mass of the coconut oil), and a phosphate buffer solution with a pH value of 7.0 is added. Hydrolysis is carried out at 550 r / min and 40°C. After 2 hours of hydrolysis, the hydrolysis is terminated, 5 mL of anhydrous ethanol is added to the round-bottom flask to terminate the reaction, 0.3 mol / L potassium hydroxide containing 5% anhydrous ethanol is used to neutralize the free fatty acids produced by hydrolysis, and the pH is adjusted to 9.2. Then, anhydrous diethyl ether and n-hexane are used to extract the hydrolyzed glyceride, and the hydrolyzed glyceride is obtained after nitrogen blowing. The lipases 10SD, TLIM, 20000L, and ADL hydrolyze glycerides, and the lipases are labeled as 10SD-AC, TLIM-AC, 20000L-AC, and ADL-AC, respectively. The coconut oil that has not been hydrolyzed is labeled as CO or CO-AC.

[0056] 1.2.2 Determination of the fatty acid composition of coconut oil and its glyceride

[0057] The method disclosed in the reference "Xia Q, Wang B, Akanbi T O, et al. Microencapsulation of lipase produced omega-3 concentrates resulted in complex coacervates with unexpectedly high oxidative stability [J]. Journal of Functional Foods, 2017, 35: 499-506. DOI (https: / / doi.org / 10.1016 / j.jff.2017.06.017)" is specifically as follows: After methyl esterification of the sample, GC-2030 type gas chromatograph is used for analysis and determination. The carrier gas is helium, the pressure is 54.2 kPa, the control mode is linear speed (31.5 cm / s), the total flow rate is 41.7 mL / min, and the column flow rate is 0.70 mL / min; split injection is used, the split ratio is 50:1, and the injection amount is 1 μL. The column temperature program is as follows: 130 ℃ for 5 min, then increased to 240 ℃ at a rate of 4 ℃ / min, and kept for 30 min until the analysis is completed.

[0058] 1.2.3 Analysis of the lipid composition of coconut oil and its glycerides

[0059] About 20 mg of sample was weighed and the weight was recorded; 1 mL of dichloromethane was added and vortexed ultrasonically until completely dissolved, and then filtered through a 0.22 μm organic filter to obtain a sample filtrate; 20 μL of the sample filtrate was added to 980 μL of dichloromethane and vortexed to obtain a diluted sample solution; 10 μL of the diluted sample solution was added to 100 μL of internal standard and placed in a 100 μL internal cannula for testing.

[0060] The internal standard used was 1 μg / mL, specifically: (DG 15:0_18:1 (d7) / TG 15:0_18:1 (d7)_15:0 / MG 18:1 (d7).

[0061] The UPLC column used was a Phenomenex Kinete C18 column (100x2.1 mm, 2.6 μm); the injection amount was 3 μL; the flow rate was 0.4 mL / min; the column temperature was 60 °C; and the sample chamber temperature was 4 °C.

[0062] 1.2.4 Determination of the sliding melting point of coconut oil

[0063] The sliding melting point was determined according to the method of GB / T 24892-2010.

[0064] 1.2.5 Fourier transform infrared spectroscopy analysis

[0065] The method disclosed in the reference “Xuan J, Wang Z, Xia Q, et al. Comparative Lipidomics Profiling of Acylglycerol from Tuna Oil Selectively Hydrolyzed by Thermomyces Lanuginosus Lipase and Candida Antarctica Lipase A [J]. Foods, 2022, 11(22). DOI: 10.3390 / foods11223664” is specifically as follows: KBr powder is thoroughly ground, and then a tablet press is used to press the powder into a transparent film. 20 μL of oil sample is applied to the center of the KBr tablet, and the tablet is placed in the instrument for measurement.

[0066] 1.2.6 Physicochemical property analysis of coconut oil and its glycerides

[0067] 1) Peroxide value determination: The peroxide value is determined according to GB 5009.227-2023.

[0068] 2) Thiobarbituric acid reagent value (TBARS value) determination: 200 mg of sample is diluted to 25 mL with n-butanol and mixed thoroughly in an ultrasonic water bath. 5 mL of the solution is taken into a dry test tube, 5 mL of freshly prepared thiobarbituric acid (TBA) solution is added, and the test tube is placed in a 95 ℃ water bath for 120 min. After cooling to room temperature, the absorbance value is measured at 532 nm. The thiobarbituric acid reagent value (TBARS) is calculated according to the following formula (1):

[0069] (1);

[0070] In formula (1), A : absorbance at 532 nm; m : sample mass / g.

[0071] 3) Conjugated diene value (CD) determination: 20-50 mg of oil sample is accurately weighed into a 50 mL test tube with a stopper, and n-hexane is added to make up the volume. Shake and dissolve thoroughly, and measure the absorbance at 232 nm. The conjugated diene value (CD) is calculated according to the following formula (2):

[0072] (2);

[0073] In formula (2), A: Absorbance value of sample at 233 nm; C: Final diluted concentration of sample (g / 100 mL); P : Length of cuvette; ε: Molar absorptivity.

[0074] 4) Determination of oxidative stability: Accelerated oxidation test was performed on coconut oil and its glycerides using Rancimat (Metrohm 743, Herison, Switzerland) oil oxidation instrument. 2 g of oil phase was heated at 90 ℃ under purified air at a flow rate of 20 L / h, and the induction time of the sample under test was recorded.

[0075] 1.2.7 Thermal stability analysis of coconut oil and its glycerides

[0076] Thermal stability analysis of coconut oil and its lipid-modified hydrolysate was performed using a simultaneous thermal analyzer (STA449F3, NETZSCH, Germany). The sample size was 10 mg, the carrier gas was high-purity nitrogen, and the gas flow rate was 30 mL / min. The initial temperature was set to 30 ℃, the heating rate was 10 ℃ / min, and the temperature was held at 600 ℃ for 5 min to obtain the DSC and TG curves.

[0077] 1.2.8 Analysis of antibacterial properties of coconut oil and glycerides

[0078] The method disclosed in the reference “Peng Y Y, Liu Z Y, Huang Q, et al. Antibacterial activity of Chinese wax tree oil on cladosporium and its mechanism [J]. Food science, 2025, 46(10): 1051-8” is as follows: Staphylococcus aureus, Escherichia coli, and Shewanella (all isolated and preserved by the applicant's laboratory) were activated, and 50 μL of the activated bacterial suspension was added to LB solid medium (Beijing Lq Technology Co., Ltd.) and evenly coated. 20 μL of coconut oil and its hydrolyzed glycerides were dropped onto sterile filter paper to completely soak, and the soaked filter paper was placed in the LB solid medium and cultured in a 37 ℃ constant temperature incubator for 48 h. The size of the inhibition zone was observed and measured, and the photograph was recorded.

[0079] The activation method is the same, that is, a ring of bacteria is picked from the refrigerated tube into nutrient broth and shaken for 8-12 h, and then a ring is picked from the nutrient broth and streaked onto LB solid medium plates. Picking a single colony from the plate is the target colony that has been activated.

[0080] The determination of growth curve refers to the method disclosed in the reference literature “Zhang J, Liu X, Zhang T, et al. Study on the bacteriostatic property and bacteriostatic mechanism of rosemarinic acid compounded bacteriostatic agent [J]. Food Bioscience, 2024, 59: 103820. DOI (https: / / doi.org / 10.1016 / j.fbio.2024.103820”), specifically: 20 μL of coconut oil and its hydrolyzed glycerides were added to 180 μL of bacterial suspension, respectively. MH broth was mixed with 180 μL of bacterial suspension as blank control (BLANK). The absorbance was measured at 600 nm, and the samples were placed in a constant temperature incubator and cultured at 37 ℃ with shaking for 24 h. The absorbance value at 600 nm was measured every 2 h, and the growth curve was plotted. Three parallel experimental groups were set for each group of experiments. The measurement time was 0, 2, 4, 6, 8, 10, 12 h.

[0081] 1.3 Statistical analysis

[0082] All experiments were performed in triplicate, and the results were expressed as “mean ± standard deviation”. One-way ANOVA and Tukey HSD multiple comparisons were performed using IBM SPSS Statistics 21 software, and differences with a confidence level of 95% were determined; all plots in the text were performed using Origin2025 software.

[0083] Example 2 Results and analysis

[0084] 2.1 Analysis of fatty acid composition of coconut oil and its hydrolyzed glycerides

[0085] The fatty acid composition changes of coconut oil after hydrolysis by four lipases (20000L, ADL, 10SD, TLIM) are shown in Table 1. The fatty acid composition and distribution affect the metabolic absorption and functional properties of lipids. Coconut oil is mainly composed of lauric acid, and the proportion of lauric acid in coconut oil before hydrolysis is 50.37±0.17%. After hydrolysis, the proportion of lauric acid decreased significantly. Especially after hydrolysis by immobilized lipases 10SD and TLIM, the content of lauric acid decreased to 34.69±0.04% and 36.89±0.11%, with a decrease of 31.13% and 26.76%. It may be because lauric acid mainly exists in the form of lauric acid triglyceride in coconut oil, and lipase will preferentially select lauric acid triglyceride as substrate during hydrolysis reaction. In addition, the factor affecting the fatty acid composition after hydrolysis is the specificity of lipase. TLIM and 10SD are 1,3 specific lipases that can accurately recognize the ester bond at sn-1 and sn-3 sites and undergo hydrolysis reaction, while ADL is a non-specific lipase that tends to randomly hydrolyze the ester bond of triglyceride. In addition to the significant change in lauric acid content, the content of medium and short chain fatty acids (C:8, C:10, C:12) also showed a downward trend after hydrolysis, from 65% to about 50%. This may be due to the smaller steric hindrance of medium chain fatty acids, which are more likely to separate from triglycerides to form diglycerides and monoglycerides.

[0086] Table 1 Fatty acid composition analysis of coconut oil and its hydrolyzed glycerides

[0087]

[0088] Note: The same row and different lowercase letters represent significant differences in fatty acid content P <0.05).

[0089] C6:0 is hexanoic acid, C8:0 is octanoic acid, C10:0 is decanoic acid, C12:0 is lauric acid, C14:0 is myristic acid, C16:0 is palmitic acid, C18:0 is stearic acid, C18:1 is oleic acid, and C18:2 is linoleic acid.

[0090] 2.2 Lipid composition and sliding melting point analysis

[0091] The lipid composition directly affects the physical properties (such as melting point and crystallization behavior) and nutritional functions of fats. The changes in the lipid composition and melting point of coconut oil after hydrolysis are shown in Table 2. Figure 1The coconut oil mainly exists in the form of triglyceride without hydrolysis, and only contains a small amount of diglyceride and monoglyceride. It can be clearly seen that the glyceride composition of coconut oil changes significantly after hydrolysis by different lipases. The content of diglyceride generated by hydrolysis of coconut oil under the action of 10SD lipase is as high as 59.81 %, while the content of diglyceride increases after hydrolysis by ADL, 20000L and TLIM, but it is still dominated by triacylglycerol. This may be because the type and action site of lipase significantly affect the lipid composition of coconut oil, and the specificity of 10SD enzyme makes it more inclined to hydrolyze the fatty acids at sn-1 and sn-3 sites. The content of monoglyceride after hydrolysis by different enzymes is still low, which may be related to the degree of hydrolysis. At a lower degree of hydrolysis, lipase preferentially binds to the ester bond of triglyceride, causing it to break down into diglyceride and free fatty acid.

[0092] The high content of medium-chain fatty acid triglyceride in coconut oil makes it exist in solid form at room temperature, while after hydrolysis by different lipases, the coconut oil exists in liquid state at room temperature, which may be related to the crystal form and molecular structure of coconut oil. Compared with unhydrolyzed coconut oil, the melting point of hydrolyzed coconut oil decreases, and the degree of decrease is positively correlated with the content of diacylglycerol. The melting point of 10SD sample group decreases from 28.2 ℃ of CO group to 22.3 ℃, with a decrease of 20.92 %. The decrease of melting point is closely related to the change of lipid composition after hydrolysis. The linear structure arrangement of triacylglycerol makes it combine closely inside the crystal, and the molecular structure is stabilized by van der Waals force. The diacylglycerol and monoacylglycerol produced after hydrolysis are irregular due to the increase of polarity, the van der Waals force decreases, and the melting point decreases.

[0093] In order to better illustrate the difference in lipid composition of coconut oil produced by the hydrolysis of four kinds of lipases, cluster heat map analysis was used to statistically analyze the lipid composition information of coconut oil and its hydrolyzed acylglycerol. Through non-targeted qualitative and quantitative analysis of lipids, it was found that 89 kinds of acylglycerols were detected in coconut oil and its hydrolyzate, including 61 kinds of TAG ( Figure 2a ), 23 kinds of DAG ( Figure 2b ), and 5 kinds of MAG ( Figure 2c ). Specifically, the content of triglyceride in unhydrolyzed coconut oil (especially TAG30:0-TAG48:3) was significantly higher than that after hydrolysis. According to the color scale of cluster heat map, for lipase 10SD, the content of 56 TAGs decreased significantly, forming medium-chain saturated fatty acid DAG and MAG, while lipases ADL, TLIM and 20000L showed different degrees of hydrolysis preference at the same degree of hydrolysis. The cluster heat map of the content and types of diacylglycerol before and after hydrolysis of coconut oil is as follows: Figure 2bAs shown, a total of 23 DAGs were generated. Among them, 13 were unsaturated fatty acid DAGs, and the clustering heat map showed that the DAG content increased significantly after 10SD hydrolysis, among which DAG 24:0, DAG 26:0, and DAG 28:0 were generated the most, which may be related to their cleavage sites. Compared with other enzymes, 10SD has stronger binding ability to sn-1 and sn-3 sites. The generation of monoglycerides may be related to the step-by-step hydrolysis of lipase. The content of monoglycerides is low when diacylglycerol is not completely hydrolyzed. A total of 5 MAGs were generated, namely MAG 12:0, MAG 16:0, MAG 18:0, MAG 18:1, and MAG 18:2. Compared with unhydrolyzed CO and other three enzymes, the content of lauric acid monoglyceride (MAG 12:0) in the 10SD-AC group increased significantly, which also indicates the tendency of 10SD enzyme to hydrolyze lauric acid glyceride.

[0094] Figure 2a The middle glycerol tristearate (TAG) is:

[0095] TG 24:0 | TG 8:0_8:0_8:0 sn-glycero-1,2,3-trioctanoate; TG 26:0 | TG 8:0_8:0_10:0 sn-glycero-1,2-dioctanoate-3-decanoate; TG 28:0 | TG 8:0_8:0_12:0 sn-glycero-1,2-dioctanoate-3-dodecanoate; TG 30:0 | TG 8:0_10:0_12:0 sn-glycero-1-octanoate-2-decanoate-3-dodecanoate; TG 32:0 | TG 8:0_12:0_12:0 sn-glycero-1-octanoate-2,3-didodecanoate; TG 34:0 | TG 8:0_12:0_14:0 sn-glycero-1-octanoate-2-dodecanoate-3-myristate; TG 34:2 | TG 8:0_8:0_18:2 sn-glycero-1,2-dioctanoate-3-linoleate; TG 35:0 | TG 10:0_11:0_14:0 sn-glycero-1-decanoate-2-undecanoate-3-myristate; TG 36:0 | TG 10:0_12:0_14:0 sn-glycero-1-decanoate-2-dodecanoate-3-myristate; TG 36:1 | TG 8:0_10:0_18:1 sn-glycero-1-octanoate-2-decanoate-3-oleate; TG 36:2 | TG 8:0_10:0_18:2 sn-glycero-1-octanoate-2-decanoate-3-linoleate; TG 37:0 | TG 11:0_12:0_14:0 sn-glycero-1-undecanoate-2-dodecanoate-3-myristate; TG 38:0 | TG 12:0_12:0_14:0 sn-glycero-1,2-didodecanoate-3-myristate; TG 38:1 | TG 8:0_12:0_18:1 sn-glycero-1-octanoate-2-dodecanoate-3-oleate; TG 38:2 | TG 8:0_12:0_18:2 sn-glycero-1-octanoate-2-dodecanoate-3-linoleate; TG 39:0 | TG 12:0_13:0_14:0 sn-glycero-1-dodecanoate-2-tridecanoate-3-myristate; TG 40:0 | TG 12:0_14:0_14:0 sn-glycero-1-dodecanoate-2,3-dimyristate; TG 40:1 | TG 10:0_12:0_18:1 sn-glycero-1-decanoate-2-dodecanoate-3-oleate; TG 40:2 | TG 10:0_12:0_18:2 sn-glycero-1-decanoate-2-dodecanoate-3-linoleate; TG 42:0 | TG 12:0_14:0_16:0 sn-glycero-1-dodecanoate-2-myristate-3-palmitate; TG 42:1 | TG 12:0_12:0_18:1 sn-glycero-1,2-didodecanoate-3-oleate;TG 42:2 | TG 12:0_12:0_18:2 sn-Glycero-1,2-dilauric-3-linoleate; TG 44:0 | TG 12:0_14:0_18:0 sn-Glycero-1-lauric-2-myristic-3-stearic ester; TG 44:1 | TG 12:0_14:0_18:1 sn-Glycero-1-lauric-2-myristic-3-oleic ester; TG 44:2 | TG 12:0_14:0_18:2 sn-Glycero-1-lauric-2-myristic-3-linoleic ester; TG 44:3 | TG 8:0_18:1_18:2 sn-Glycero-1-octanoic-2-oleic-3-linoleic ester; TG 46:0 | TG 12:0_16:0_18:0 sn-Glycero-1-lauric-2-palmitic-3-stearic ester; TG 46:1 | TG 12:0_16:0_18:1 sn-Glycero-1-lauric-2-palmitic-3-oleic ester; TG 46:2 | TG 12:0_16:0_18:2 sn-Glycero-1-lauric-2-palmitic-3-linoleic ester; TG 46:3 | TG 10:0_18:1_18:2 sn-Glycero-1-decanoic-2-oleic-3-linoleic ester; TG 47:0 | TG 15:0_16:0_16:0 sn-Glycero-1-pentadecanoic-2,3-dipalmitic ester; TG 47:1 | TG 15:0_16:0_16:1 sn-Glycero-1-pentadecanoic-2-palmitic-3-palmitoleic ester; TG 48:0 | TG 14:0_16:0_18:0 sn-Glycero-1-myristic-2-palmitic-3-stearic ester; TG 48:1 | TG 14:0_16:0_18:1 sn-Glycero-1-myristic-2-palmitic-3-oleic ester; TG 48:2 | TG 12:0_18:1_18:1 sn-Glycero-1-lauric-2,3-dioleic ester; TG 48:3 | TG 12:0_18:1_18:2 sn-Glycero-1-lauric-2-oleic-3-linoleic ester; TG 48:4 | TG 12:0_18:2_18:2 sn-Glycero-1-lauric-2,3-dilinoleic ester; TG 49:0 | TG 15:0_16:0_18:0 sn-Glycero-1-pentadecanoic-2-palmitic-3-stearic ester; TG 50:0 | TG 16:0_16:0_18:0 sn-Glycero-1,2-dipalmitic-3-stearic ester; TG 50:1 | TG 16:0_16:0_18:1 sn-Glycero-1,2-dipalmitic-3-oleic ester; TG 50:2 | TG 14:0_18:1_18:1 sn-Glycero-1-myristic-2,3-dioleic ester;TG 50:3 | TG 14:0_18:1_18:2 sn-glycero-1-myristic acid-2-oleic acid-3- linoleic ester; TG 50:4 | TG 14:0_18:2_18:2 sn-glycero-1-myristic acid-2,3- linoleic ester; TG 52:0 | TG 16:0_18:0_18:0 sn-glycero-1-palmitic acid-2,3- distearic ester; TG 52:1 | TG 16:0_18:0_18:1 sn-glycero-1-palmitic acid-2-stearic acid- 3-oleic ester; TG 52:2 | TG 16:0_18:1_18:1 sn-glycero-1-palmitic acid-2,3-dioleic ester; TG 52:3 | TG 16:0_18:1_18:2 sn-glycero-1-palmitic acid-2-oleic acid-3-linoleic ester; TG 52:4 | TG 16:0_18:2_18:2 sn-glycero-1-palmitic acid-2,3-dilinoleic ester; TG 54:2 | TG 18:0_18:1_18:1 sn-glycero-1-stearic acid-2,3-dioleic ester; TG 54:3 | TG 18:1_18:1_18:1 sn-glycero-1,2,3-trioleic ester; TG 54:4 | TG 18:0_18:2_18:2 sn-glycero-1-stearic acid-2,3-dilinoleic ester; TG 54:5 | TG 18:1_18:2_18:2 sn-glycero-1-oleic acid-2,3-dilinoleic ester; TG 54:6 | TG 18:2_18:2_18:2 sn-glycero-1,2,3-trilinoleic ester; TG 54:7 | TG 18:2_18:2_18:3 sn-glycero-1,2-dilinoleic acid-3-linolenic ester; TG 56:1 | TG 16:0_22:0_18:1 sn-glycero-1-palmitic acid-2-behenic acid-3-oleic ester; TG 56:2 | TG 20:0_18:1_18:1 sn-glycero-1-arachidic acid-2,3-dioleic ester; TG 56:3 | TG 18:1_18:1_20:1 sn-glycero-1,2-dioleic acid-3-eicosenoic ester; TG 56:4 | TG 18:1_20:1_18:2 sn-glycero-1-oleic acid-2-eicosenoic acid-3-linoleic ester; TG 58:1 | TG 16:0_24:0_18:1 sn-glycero-1-palmitic acid-2-tetracosanoic acid-3-oleic ester; TG 58:2 | TG 22:0_18:1_18:1 sn-glycero-1-behenic acid-2,3-dioleic ester; TG 58:3 | TG 22:0_18:1_18:2 sn-glycero-1-behenic acid-2-oleic acid-3-linoleic ester.

[0096] Figure 2bDiacylglycerols (DAGs) are:

[0097] DG 36:4 | DG 18:2_18:2 sn-glycerol-1,2-dilinoleate; DG 36:0 | DG 18:0_18:0 sn- glycerol-1,2-distearate; DG 34:0 | DG 16:0_18:0 sn-glycerol-1-palmitate-2-stearate; DG 36:3 | DG 18:1_18:2 sn-glycerol-1-oleate-2-linoleate; DG 36:2 | DG 18:1_18:1 sn- glycerol-1,2-dioleate; DG 32:0 | DG 14:0_18:0 sn-glycerol-1-myristate-2-stearate; DG 26:1 | DG 8:0_18:1 sn-glycerol-1-octanoate-2-oleate; DG 30:0 | DG 15:0_15:0 sn-glycerol-1,2- pentacosanate; DG 28:0 | DG 12:0_16:0 sn-glycerol-1-laurate-2-palmitate; DG 26:0 | DG 12:0_14:0 sn-glycerol-1-laurate-2-myristate; DG 36:1 | DG 18:0_18:1 sn-glycerol-1- stearate-2-oleate; DG 24:0 | DG 12:0_12:0 sn-glycerol-1,2-dilaurate; DG 22:0 | DG 10:0_12:0 sn-glycerol-1-decanoate-2-laurate; DG 30:1 | DG 12:0_18:1 sn-glycerol-1- laurate-2-oleate; DG 34:1 | DG 16:0_18:1 sn-glycerol-1-palmitate-2-oleate; DG 32:1 | DG 14:0_18:1 sn-glycerol-1-myristate-2-oleate; DG 30:2 | DG 12:0_18:2 sn-glycerol-1- laurate-2-linoleate; DG 28:2 | DG 10:0_18:2 sn-glycerol-1-decanoate-2-linoleate; DG 28:1 | DG 10:0_18:1 sn-glycerol-1-decanoate-2-oleate; DG 26:2 | DG 8:0_18:2 sn-glycerol-1- octanoate-2-linoleate; DG 32:2 | DG 14:0_18:2 sn-glycerol-1-myristate-2-linoleate; DG 20:0 | DG 8:0_12:0 sn-glycerol-1-octanoate-2-laurate; DG 18:0 | DG 8:0_10:0 sn-glycerol-1- octanoate-2-decanoate.

[0098] Figure 2c Monoglycerides (MAGs) are:

[0099] MG 12:0: sn-glycerol-1-laurate (1-lauric acid-sn-glyceryl ester); MG 16:0: sn-glycerol-1-palmitate (1-palmitic acid-sn-glyceryl ester); MG 18:0: sn-glycerol-1-stearate (1-stearic acid-sn-glyceryl ester); MG 18:1: sn-glycerol-1-oleate (1-oleic acid-sn-glyceryl ester); MG 18:2: sn-glycerol-1-linoleate (1-linoleic acid-sn-glyceryl ester).

[0100] 2.3 Fourier Transform Infrared Spectroscopy Analysis

[0101] Infrared spectra of coconut oil and its hydrolyzed acylglycerols, as follows Figure 3 As shown, PEAK1-PEAK8 correspond to vibrational absorptions of different functional groups. PEAK1 represents the stretching vibration of the hydroxyl group (-OH); PEAK4 represents the stretching vibration of the carbonyl group (C=O); PEAK7 is an absorption peak resulting from the stretching vibration of the ester bond due to the ether bond; and the remaining peaks (PEAK2, 3, 5, 6, 8) are related to the stretching and bending vibrations of the methylene group (-CH2-). Pure coconut oil does not show a significant absorption peak at peak 1 (PEAK1), while hydrolyzed coconut oil glycerides all show broad and strong absorption peaks at peak 1, indicating the production of hydroxyl (-OH) groups after lipase hydrolysis. This may be because coconut oil exists mainly in the form of TAGs before hydrolysis, while DAG and MAG are generated after hydrolysis. Furthermore, the signal intensity of coconut oil at peak 4 (PEAK4) is greater than 10 SD-AC and 20000 L-AC. This may be because the lipase cleaves the esterified fatty acids on the glycerol backbone during coconut oil hydrolysis, reducing the original ester bond (-COO-) content. The peaks PEAK2, 3, 5, 6, and 8 (-CH2-related peaks) of the four enzymatic hydrolysis products (20000L-AC, ADL-AC, 10SD-AC, and TLIM-AC) showed no significant shift or intensity change. This is because the main fatty acids in coconut oil are medium- and short-chain saturated fatty acids such as lauric acid (C12:0), and the arrangement of their methylene groups (-CH2-) did not change significantly before and after enzymatic hydrolysis. The number of methylene groups decreased slightly due to the reduction in the number of fatty acid chains, but they still dominated overall.

[0102] 2.4 Oxidative Stability Analysis of Coconut Oil and its Hydrolyzed Glycerides

[0103] The primary and secondary oxidation products of oil and fat are generated under the influence of factors such as light and oxygen. The oxidation degree of oil and fat is usually represented by physical and chemical indicators such as peroxide value, conjugated diene value, and thiobarbituric acid value. The peroxide value, thiobarbituric acid value, conjugated diene value, and oxidation induction time of coconut oil and its enzymatic glycerol products are shown in Table 2. The peroxide value and thiobarbituric acid value represent the primary product (hydroperoxide) and secondary product (aldehyde, ketone, and epoxide) of the oil oxidation stage, respectively. The results show that CO has good oxidation stability and is not prone to oxidation during storage, which is related to the high saturation of coconut oil, which is mainly composed of medium and short chain saturated fatty acids. The medium and short carbon chains have stronger resistance. The peroxide value, thiobarbituric acid value, and conjugated diene value of the enzymatic glycerol ester are all increased, among which 10SD has the deepest oxidation degree, with a peroxide value of 15.41±0.22 mg / g, a thiobarbituric acid value of 5.29±0.13 μmol / g, and a CD value of 7.52±0.22 mmol / 100g. The reason for the change in the oxidation degree of the enzymatic glycerol ester of coconut oil after enzymatic hydrolysis may be the change in the composition of glycerol ester and fatty acid.

[0104] Oxidation induction time can more directly reflect the oxidation stability of coconut oil. The Metrohm 743 type oil oxidation stability analyzer was used to perform accelerated oxidation test on coconut oil and its glycerol ester, construct an oxidation model under high temperature environment, and reflect the oxidation degree by collecting the volatile products after oxidation to change the conductivity of ultrapure water. The endpoint of the test is the rapid increase of water conductivity, and is defined as the induction period represented by h. According to the results, the oxidation induction time of CO is the longest, reaching 42.70±3.21 h, indicating that the oxidation rate of CO is small during this process, showing a slow growth trend. The oxidation induction time of the enzymatic glycerol ester after hydrolysis (hydrolyzed glycerol ester) is greatly shortened, and that of 10SD-AC is reduced from 42.70±3.21 h to 26.82±1.34 h. In the follow-up study, attention should be paid to the optimization and conditions of the hydrolysis conditions, further reducing the risk of oxidation of coconut oil, and providing a good theoretical basis for its application in the food field.

[0105] Table 2 Physical and chemical properties and oxidation stability of coconut oil and its hydrolyzed glycerol ester

[0106]

[0107] Note: The same row and different lowercase letters represent significant differences in the relative content of fatty acids P <0.05).

[0108] 2.5 Thermal stability analysis of coconut oil and its hydrolyzed glycerol ester

[0109] Differential scanning calorimetry (DSC) is a highly efficient method for studying the thermal stability behavior of oils and fats and evaluating their melting properties. The thermal stability (DSC curves) of coconut oil and its hydrolyzed glycerides are shown below. Figure 4a As shown, the DSC curves of coconut oil and different enzymatically hydrolyzed oil samples at the same heating rate are similar. Around 100 °C, all four types of enzymatically hydrolyzed coconut oil showed significant endothermic peaks. Combined with TG thermogravimetric analysis, it was found that the sample mass did not change significantly at 100 °C, suggesting that the oil phase may undergo a first phase transition at 100 °C, at which point coconut oil and its acylglycerols are relatively stable. With increasing temperature, a second significant endothermic reaction occurred, with the endothermic peak temperature for CO reaching 437.4 °C, while the peak temperatures of the enzymatically hydrolyzed acylglycerols were lower than this. This indicates that the stability of coconut oil glycerols decreased after enzymatic hydrolysis. This may be because the lipid composition of coconut oil after hydrolysis changes from triesters to diesters and monoesters, disrupting the original crystal structure and leading to reduced stability. Simultaneously, the fatty acid composition affects the carbon chain arrangement, causing a shift in the original spatial configuration. After the peak, all oil samples showed significant exothermic reactions, indicating degradation under thermal conditions, resulting in the loss of their original stability and physicochemical properties.

[0110] Thermogravimetric analysis (TG) is used to reflect changes in the mass of a substance during a controlled heating process. Figure 4b During the temperature range from 30 ℃ to 600 ℃, all oil samples exhibited similar mass loss patterns. From 30 ℃ to 200 ℃, coconut oil and its glycerides showed good stability. Almost no loss of mass was observed, indicating low water content and the absence of other impurities in the oil samples. Before 431.79 ℃, the CO thermogravimetric loss was consistently less than that of enzymatically hydrolyzed glycerides at the same temperature, indicating that natural coconut oil exhibited good stability and minimal heat loss at this temperature. The temperature at which the loss rate reached its maximum coincided with the temperature at which the endothermic peak appeared, a finding corroborated by DSC and TG analyses.

[0111] 2.6 Analysis of the antibacterial properties of coconut oil and glycerides

[0112] To investigate the antibacterial effects of coconut oil and its enzymatically hydrolyzed glycerol esters against pathogenic and putrefactive bacteria, sterile filter paper discs containing coconut oil and its hydrolyzed glycerol esters were placed in culture media containing Staphylococcus aureus, Escherichia coli, and Shewanella for inhibition zone experiments. The experimental results are as follows: Figure 5 As shown in Table 3, pure coconut oil had no inhibitory effect on *E. coli*, and no inhibition zone was observed. After hydrolysis modification, its inhibitory effect on *E. coli* was significantly enhanced. Comparing the antibacterial effects of the four hydrolyzed glycerides, 10SD-AC showed the strongest inhibitory effect on all three bacteria, with an inhibition zone of 12.96 ± 0.48 mm against *E. coli*. The growth curves of the three bacteria are shown below. Figure 6a- Figure 6cAs shown, coconut oil and its hydrolyzed glycerol esters had a significant inhibitory effect on Staphylococcus aureus and Shewanella throughout their growth process, exhibiting a trend of initial decrease followed by near-constancy. This indicates that the activity of these two bacteria was rapidly inhibited or even inactivated after the addition of coconut oil and its hydrolyzed glycerol esters, leading to a decrease in absorbance. Meanwhile... Figure 6b The results show that hydrolyzed glycerol esters still have a significant inhibitory effect on *Escherichia coli*, while the growth curve of the CO group shows an upward trend. Combining the effects of coconut oil and its hydrolyzed glycerol esters on the growth curves of the three bacteria (*Staphylococcus aureus*, *Escherichia coli*, and *Shewanella*), it can be concluded that hydrolyzed coconut oil glycerol esters have excellent antibacterial properties. This may be because the abundance of lauric acid glycerides in the diglycerides and monoglycerides produced after enzymatic hydrolysis increases. Lauric acid monoglycerides have good bactericidal ability and inhibit bacteria, fungi, and enveloped viruses. Simultaneously, the medium- and short-chain fatty acid derivatives (caprylic acid, capric acid, and lauric acid) produced during hydrolysis can disrupt the lipid membrane of bacteria, thus achieving a bactericidal effect. Based on the above fatty acid composition and glycerol ester composition, it can be found that enzymatic hydrolysis effectively improves the antibacterial properties of coconut oil by altering the structure and abundance of coconut oil lipids. This provides a new approach for the application of enzymatically hydrolyzed coconut oil in antibacterial applications.

[0113] Table 3. Diameter of inhibition zones of coconut oil and its hydrolyzed glycerides against Staphylococcus aureus, Escherichia coli, and Shewanella.

[0114]

[0115] Note: Different lowercase letters in the same row indicate significant differences in the diameter of the inhibition zone. P <0.05).

[0116] It should be understood that the disclosed invention is not limited to the specific methods, schemes, and substances described, as these are all subject to variation. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the invention, which is limited only by the appended claims.

Claims

1. The application of hydrolyzed glycerides in the preparation of agents with enhanced antibacterial activity, characterized in that, The method for preparing the hydrolyzed glycerol ester includes the following steps: (1) Place cold-pressed coconut oil in a reaction vessel, add lipase and phosphate buffer solution in sequence, and hydrolyze for 1-2 h at 35-45 ℃ and 500-600 r / min to obtain hydrolysate; (2) The reaction was terminated by adding anhydrous ethanol to the hydrolysate. After neutralization with alkali, extraction with organic solvent, and nitrogen blowing, hydrolyzed glycerol esters were obtained. The lipase is 10 SD; The amount of lipase added is 5-6% of the mass of the cold-pressed coconut oil; The mass-to-volume ratio of the cold-pressed coconut oil to the phosphate buffer solution is 1-1.2:2; The pH of the phosphate buffer solution is 7.0; The hydrolyzed glycerol ester contains 37% triglycerides, 59.81% diglycerides, 34.69% lauric acid, and has a sliding melting point of 22.3 °C. The method for neutralization is as follows: use 0.3 mol / L potassium hydroxide containing 5% anhydrous ethanol to neutralize the free fatty acids produced by hydrolysis, and adjust the pH to 9.2; The organic solvent extraction method is as follows: first, anhydrous diethyl ether of the same volume as the hydrolysate is added for the first extraction at 30 °C, and then n-hexane of the same volume as the hydrolysate is added for the second extraction at 45 °C.

2. The application of hydrolyzed glycerides in the preparation of antibacterial agents, characterized in that, The antibacterial agent has an inhibitory effect on Shewanella. The method for preparing the hydrolyzed glycerol ester includes the following steps: (1) Place cold-pressed coconut oil in a reaction vessel, add lipase and phosphate buffer solution in sequence, and hydrolyze for 1-2 h at 35-45 ℃ and 500-600 r / min to obtain hydrolysate; (2) The reaction was terminated by adding anhydrous ethanol to the hydrolysate. After neutralization with alkali, extraction with organic solvent, and nitrogen blowing, hydrolyzed glycerol esters were obtained. The lipase is 10 SD; The amount of lipase added is 5-6% of the mass of the cold-pressed coconut oil; The mass-to-volume ratio of the cold-pressed coconut oil to the phosphate buffer solution is 1-1.2:2; The pH of the phosphate buffer solution is 7.0; The hydrolyzed glycerol ester contains 37% triglycerides, 59.81% diglycerides, 34.69% lauric acid, and has a sliding melting point of 22.3 °C. The method for neutralization is as follows: use 0.3 mol / L potassium hydroxide containing 5% anhydrous ethanol to neutralize the free fatty acids produced by hydrolysis, and adjust the pH to 9.2; The organic solvent extraction method is as follows: first, anhydrous diethyl ether of the same volume as the hydrolysate is added for the first extraction at 30 °C, and then n-hexane of the same volume as the hydrolysate is added for the second extraction at 45 °C.

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