Dietary triglyceride oil for preventing fatty liver, and preparation method and use thereof

Abstract

The application belongs to the technical field of oil and fat chemistry, and particularly relates to a diglyceride oil capable of preventing fatty liver, a preparation method and application of the diglyceride oil, and the preparation method comprises the following steps: (1) mixing raw oil with glycerol, adding a lipase preparation, and performing enzymatic reaction in a solvent-free system; the content of monoglyceride in the raw oil is 40-80 wt%; (2) separating the reaction product of step (1), collecting the upper oil phase, and then performing secondary molecular distillation purification on the oil phase to obtain an oil product with a diglyceride content of greater than or equal to 80 wt%. The technical scheme of the application uses raw oil with a monoglyceride component content greater than 40% as raw material to synthesize an oil product with a high diglyceride content, and the amount of the lipase preparation added is low, so that the production cost can be effectively reduced, and the obtained product has high diglyceride and sterol contents and has a significant effect in preventing fatty liver.

Description

Technical Field

[0001] This invention relates to the field of oil modification and processing technology, specifically to a diglyceride oil that can prevent fatty liver, its preparation method, and its application. Background Technology

[0002] Diacylglycerol (DAG) is a structural lipid composed of glycerol molecules linked to two fatty acid chains via ester bonds. It is naturally found in animal and vegetable oils, but its content in conventional edible oils is usually less than 5%. Diacylglycerol oil was approved as a new resource food by the Chinese Ministry of Health in 2009, and in 2021, the National Health Commission of China further standardized its quality standards (diacylglycerol content ≥40%, daily consumption ≤30 grams). However, the full realization of its health benefits still depends on products with higher purity.

[0003] Currently, the industrial preparation methods for diglycerides mainly fall into three categories: chemical synthesis, high-temperature esterification, and enzymatic methods. Chemical synthesis typically involves reacting protected glycerol derivatives (such as benzyl glycidyl ether) with fatty acids, followed by purification steps such as deprotection and recrystallization. While this can yield 1,2-diglycerides with 85-95% purity, the process involves organic solvents (such as n-hexane and diethyl ether) and heavy metal catalysts, posing a risk of solvent residue and incurring high environmental costs. High-temperature esterification catalyzes the reaction of oils or fatty acids with glycerol at 150–250°C and high pressure. Although simple to operate, the high-temperature environment easily leads to the formation of carcinogens such as glycidyl esters (GE) and 3-chloropropanol esters (3-MCPD), raising concerns about food safety. Enzymatic methods, due to their mild conditions and high selectivity, are considered the mainstream approach and can be further divided into:

[0004] 1. Transesterification method: Immobilized lipase catalyzes the reaction of triglycerides with glycerol, and diglycerides are generated through glycerolysis;

[0005] 2. Hydrolysis method: Triglycerides are partially hydrolyzed using lipase, followed by esterification and recombination;

[0006] 3. Direct esterification: Synthesis using free fatty acids and glycerol as substrates. Enzymatic methods can theoretically achieve highly selective synthesis; for example, when using immobilized enzymes such as Lipozyme 435, product purity can reach over 90%.

[0007] However, several bottlenecks remain in actual industrial production: enzyme catalysis requires precise control of substrate ratios (such as the glycerol / oil molar ratio), water activity, and reaction time; high-viscosity systems result in low mass transfer efficiency, often necessitating the addition of organic solvents (such as ethanol) to improve flowability, which in turn introduces a purification burden. Furthermore, in existing experiments, the amount of enzyme added is typically around 15% (as a percentage of the monoglyceride mass), thus hindering industrial-scale production.

[0008] In summary, while current diglyceride preparation technologies offer diverse pathways, they are all limited by trade-offs in efficiency, cost, safety, and product functionality. The industry urgently needs a method that can simultaneously achieve low enzyme consumption, solvent-free operation, high-purity DAG synthesis, and sufficient retention of active ingredients such as sterols, to fully realize its potential in preventing metabolic diseases and meet the demand for upgraded healthy fats. Summary of the Invention

[0009] The present invention aims to overcome the shortcomings of existing methods for preparing diglyceride oil in terms of efficiency, cost, and safety, and provides a diglyceride oil that can prevent fatty liver, as well as its preparation method and application, to overcome the above-mentioned deficiencies.

[0010] To achieve the above-mentioned objectives, the present invention is implemented through the following technical solution:

[0011] In a first aspect, the present invention provides a method for preparing diglyceride oil for preventing fatty liver, comprising the following steps:

[0012] (1) Mix the raw oil with glycerin, add lipase preparation, and carry out enzymatic hydrolysis in a solvent-free system;

[0013] The raw oil contains 40-80 wt% monoglycerides.

[0014] The lipase preparation is a complex enzyme containing Rhizopus oryzae lipase and Candida albicans lipase.

[0015] (2) Separate the reaction product of step (1), collect the upper oil phase, and then purify the oil phase by secondary molecular distillation to obtain an oil product with a diglyceride content ≥80wt%.

[0016] Diglycerides, as functional components of natural oils, have long attracted academic attention due to their unique metabolic characteristics compared to ordinary triglycerides, particularly in their unique value in regulating visceral fat and maintaining liver health. However, existing preparation technologies have long been constrained by multiple contradictions: while chemical methods can synthesize high-purity products, they are limited to laboratory settings due to organic solvents and heavy metal residues; enzymatic methods, although under mild conditions, suffer from mass transfer resistance in high-viscosity systems, forcing industry to rely on solvents to improve flowability, which not only increases the purification burden but also poses food safety risks. More importantly, traditional processes have poor adaptability to raw materials; high-acid-value oils require pre-esterification, resulting in the loss of active ingredients, while low-saturation oils lose their commercial value due to turbidity at low temperatures.

[0017] Faced with this impasse, researchers began to re-examine the nature of the reaction: Could they completely get rid of solvent dependence through raw material innovation? And could they break through the constraints of "high temperature damaging activity and low temperature reducing purity" in the purification process? This thinking directly led to a shift in the technical solution of this application, namely, instead of continuing to optimize the triglyceride conversion pathway, it is better to explore a new path to develop a direct enzymatic hydrolysis route for high monoglyceride feedstock oil.

[0018] Theoretically, since monoglycerides only require one acylation step to generate diglycerides, using monoglycerides as a reactant can significantly shorten the reaction chain and lower the enzyme catalytic energy barrier. However, the challenge lies in the fact that monoglycerides have high melting points and are prone to crystallization, making them more likely to form gels in solvent-free systems, hindering mass transfer. To address this, this application creatively targets specific feedstock oils with a monoglyceride content of 40-80 wt%, and breaks through conventional single-enzyme catalysis thinking, discovering that a specific compound lipase preparation containing Rhizopus oryzae lipase and Candida albicans lipase can form a synergistic effect in a solvent-free environment. Specifically, the Rhizopus oryzae lipase primarily targets acyl transfer to construct the backbone, preferentially hydrolyzing monoglycerides to generate free fatty acids, instantly reducing the local viscosity in the reaction system. The Candida albicans lipase then catalyzes the esterification of free fatty acids with the remaining monoglycerides to synthesize diglycerides, thereby precisely controlling the selection of the diglyceride reaction activity potential. Therefore, during this process, "viscosity oscillations" can be generated at the microscopic scale, continuously disrupting the gel precursor structure of monoglycerides, thereby drastically reducing the enzyme addition amount to ≤1% (compared to 3-15% in traditional processes) and compressing the reaction time to 3-10 hours. Thus, this design completely avoids solvent recovery costs while simultaneously solving the problem of enzyme inactivation caused by high viscosity.

[0019] Finally, this application innovatively uses two-stage molecular distillation as the core purification step: the first stage is low-temperature distillation to remove free fatty acids and water, and the second stage is precise temperature control to achieve efficient separation of diglycerides from monoglycerides / triglycerides. This process chain design completely avoids the destruction of diglyceride activity by high temperature.

[0020] In summary, this solution, through innovation in raw material composition, adaptation of complex enzyme systems, and reconstruction of distillation and purification processes, forms a complete technical closed loop of "highly active raw materials - low-consumption, high-efficiency catalysis - targeted activity retention." Its innovation lies not only in overcoming technical biases at each stage (such as the previously considered difficulty in direct reaction of high monoglyceride raw materials and insufficient enzyme activity in solvent-free systems), but also in the fact that the overall solution is the first to achieve a leap from "ordinary dietary oil" to "targeted fatty liver prevention functional oil," providing the industry with a novel solution that combines economic efficiency with significant biological effects.

[0021] Preferably, the mass ratio of the raw oil to glycerin in step (1) is 60:40 to 95:5.

[0022] This application innovatively solves the mass transfer barrier caused by monoglyceride crystallization in solvent-free enzymatic hydrolysis systems by precisely controlling the mass ratio of feedstock oil to glycerol from 60:40 to 95:5. Specifically, the glycerol ratio setting has a dual function: when the monoglyceride content in the feedstock oil is low (40 wt%), a higher glycerol content (up to 40%) forms a hydrogen bond network through its polyhydroxy structure, disrupting the orderly stacking of monoglyceride molecules and inhibiting crystal nucleation; while when the monoglyceride content is high (80 wt%), a lower glycerol ratio (5%) avoids excessive free hydroxyl groups from initiating phase separation, while ensuring stoichiometric balance of the reaction. This dynamic ratio design keeps the reaction system in a liquid crystal state rather than a gel state, reducing viscosity by more than 60% compared to traditional solvent-free systems. This not only makes it possible to reduce the enzyme addition to ≤1%, but also compresses the reaction time to 3-10 hours, achieving mass transfer efficiency comparable to traditional solvent methods while avoiding organic solvents, laying a key foundation for continuous industrial production.

[0023] Preferably, the raw oil in step (1) also contains 8,000 ppm to 16,000 ppm of sterols.

[0024] Traditional processes generally overlook the functional value of trace sterols in raw oils. Existing technologies either use ordinary vegetable oils (sterol content is usually below 6000 ppm) or cause significant sterol degradation during purification due to high temperatures. This invention discovers that sterols within a specific range (8000–16000 ppm) in raw oils can be efficiently retained in solvent-free enzymatic hydrolysis and low-temperature molecular distillation processes, forming a unique synergistic effect with high-purity diglycerides in metabolic regulation. This design overturns the traditional approach of "only pursuing diglyceride purity," creatively discovering that sterols, as regulators of hepatic cholesterol metabolism, can indirectly reduce hepatic lipid deposition by inhibiting intestinal cholesterol absorption, while diglycerides directly promote hepatic fatty acid oxidation. The two produce a biological synergistic effect of "1+1>2" within a specific content range (when the raw sterol content is below 8000 ppm, the final product activity is insufficient; above 16000 ppm, it is easily thermally decomposed during distillation, producing byproducts).

[0025] Furthermore, this application also discovers that because sterol molecules consist of a rigid sterol core and flexible side chains, their spatial structure can be embedded in the interlayer of monoglyceride molecules, thereby acting as a structure modifier to inhibit the crystallization of monoglycerides. Specifically, the hydrophobic ring of the sterol core can bind to the monoglyceride fatty acid chain through van der Waals forces, thereby disrupting the orderly stacking of monoglyceride molecules, while the hydroxyl groups in the sterol can form a hydrogen bond network with the glycerol backbone, increasing the intermolecular distance. Therefore, this "molecular wedge" effect significantly reduces the crystallization tendency of the system, allowing the reaction to proceed in an orderly manner under solvent-free conditions.

[0026] Preferably, the preparation method of the raw material oil is as follows: vegetable oil and glycerol are mixed and reacted under the catalysis of Aspergillus niger lipase preparation. After the reaction is completed, the reactants are centrifuged and the upper oil layer is taken for two-stage molecular distillation. The temperature of the first-stage molecular distillation is 160-175℃ and the vacuum degree is 20-100Pa. The temperature of the second-stage molecular distillation is 180-195℃ and the vacuum degree is 10-20Pa.

[0027] Preferably, the mass ratio of Rhizopus oryzae lipase to Candida lipase in the lipase preparation in step (1) is 1:1 to 4:1.

[0028] This invention is the first to discover a significant synergistic catalytic mechanism between Rhizopus oryzae lipase and Candida albicans lipase in a specific ratio. Rhizopus oryzae lipase preferentially catalyzes the esterification of glycerol with fatty acids to generate monoglyceride intermediates, while Candida albicans lipase specifically promotes the transesterification of monoglycerides with sterol esters in the feedstock oil. This dual-enzyme relay effect significantly optimizes the diglyceride synthesis pathway. More importantly, this ratio range directly correlates with product activity: when the enzyme ratio is between 1:1 and 4:1, the dual-enzyme synergy not only achieves ultra-low enzyme consumption under solvent-free conditions but also directionally retains sterol active groups (final product sterol ≥15000ppm), allowing diglycerides and sterols to form spatially complementary molecular assemblies (resulting in a more than 2-fold increase in liver TAI index improvement compared to the control group). This "directed coupling of dual enzymes - self-assembly of active molecules" effect essentially reconstructs the biosynthetic pathway of functional lipids, providing a novel material basis for the prevention of fatty liver.

[0029] Preferably, the amount of lipase preparation added in step (1) is 0.2 to 1 wt% of the raw oil mass.

[0030] Preferably, the enzymatic hydrolysis reaction temperature in step (1) is 40–75°C and the reaction time is 3–10 h.

[0031] Preferably, the conditions for the secondary molecular distillation in step (2) include:

[0032] First-stage distillation temperature: 100–150℃, vacuum degree: <50Pa;

[0033] Secondary distillation temperature: 150–180℃, vacuum degree: <10Pa.

[0034] Traditional molecular distillation, aiming for DAG purity >90%, often pushes the secondary distillation temperature above 230℃, leading to the decomposition or isomerization of heat-sensitive sterols. This method innovatively employs a gradient temperature control strategy: primary distillation at ≤150℃ / 50Pa removes light components, while secondary distillation at ≤180℃ / 10Pa retains the heavy phase. This low-temperature environment not only increases sterol retention to ≥15000ppm (2.5 times higher than traditional processes) but also avoids DAG loss. In practical applications, this process maintains a stable DAG purity of ≥80% in the final product, while maintaining an acid value ≤0.15mgKOH / g. These indicators collectively form the functional basis for preventing fatty liver: high-purity DAG promotes β-oxidation of fatty acids in the liver, while sufficient sterols inhibit cholesterol esterification and deposition, thus blocking abnormal lipid accumulation in hepatocytes through a dual-pathway approach.

[0035] Preferably, the triglyceride content in the raw oil is ≤10wt%.

[0036] Secondly, this application also provides a diglyceride oil, which is prepared by the method described in any of the preceding claims, having a diglyceride content of ≥80wt% and a sterol content of ≥15000ppm.

[0037] Thirdly, this application also provides the use of the diglyceride oil described above in the preparation of a medicament for the prevention of fatty liver.

[0038] Therefore, this application has the following beneficial effects:

[0039] This invention can prepare diglyceride oil products with a diglyceride content greater than 80% and a sterol content higher than 15,000 ppm, showing significant clinical efficacy in preventing fatty liver, an effect that cannot be achieved in comparative studies. Compared with existing technologies, the technical solution of this invention can use raw oil with a monoglyceride component content greater than 40% as a raw material to synthesize high-content diglyceride oil products. The operation is simple, the amount of lipase preparation added is low, which can effectively reduce production costs; the obtained diglycerides have high purity and high sterol content, showing significant effects in preventing fatty liver. Attached Figure Description

[0040] Figure 1 This represents the change in visceral fat area (VFA) relative to pre-intervention values ​​during the fatty liver prevention test in this application.

[0041] Figure 2 This represents the change in total body fat (TBF) relative to pre-intervention levels during the fatty liver prevention test in this application. Detailed Implementation

[0042] The present invention will be further described below with reference to specific embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0043] The feedstock oils used in the following embodiments of this application can be obtained through external purchase or laboratory preparation. The method for preparing feedstock oils with different monoglyceride contents through laboratory preparation is as follows:

[0044] Preparation of feedstock oil

[0045] Preparation Example 1: Rapeseed oil and glycerol were mixed at a mass ratio of 100:10. Aspergillus niger lipase preparation AP6 (purchased from Sigma 62301 200U / g, the same lipase used in the following preparation examples) was added to the mixture at 1% of the rapeseed oil mass for catalytic reaction. The reaction temperature was 60℃ and the reaction time was 8 hours. After the reaction, the reactants were centrifuged, and the upper oil layer was subjected to two-stage molecular distillation. The first-stage molecular distillation temperature was 175℃ and the vacuum degree was 50Pa, while the second-stage molecular distillation temperature was 180℃ and the vacuum degree was 20Pa, yielding purified light components, i.e., the raw oil (the raw oil contained 81.13% monoglycerides, 0.12% triglycerides, and 10021ppm sterols).

[0046] Preparation Example 2: Rice bran oil and glycerol were mixed at a mass ratio of 100:10. Aspergillus niger lipase preparation AP6 was added to the mixture at 1% of the rice bran oil mass for catalytic reaction. The reaction temperature was 60℃ and the reaction time was 8 hours. After the reaction, the reactants were centrifuged, and the upper oil layer was subjected to two-stage molecular distillation. The first-stage molecular distillation was carried out at 165℃ under a vacuum of 20 Pa, and the second-stage molecular distillation was carried out at 190℃ under a vacuum of 10 Pa, yielding purified light components, i.e., the raw oil (the raw oil contained 61.92% monoglycerides, 4.33% triglycerides, and 14988 ppm sterols).

[0047] Preparation Example 3: Peanut oil (sterol content 3850 ppm, supplemented with 4200 ppm sterols) and glycerol were mixed at a mass ratio of 100:10. Aspergillus niger lipase preparation AP6, at 1% of the peanut oil mass, was added to the mixture for catalytic reaction at 60℃ for 8 hours. After the reaction, the reactants were centrifuged, and the upper oil layer was subjected to two-stage molecular distillation. The first-stage molecular distillation was carried out at 160℃ under a vacuum of 100 Pa, and the second-stage molecular distillation was carried out at 195℃ under a vacuum of 10 Pa, yielding purified light components, i.e., the raw oil (the raw oil contained 40.65% monoglycerides, 9.71% triglycerides, and 8111 ppm sterols).

[0048] Preparation Example 4: Corn oil and glycerol were mixed at a mass ratio of 100:10. Aspergillus niger lipase preparation AP6, at 1% of the corn oil mass, was added to the mixture for catalytic reaction at 60°C for 8 hours. After the reaction, the reactants were centrifuged, and the upper oil layer was subjected to two-stage molecular distillation. The first-stage molecular distillation was carried out at 170°C under a vacuum of 50 Pa, and the second-stage molecular distillation was carried out at 190°C under a vacuum of 20 Pa, yielding purified light components, i.e., the feedstock oil (the feedstock oil contained 70.55% monoglycerides, 3.18% triglycerides, and 12310 ppm sterols).

[0049] Preparation Example 5: Soybean oil and glycerol were mixed at a mass ratio of 100:10. Aspergillus niger lipase preparation AP6, at 1% of the soybean oil mass, was added to the mixture for catalytic reaction at 60°C for 8 hours. After the reaction, the reactants were centrifuged, and the upper oil layer was subjected to two-stage molecular distillation. The first-stage molecular distillation was carried out at 150°C under a vacuum of 100 Pa, and the second-stage molecular distillation was carried out at 205°C under a vacuum of 10 Pa, yielding purified light components, i.e., the raw oil (the raw oil contained 53.22% monoglycerides, 20.76% triglycerides, and 11020 ppm sterols).

[0050] Example 1

[0051] A method for preparing diglyceride oil for preventing fatty liver includes the following steps:

[0052] (1) The raw material oil (containing 81.13% monoglyceride, 0.12% triglyceride, and 10021 ppm sterol) and glycerol prepared in Preparation Example 1 were mixed at a mass ratio of 95:5. Then, 5.1% of a compound enzyme preparation by mass of the raw material oil was added to catalyze the reaction at 60°C for 5 hours to obtain the reactants. The compound enzyme preparation was a 1:1 mass ratio of Rhizopus oryzae lipase (purchased from Sigma 80612 30,000 U / g) and Candida albicans lipase (purchased from Sigma 62316 2000 U / g).

[0053] (2) After centrifuging the reactants to obtain the supernatant, a second molecular distillation was performed. The temperature of the first molecular distillation was 150℃ and the vacuum degree was 50Pa. The temperature of the second molecular distillation was 180℃ and the vacuum degree was 10Pa. The heavy phase product was collected and decolorized and deodorized to obtain diglyceride oil.

[0054] Example 2

[0055] A method for preparing diglyceride oil for preventing fatty liver includes the following steps:

[0056] (1) The raw material oil (containing 61.92% monoglyceride, 4.33% triglyceride, and 14988 ppm sterol) prepared in Preparation Example 2 and glycerol were mixed at a mass ratio of 60:40. Then, 1% of the mass of the raw material oil was added to a compound enzyme preparation for catalytic reaction at a reaction temperature of 75°C for 3 hours to obtain the reactants. The compound enzyme preparation was a combination of Rhizopus oryzae lipase and Candida albicans lipase at a mass ratio of 4:1.

[0057] (2) After centrifuging the reactants to obtain the supernatant, a second molecular distillation was performed. The temperature of the first molecular distillation was 150℃ and the vacuum degree was 10Pa. The temperature of the second molecular distillation was 170℃ and the vacuum degree was 10Pa. The heavy phase product was collected and decolorized and deodorized to obtain diglyceride oil.

[0058] Example 3

[0059] A method for preparing diglyceride oil for preventing fatty liver includes the following steps:

[0060] (1) The raw material oil (containing 40.65% monoglyceride, 9.71% triglyceride, and 8111 ppm sterol) prepared in Preparation Example 3 and glycerol were mixed at a mass ratio of 70:30. Then, 0.2% of a compound enzyme preparation by mass of the raw material oil was added for catalytic reaction at 40°C for 10 hours to obtain the reactant. The compound enzyme preparation was a combination of Rhizopus oryzae lipase and Candida albicans lipase at a mass ratio of 3:1.

[0061] (2) After centrifuging the reactants to obtain the supernatant, a second molecular distillation was performed. The temperature of the first molecular distillation was 120℃ and the vacuum degree was 10Pa. The temperature of the second molecular distillation was 150℃ and the vacuum degree was 0.1Pa. The heavy phase product was collected and decolorized and deodorized to obtain diglyceride oil.

[0062] Example 4

[0063] A method for preparing diglyceride oil for preventing fatty liver includes the following steps:

[0064] (1) The raw material oil (containing 70.55% monoglyceride, 3.18% triglyceride, and 12310 ppm sterol) and glycerol prepared in Preparation Example 4 were mixed at a mass ratio of 85:15. Then, 0.5% of a compound enzyme preparation by mass of the raw material oil was added to catalyze the reaction at 75°C for 4 hours to obtain the reactants. The compound enzyme preparation was a combination of Rhizopus oryzae lipase and Candida albicans lipase at a mass ratio of 2:1.

[0065] (2) After centrifuging the reactants to obtain the supernatant, a second molecular distillation was performed. The temperature of the first molecular distillation was 150℃ and the vacuum degree was 30Pa. The temperature of the second molecular distillation was 180℃ and the vacuum degree was 10Pa. The heavy phase product was collected and decolorized and deodorized to obtain diglyceride oil.

[0066] Comparative Example 1 (different raw materials, high triglyceride content: lower DAG purity, lower sterol content)

[0067] The only difference from Example 1 is the raw material oil used to prepare diglycerides; all other conditions are the same, as follows:

[0068] The raw material for preparing diglycerides is commercially available sunflower seed oil.

[0069] Comparative Example 2 (Different raw materials, higher triglyceride content: lower DAG purity)

[0070] The only difference from Example 3 is the raw material oil used to prepare diglycerides; all other conditions are the same. The raw material oil used is the same as that prepared in Example 5.

[0071] Comparative Example 3 (Different molecular distillation purification processes: low sterol content)

[0072] The only difference from Example 1 is the molecular distillation process in step (2), while the other conditions are the same, as follows:

[0073] After centrifuging the reactants to obtain the supernatant, a two-stage molecular distillation was performed. The first-stage molecular distillation was carried out at a temperature of 150℃ and a vacuum of 10 Pa; the second-stage molecular distillation was carried out at a temperature of 230℃ and a vacuum of 0.1 Pa. The heavy phase product was collected and decolorized and deodorized to obtain diglyceride oil.

[0074] Comparative Example 4 (same raw materials, single lipase: DAG purity insufficient)

[0075] The only difference from Example 3 is that the total amount and composition of the lipase preparation remain the same, except that it is only Rhizopus oryzae lipase, while the other conditions are the same.

[0076] Comparative Example 5 (same raw materials, single lipase: DAG purity insufficient)

[0077] The only difference from Example 3 is that the total amount and composition of the lipase preparation remain the same, except that it is only Candida lipase, while the other conditions are the same.

[0078] Detection method:

[0079] The detection methods for triglycerides, diglycerides, monoglycerides, and free fatty acids are in accordance with GB / T 26636-2011 Determination of Polymeric Triglycerides in Animal and Vegetable Oils by High Performance Size Exclusion Chromatography (HPSEC).

[0080] The acid value test method refers to GB 5009.229-2016 National Food Safety Standard - Determination of Acid Value in Food;

[0081] The detection method for sterols refers to GB / T 25223-2013 Determination of Sterol Composition and Total Sterols in Animal and Vegetable Oils by Gas Chromatography.

[0082] The test results are shown in Table 1 below:

[0083] Table 1. Diglyceride and sterol content of the diglyceride oils prepared by the above methods.

[0084]

[0085] As shown in Table 1 above, the preparation method described in this application enables the targeted synthesis of oil products with high diglyceride and sterol content using raw oil containing more than 40% monoglyceride as the raw material and a specific compound lipase preparation containing Rhizopus oryzae lipase and Candida albicans lipase as the catalyst. Furthermore, compared to existing technologies, the content of the added lipase preparation is significantly reduced, and the reaction process is always carried out in a solvent-free system. This eliminates the dependence on solvents in existing technologies, thereby reducing the burden of subsequent purification, effectively lowering production costs, and simplifying the operation process, thus facilitating industrial implementation.

[0086] Verification of the effectiveness of fatty liver prevention test:

[0087] Study Design: This is a randomized, placebo-controlled, double-blind study. Participants must meet the inclusion criteria and provide written informed consent before enrollment.

[0088] The inclusion criteria were: (1) age 18-50 years, (2) body mass index (BMI) ≥22 kg / m2 or waist circumference ≥85 cm for men and ≥80 cm for women, (3) no history of food allergies, and (4) willingness to complete the questionnaire for this trial.

[0089] People with the following conditions are excluded: (1) weight loss of more than 4.5 kg in the past 2 months; (2) impaired liver and kidney function; (3) endocrine diseases under medication (such as diabetes, thyroid disease); (4) diagnosed with hyperlipidemia; (5) diagnosed with cancer; (6) heavy drinking in the past year; (7) currently smoking; (8) breastfeeding or pregnant.

[0090] Based on a computer-generated list of participants with BMI and weight, eligible participants (n = 103,61 men) were randomly assigned to different diglyceride intervention groups in groups of five. Participants underwent clinical analysis at weeks 0, 4, and 8 of the treatment period. They were required to maintain their lifestyle habits, such as diet and physical activity, during the testing period.

[0091] Diet for testing

[0092] Given the limited use of cooking oil in Chinese breakfasts, participants were provided with only lunch and dinner during the intervention period (limited to 30 grams of diglyceride oil per person per day). Foods made from DAG-rich cooking oils (the diglyceride oils prepared in the examples or comparative examples) were provided to the diglyceride intervention participants, with an average daily oil intake of 22.39 g per person. These foods were prepared uniformly by the canteen, and the recipes and food preparation were identical except for the use of different cooking oils. There was no restriction on carbohydrate intake during the intervention. After the meals were prepared, the researchers divided them into four portions, with four dishes per meal for each group. Petri dishes for different groups were prepared, packaged, and labeled for differentiation.

[0093] Before the intervention (week 0) and after the intervention (week 8) for three consecutive days, the weight of raw materials (including cooking oil and seasonings) and each meal was objectively recorded before cooking. All ingredients were prepared solely for the intervention and had no other use. Participants were provided with pre-weighed dishes and staple foods in the cafeteria. At the end of each meal, the remaining dishes and staple foods were weighed using an electronic scale and recorded (accurate to 0.005 kg). Participants' nutritional expenditure was assessed for three consecutive days by integrating their self-reported intake of additional food or beverages with the expenditure of their provided meals. There was no statistically significant difference in energy intake between the two groups before and after the intervention.

[0094] Results Analysis

[0095] Tissue attenuation imaging (TAI) and tissue scattering distribution imaging (TSI) were used to quantitatively examine and diagnose hepatic steatosis. Visceral fat area (VFA) and total body fat (TBF) were measured using dual-energy X-ray ablation (HORIZON-A-CN, HOLOGIC Inc., Marlborough, MA, USA). All measurements were taken under fasting conditions, with participants removing outer clothing and metal objects. All measurements were performed by a licensed physician.

[0096] Test results are as follows Figure 1 , Figure 2 And as shown in Table 2 below. In Table 2: ∆TSI represents the change in liver TSI relative to pre-intervention levels; ∆TAI represents the change in liver TAI relative to pre-intervention levels.

[0097] ∆VFA represents the change in visceral fat area (VFA) relative to before intervention; ∆TBF represents the change in total body fat (TBF) relative to before intervention.

[0098] Table 2

[0099]

[0100] In the table above, * indicates that there was a significant difference between the diglyceride intervention group and the pre-intervention group in the examples. (Analysis of variance, *P<0.05, **P<0.01, ***P<0.001).

[0101] From above Figure 1 , Figure 2 As shown in Table 2, after 8 weeks of intervention with the diglyceride oil prepared in this application, the TBF and VFA of the high-purity diglyceride oil rich in sterols in the examples decreased more significantly than those of the other proportion groups, indicating that the diglyceride oil prepared in each example has outstanding effects in preventing fatty liver.

[0102] The specific embodiments described herein are merely illustrative of the spirit of the invention. Those skilled in the art to which this invention pertains may make various modifications or additions to the described specific embodiments or use similar methods to substitute them, without departing from the spirit of the invention or exceeding the scope defined by the appended claims.

Claims

1. A method for preparing a diglyceride oil for preventing fatty liver, characterized in that, Includes the following steps: (1) Mix the raw oil with glycerin, add lipase preparation, and carry out enzymatic hydrolysis in a solvent-free system; The raw material oil contains 40-80 wt% monoglycerides and ≤10 wt% triglycerides. The lipase preparation is a compound enzyme containing Rhizopus oryzae lipase and Candida albicans lipase, with the mass ratio of Rhizopus oryzae lipase to Candida albicans lipase being 1:1 to 4:1, and the amount of lipase preparation added being 0.2 to 1 wt% of the raw oil mass; (2) Separate the reaction product of step (1), collect the upper oil phase, and then purify the oil phase by secondary molecular distillation to obtain an oil product with a diglyceride content ≥80wt%. The conditions for the second-order molecular distillation include, First-stage distillation temperature: 100–150℃, vacuum degree: ≤50Pa; Secondary distillation temperature: 150–180℃, vacuum degree: ≤10Pa.

2. The preparation method according to claim 1, characterized in that, The mass ratio of the raw oil to glycerin in step (1) is 60:40 to 95:

5.

3. The preparation method according to claim 1 or 2, characterized in that: The raw material oil mentioned in step (1) also contains 8,000 ppm to 16,000 ppm of sterols.

4. The preparation method according to claim 3, characterized in that: The preparation method of the raw material oil is as follows: vegetable oil and glycerol are mixed and reacted under the catalysis of Aspergillus niger lipase preparation. After the reaction is completed, the reactants are centrifuged and the upper oil layer is taken for two-stage molecular distillation. The temperature of the first-stage molecular distillation is 160-175℃ and the vacuum degree is 20-100Pa. The temperature of the second-stage molecular distillation is 180-195℃ and the vacuum degree is 10-20Pa.

5. The preparation method according to claim 1, characterized in that, The enzymatic hydrolysis reaction temperature in step (1) is 40-75℃ and the reaction time is 3-10h.

6. The use of a diglyceride oil prepared by the method according to any one of claims 1-5 in the preparation of a medicament for the prevention of fatty liver.

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