Method for enzymatic preparation of lactulose fatty acid monoester and use thereof

Abstract

This invention provides a method and application for the enzymatic preparation of lactulose fatty acid monoesters, belonging to the field of biosynthesis technology. By preparing a series of lactulose monoesters with different fatty acid chain lengths (C8 to C18), this invention systematically reveals for the first time their surface activity, emulsifying properties, emulsion stability, and in vitro digestion behavior, and establishes a clear structure-activity relationship. The provided lactulose esters not only possess excellent, tunable emulsifying properties but also exhibit the prebiotic potential of the lactulose group, thus providing a novel, predictable, bifunctional, green surfactant for the food, pharmaceutical, and cosmetic industries.

Description

Technical Field

[0001] This invention belongs to the field of biosynthesis technology, specifically relating to a method and application for the enzymatic preparation of lactulose fatty acid monoesters. Background Technology

[0002] Sugar esters are an important class of nonionic surfactants, typically prepared by acylation of the hydroxyl groups of sugars with fatty acids or their derivatives. Their molecular structure combines a hydrophilic sugar head with a hydrophobic fatty acid tail, a unique amphiphilic structure that endows them with excellent surface activity, significantly reducing the tension at the oil-water interface. Therefore, they are widely used as emulsifiers, dispersants, solubilizers, and wetting agents. Furthermore, sugar ester surfactants are usually derived from renewable resources, exhibiting good biodegradability and low toxicity, and are considered environmentally friendly "green chemicals," attracting significant attention in the food, pharmaceutical, cosmetic, and fine chemical industries. For example, sucrose esters, as mature commercial products, are widely used as food-grade emulsifiers.

[0003] However, despite the increasingly widespread development and application of glycolipid surfactants, current research mainly focuses on improving their emulsifying properties and environmental friendliness. Research on developing novel functional glycolipids with unique nutritional value or health benefits remains very limited. In particular, the exploration of glycolipid surfactants capable of exerting prebiotic or other specific nutritional effects is still in its early stages, yet these products possess enormous market potential, meeting the growing consumer demand for healthy, natural, and multifunctional products.

[0004] Lactulose (4- O - β -D-galactopyranosyl- β Fructose-D-furanose is a synthetic disaccharide known for its significant prebiotic activity. Unlike common dietary disaccharides such as sucrose and lactose, which are hydrolyzed into monosaccharides and provide energy in the human body, lactulose resists degradation by digestive enzymes and reaches the colon mostly intact. In the colon, it can be selectively fermented and utilized by beneficial gut bacteria such as Bifidobacteria and Lactobacilli, thereby significantly promoting the growth of these bacteria and their beneficial metabolic activities. Based on this characteristic, lactulose has long been successfully used as a "bifidus factor" in infant formula, and its industrial production technology is quite mature, providing a reliable raw material source for the preparation of functional ester derivatives.

[0005] Despite the clear health benefits of lactulose, current research on lactulose fatty acid monoesters is limited. Existing technologies primarily focus on the development of sugar esters using common sugars such as sucrose as raw materials, with relatively insufficient research on novel functional sugar esters that combine emulsifying properties with specific health benefits (such as prebiotic effects). For example, Chinese invention patent CN103874765A discloses a method for hydrolyzing and modifying a mixture of sucrose esters using lipase to regulate its esterification degree distribution; Chinese invention patent CN102850413A discloses a new process for synthesizing sucrose esters using a three-phase phase transfer catalyst; and Chinese invention patent CN110229197A discloses a solvent-free green synthesis method for sucrose esters. Although these technologies have made some progress in the preparation process, they still have common limitations: First, existing research focuses on conventional sugar esters such as sucrose esters, and lacks the development of functional surfactants that also have the prebiotic properties of lactulose; Second, existing technologies are limited to improving synthesis yield and optimizing green processes, while systematic characterization of the interfacial chemical properties and emulsifying performance of the products is relatively lacking, especially the structure-activity relationship between molecular structure and emulsifying performance, interfacial activity and in vitro digestion behavior is still unclear.

[0006] Therefore, it is urgent to construct an efficient enzymatic preparation system for lactulose fatty acid monoesters and to conduct in-depth research on its surface activity, emulsifying properties and in vitro digestion behavior, so as to lay a theoretical foundation for the development of novel functional food ingredients. Summary of the Invention

[0007] Addressing the shortcomings of existing technologies, this invention aims to provide a novel lactulose fatty acid monoester, its preparation method, and its applications, overcoming the limitations of existing technologies in terms of the single function of emulsifiers and the lack of systematic research on the emulsifying properties of lactulose esters. This invention, through the preparation of a series of lactulose monoesters with different fatty acid chain lengths (C8 to C18), systematically reveals for the first time their surface activity, emulsifying properties, emulsion stability, and in vitro digestion behavior, and establishes a clear structure-activity relationship. The lactulose esters provided by this invention not only possess excellent, tunable emulsifying properties but also exhibit the prebiotic potential of the lactulose group, thus providing a novel, predictable, bifunctional, green surfactant for the food, pharmaceutical, and cosmetic fields.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: A method for preparing lactulose fatty acid monoesters by enzymatic method, comprising the following steps: Lactulose and ethylene fatty acid esters were mixed, and then a mixed solvent of anhydrous pyridine and tert-butanol was added. The mixture was stirred until completely dissolved to obtain a mixed solution. An enzyme catalyst was added to the mixed solution and the reaction was continued. The reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was completed, the enzyme catalyst was removed while hot, the filtrates were combined, and the solution was concentrated under reduced pressure to obtain a yellow oily crude product. Then, rapid column chromatography was performed using a dichloromethane / methanol gradient elution to separate the target fraction. The target fraction was collected and concentrated to obtain purified lactulose monoester.

[0009] The molar ratio of lactulose to ethylene fatty acid esters is 1:3.

[0010] The volume ratio of anhydrous pyridine to tert-butanol is 1:1.

[0011] The volume-to-mass ratio of the mixed solvent to lactulose is 20 mL: 1 g.

[0012] The enzyme catalyst is Novozym 435; the amount of enzyme catalyst added is 30% of the mass of lactulose.

[0013] The reaction temperature of the above reaction system is 50℃.

[0014] The mobile phase for the thin-layer chromatography method is dichloromethane and methanol in a volume ratio of 9:1.

[0015] The specific steps for rapid column chromatography separation using dichloromethane / methanol gradient elution are as follows: The reaction mixture is filtered and concentrated under reduced pressure to obtain an oily substance, which is then transferred to a chromatography column packed with 200-300 mesh silica gel. Gradient elution is performed using a mixed solvent with dichloromethane to methanol volume ratios of 100:1, 47:3, 22:3, and 41:9, respectively. The collected samples are analyzed by thin-layer chromatography to detect R. f The fraction with a value of 0.3 was concentrated under reduced pressure to obtain the target product, wherein the methanol was an infinite amount of methanol.

[0016] The aforementioned fatty acid vinyl ester is selected from one of vinyl octanoate, vinyl decanoate, vinyl laurate, vinyl myristate, vinyl palmitate, and vinyl stearate.

[0017] The lactulose monoesters include lactulose caprylate, lactulose decanoate, lactulose laurate, lactulose myristate, lactulose palmitate, or lactulose stearate.

[0018] Preferably, the present invention provides a method for preparing lactulose fatty acid monoesters by an enzymatic method, comprising the following steps: Lactulose (3 g, 8.8 mmol) and ethylene esters of fatty acids (26.4 mmol) were placed in a 200 mL round-bottom flask. A mixed solvent of anhydrous pyridine and tert-butanol (60 mL, 1:1, v / v) was added to the flask, and the mixture was magnetically stirred at 50 °C until the starting materials were completely dissolved. Novozym 435 (0.9 g) was then added to the reaction system to initiate the reaction, and the resulting mixture was continuously stirred at 50 °C. The reaction progress was monitored by thin-layer chromatography (TLC). After 24 hours, the reaction was terminated, and Novozym 435 was removed by hot filtration. The filter cake was washed with methanol, and the filtrate and washings were combined and concentrated under reduced pressure to obtain a yellow, oily crude product. Then, rapid column chromatography was performed using a gradient elution of dichloromethane / methanol (100% DCM, 47:3, 22:3, 41:9, v / v). R was collected. f The target fraction of 0.3 was extracted and concentrated to obtain purified lactulose monoester.

[0019] A lactulose fatty acid monoester prepared by the above method.

[0020] The present invention also provides the application of the above-mentioned lactulose fatty acid monoester in food, medicine or cosmetics.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention, through the preparation of a series of lactulose monoesters with different fatty acid chain lengths (C8 to C18), systematically reveals for the first time their surface activity, emulsifying properties, and in vitro digestion behavior, and establishes a clear structure-activity relationship. The lactulose esters provided by this invention not only possess excellent, tunable emulsifying properties but also exhibit the prebiotic potential of the lactulose group, thus offering a novel, predictable, bifunctional, green surfactant for the food, pharmaceutical, and cosmetic industries. Attached Figure Description

[0022] Figure 1 Synthetic routes for a series of lactulose fatty acid monoesters.

[0023] Figure 2 Lactulose caprylate (E-C8) 1 1H NMR spectrum (CD3OD solvent).

[0024] Figure 3 Lactulose caprylate (E-C8) 13 C10 NMR spectrum (CD3OD solvent).

[0025] Figure 4 HMBC spectrum of lactulose caprylate (E-C8) (CD3OD solvent).

[0026] Figure 5Lactulose decanoate (E-C10) 1 1H NMR spectrum (CD3OD solvent).

[0027] Figure 6 Lactulose decanoate (E-C10) 13 C10 NMR spectrum (CD3OD solvent).

[0028] Figure 7 HMBC spectrum of lactulose decanoate (E-C10) (CD3OD solvent).

[0029] Figure 8 Lactulose lauryl ester (E-C12) 1 1H NMR spectrum (CD3OD solvent).

[0030] Figure 9 Lactulose lauryl ester (E-C12) 13 C10 NMR spectrum (CD3OD solvent).

[0031] Figure 10 Lactulose myristate (E-C14) 1 1H NMR spectrum (CD3OD solvent).

[0032] Figure 11 Lactulose myristate (E-C14) 13 C10 NMR spectrum (CD3OD solvent).

[0033] Figure 12 Lactulose palmitate (E-C16) 1 1H NMR spectrum (CD3OD solvent).

[0034] Figure 13 Lactulose palmitate (E-C16) 13 C10 NMR spectrum (CD3OD solvent).

[0035] Figure 14 Lactulose stearate (E-C18) 1 1H NMR spectrum (CD3OD solvent).

[0036] Figure 15 Lactulose stearate (E-C18) 13 C10 NMR spectrum (CD3OD solvent).

[0037] Figure 16 Surface tension diagrams of lactulose monoester solutions at different concentrations.

[0038] Figure 17 Interfacial tension diagrams of lactulose monoester at concentrations of 2.5 μM (left) and 25 μM (right) in an oil-water system.

[0039] Figure 18 Dynamic interfacial pressure (π) plots of lactulose monoester at concentrations of 2.5 μM (left) and 25 μM (right) in an oil / water system.

[0040] Figure 19 The appearance of aqueous solutions of E-C18 in the concentration range of 0 to 2.0 mg / mL (left) and the particle size distribution of particles formed by compound E-C18 in water at a concentration of 2.0 mg / mL (right).

[0041] Figure 20 Bright-field, polarized light microscopy, and transmission electron microscopy images of compound E-C18 in water at a concentration of 2.0 mg / mL (from left to right).

[0042] Figure 21 The three-phase contact angle (θ) of compound E-C18 in oil and deionized water was measured over 10 seconds.

[0043] Figure 22 Appearance of emulsions formed from E-C8 to E-C18 after standing for 2 hours.

[0044] Figure 23 Particle size distribution of particles in emulsions formed from E-C8 to E-C18 (purple area represents droplet size).

[0045] Figure 24 The average particle size (A) and particle size distribution (B) of the emulsions formed from E-C8 to E-C18 at different stages of the in vitro digestion process.

[0046] Figure 25 Laser confocal scanning electron microscopy (CLSM) images of E-C8 to E-C18 stable emulsions at three different stages of in vitro digestion (A) and free fatty acid (FFA) release rate of E-C8 to E-C18 stable emulsions during small intestinal digestion (B). Detailed Implementation

[0047] To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention will be described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit its scope. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0048] Example 1: Synthesis of lactulose caprylate (E-C8) Includes the following steps: Lactulose (3 g, 8.8 mmol) and vinyl octanoate (4.49 g, 26.4 mmol) were placed in a 200 mL round-bottom flask. A mixed solvent of anhydrous pyridine and tert-butanol (60 mL, 1:1, v / v) was added to the flask, and the mixture was magnetically stirred at 50 °C until the starting materials were completely dissolved. Novozym 435 (0.9 g) was then added to the reaction system to initiate the reaction, and the resulting mixture was continuously stirred at 50 °C. The reaction progress was monitored by thin-layer chromatography (TLC). After 24 hours, the reaction was terminated, and Novozym 435 was removed by hot filtration. The filter cake was washed with methanol, and the filtrate and washings were combined and concentrated under reduced pressure to obtain a yellow, oily crude product. Then, rapid column chromatography was performed using a gradient elution of dichloromethane / methanol (100% DCM, 47:3, 22:3, 41:9, v / v). R was collected. f The target fraction of 0.3 was extracted and concentrated to obtain purified lactulose caprylate (E-C8).

[0049] Yield: 61.1%. A mixture of 87.5% 1- O -, 4.7% 6- O - and 7.8% 6'- O -isomers. 1 H NMR (500 MHz, Methanol- d 4) δ 4.39 (d, J=7.6 Hz, 1H), 4.31 (dd, J=11.7, 7.7 Hz, 1H), 4.25-4.10 (m, 3H), 4.06-3.95 (m, 2H), 3.92 (d, J = 9.2 Hz,1H), 3.87-3.77 (m, 2H), 3.71 (ddt, J=18.8, 11.4, 6.4 Hz, 3H), 3.64-3.53 (m,2H), 3.50 (dt, J=10.4, 5.1 Hz, 1H), 2.36 (q, J=7.6 Hz, 2H), 1.63 (s, 2H),1.37-1.26 (m, 8H), 0.91 (t, J=6.7 Hz, 3H). 13 C NMR (125 MHz, Methanol- d 4) δ173.67 (d, J=35.9 Hz), 103.68, 101.65,101.01, 96.80, 85.16, 80.86, 77.94, 76.18, 75.88, 73.53, 73.29, 71.11, 69.10,68.94, 67.15, 66.56, 65.14, 63.85, 62.88, 62.80, 61.43, 61.34, 33.54, 31.46,28.79, 28.69, 24.58, 22.26, 13.00. MS (ESI, +ve): m / z 491.21 [M+Na + ] Example 2: Synthesis of lactulose decanoate (E-C10) Includes the following steps: Lactulose (3 g, 8.8 mmol) and vinyl decanoate (5.24 g, 26.4 mmol) were placed in a 200 mL round-bottom flask. A mixed solvent of anhydrous pyridine and tert-butanol (60 mL, 1:1, v / v) was added to the flask, and the mixture was magnetically stirred at 50 °C until the starting materials were completely dissolved. Novozym 435 (0.9 g) was then added to the reaction system to initiate the reaction, and the resulting mixture was continuously stirred at 50 °C. The reaction progress was monitored by thin-layer chromatography (TLC). After 24 hours, the reaction was terminated, and Novozym 435 was removed by hot filtration. The filter cake was washed with methanol, and the filtrate and washings were combined and concentrated under reduced pressure to obtain a yellow, oily crude product. Then, rapid column chromatography was performed using a gradient elution of dichloromethane / methanol (100% DCM, 47:3, 22:3, 41:9, v / v). R was collected. f The target fraction of 0.3 was extracted and concentrated to obtain purified lactulose decanoate (E-C10).

[0050] Yield: 57.6%. A mixture of 87.5% 1- O -, 6.2% 6- O - and 6.3% 6'- O - isomer. 1 H NMR (500 MHz, Methanol- d 4) δ4.39 (d, J=7.6 Hz, 1H), 4.31 (dd, J=12.0, 7.7 Hz, 1H), 4.25-4.10 (m, 3H), 4.06-3.95 (m, 2H), 3.92 (d, J=9.3 Hz,1H), 3.86-3.77 (m, 2H), 3.76-3.64 (m, 2H), 3.63-3.54 (m, 2H), 3.50 (td, J=10.2, 3.3 Hz, 1H), 2.36 (q, J=7.6 Hz, 2H), 1.62 (d, J=7.0 Hz, 2H), 1.30 (s,12H), 0.90 (t, J=6.9 Hz, 3H). 13 C NMR (125 MHz, Methanol- d 4) δ 173.82, 173.54, 103.68, 101.65,101.00, 96.80, 85.15, 80.86, 77.93, 76.19, 75.88, 75.61, 73.53, 73.29, 71.11,69.11, 68.94, 67.15, 66.56, 65.14, 63.86, 62.83 (d, J = 8.6 Hz), 61.43,61.34, 33.55, 31.63, 29.18, 29.02, 28.83, 24.58, 22.32, 13.02. MS (ESI, +ve): m / z 519.24 [M+Na + ]. Example 3: Synthesis of Lactulose Laurate (E-C12) Includes the following steps: Lactulose (3 g, 8.8 mmol) and vinyl lauryl ester (5.98 g, 26.4 mmol) were placed in a 200 mL round-bottom flask. A mixed solvent of anhydrous pyridine and tert-butanol (60 mL, 1:1, v / v) was added to the flask, and the mixture was magnetically stirred at 50 °C until the starting materials were completely dissolved. Novozym 435 (0.9 g) was then added to the reaction system to initiate the reaction, and the resulting mixture was continuously stirred at 50 °C. The reaction progress was monitored by thin-layer chromatography (TLC). After 24 hours, the reaction was terminated, and Novozym 435 was removed by hot filtration. The filter cake was washed with methanol, and the filtrate and washings were combined and concentrated under reduced pressure to obtain a yellow, oily crude product. Then, rapid column chromatography was performed using a gradient elution of dichloromethane / methanol (100% DCM, 47:3, 22:3, 41:9, v / v). R was collected. f The target fraction of 0.3 was extracted and concentrated to obtain purified lactulose laurate (E-C12).

[0051] Yield: 54.5%. A mixture of 82.4% 1- O -, 13.2% 6- O - and 4.4% 6'- O -isomers. 1 H NMR (500 MHz, Methanol- d 4) δ 4.42 (d, J=7.6 Hz, 1H), 4.25-4.13 (m,2H), 4.08-3.97 (m, 2H), 3.94 (d, J=9.3 Hz, 1H), 3.87-3.79 (m, 2H), 3.77-3.67(m, 2H), 3.66-3.56 (m, 2H), 3.52 (dtd, J=10.6, 5.2, 3.1 Hz, 1H), 2.38 (q, J=7.6 Hz, 2H), 1.70-1.58 (m, 2H), 1.33 (d, J=11.8 Hz, 16H), 0.92 (t, J=6.8 Hz,3H). 13 C NMR (125 MHz, Methanol- d 4) δ175.25, 103.06, 98.22, 79.34, 77.60, 77.29, 77.02, 74.93, 74.70, 72.53, 70.52, 68.57, 67.98, 66.57, 65.28, 64.29, 64.22, 62.85, 62.76, 34.97, 34.94, 33.09, 30.77, 30.65, 30.50, 30.45, 30.26, 25.98, 23.76, 14.47. MS (ESI, +ve): m / z 547.27 [M+Na + ]. Example 4: Synthesis of lactulose myristate (E-C14) Includes the following steps: Lactulose (3 g, 8.8 mmol) and vinyl myristate (6.72 g, 26.4 mmol) were placed in a 200 mL round-bottom flask. A mixed solvent of anhydrous pyridine and tert-butanol (60 mL, 1:1, v / v) was added to the flask, and the mixture was magnetically stirred at 50 °C until the starting materials were completely dissolved. Novozym 435 (0.9 g) was then added to the reaction system to initiate the reaction, and the resulting mixture was continuously stirred at 50 °C. The reaction progress was monitored by thin-layer chromatography (TLC). After 24 hours, the reaction was terminated, and Novozym 435 was removed by hot filtration. The filter cake was washed with methanol, and the filtrate and washings were combined and concentrated under reduced pressure to obtain a yellow, oily crude product. The crude product was then separated by rapid column chromatography using a gradient elution of dichloromethane / methanol (100% DCM, 47:3, 22:3, 41:9, v / v). R was collected. f The target fraction of 0.3 was extracted and concentrated to obtain purified lactulose myristate (E-C14).

[0052] Yield: 66.0%. A mixture of 89.1% 1- O -, 6.3% 6- O - and 4.6% 6'- O - isomers. 1 H NMR (500 MHz, Methanol- d 4) δ4.39 (d, J=7.6 Hz, 1H), 4.22-4.10 (m,2H), 4.06-3.95 (m, 2H), 3.92 (d, J=9.1 Hz, 1H), 3.86-3.77 (m, 2H), 3.70(dddd, J=20.6, 11.9, 8.6, 4.3 Hz, 2H), 3.63-3.54 (m, 2H), 3.50 (ddd, J=13.2,9.5, 3.4 Hz, 1H), 2.36 (dt, J=9.5, 7.3 Hz, 2H), 1.67-1.57 (m, 2H), 1.35-1.26(m, 20H), 0.90 (t, J = 6.9 Hz, 3H). 13 C NMR (125 MHz, Methanol- d 4) δ 175.24, 103.06, 98.22, 82.27, 79.34, 77.61, 77.29, 74.70, 72.53, 72.49, 70.52, 70.36, 68.57, 67.98, 66.56, 65.29, 64.22, 62.85, 62.76, 34.97, 33.10, 30.83, 30.79, 30.76, 30.66, 30.50, 30.46, 30.27, 30.23, 25.98, 23.76, 14.47. MS (ESI, +ve): m / z 575.30 [M+Na + ]. Example 5: Synthesis of lactulose palmitate (E-C16) Includes the following steps: Lactulose (3 g, 8.8 mmol) and vinyl palmitate (7.46 g, 26.4 mmol) were placed in a 200 mL round-bottom flask. A mixed solvent of anhydrous pyridine and tert-butanol (60 mL, 1:1, v / v) was added to the flask, and the mixture was magnetically stirred at 50 °C until the starting materials were completely dissolved. Novozym 435 (0.9 g) was then added to the reaction system to initiate the reaction, and the resulting mixture was continuously stirred at 50 °C. The reaction progress was monitored by thin-layer chromatography (TLC). After 24 hours, the reaction was terminated, and Novozym 435 was removed by hot filtration. The filter cake was washed with methanol, and the filtrate and washings were combined and concentrated under reduced pressure to obtain a yellow, oily crude product. Then, rapid column chromatography was performed using a gradient elution of dichloromethane / methanol (100% DCM, 47:3, 22:3, 41:9, v / v). R was collected. f The target fraction of 0.3 was extracted and concentrated to obtain purified lactulose palmitate (E-C16).

[0053] Yield: 70.7%. A mixture of 76.5% 1- O -, 14.7% 6- O - and 8.8% 6'- O -isomers. 1 H NMR (500 MHz, Methanol- d 4) δ 4.39 (d, J=7.6 Hz, 1H), 4.22-4.11 (m,2H), 4.06-3.95 (m, 2H), 3.92 (d, J=9.3 Hz, 1H), 3.86-3.77 (m, 2H), 3.70(dddd, J=17.5, 8.5, 1.29 (s,24H), 0.90 (t, J=6.8 Hz, 3H). 13 C NMR (125 MHz, Methanol- d 4) δ175.24, 174.95, 105.09, 103.06, 98.22, 79.34, 77.30, 77.02, 74.70, 72.53, 70.52, 68.57, 67.98, 66.57, 64.29, 64.22, 62.85, 62.76, 34.97, 34.94, 33.10, 30.82, 30.79, 30.77, 30.66, 30.50, 30.46, 30.27, 30.23, 26.03, 25.98, 23.76, 14.47. MS (ESI, +ve): m / z 603.34 [M+Na + ]. Example 6: Synthesis of lactulose stearate (E-C18) Includes the following steps: Lactulose (3 g, 8.8 mmol) and vinyl stearate (8.20 g, 26.4 mmol) were placed in a 200 mL round-bottom flask. A mixed solvent of anhydrous pyridine and tert-butanol (60 mL, 1:1, v / v) was added to the flask, and the mixture was magnetically stirred at 50 °C until the starting materials were completely dissolved. Novozym 435 (0.9 g) was then added to the reaction system to initiate the reaction, and the resulting mixture was continuously stirred at 50 °C. The reaction progress was monitored by thin-layer chromatography (TLC). After 24 hours, the reaction was terminated, and Novozym 435 was removed by hot filtration. The filter cake was washed with methanol, and the filtrate and washings were combined and concentrated under reduced pressure to obtain a yellow, oily crude product. Then, rapid column chromatography was performed using a gradient elution of dichloromethane / methanol (100% DCM, 47:3, 22:3, 41:9, v / v). R was collected. f The target fraction of 0.3 was extracted and concentrated to obtain purified lactulose stearate (E-C18).

[0054] Yield: 64.5%. A mixture of 89.2% 1- O -, 4.6% 6- O - and 6.2% 6'- O - isomers. 1 H NMR (500 MHz, Methanol- d 4) δ4.39 (d, J=7.6 Hz, 1H), 4.31 (dd, J=12.0, 7.7 Hz, 1H), 4.22-4.10 (m, 2H), 4.06-3.95 (m, 2H), 3.95-3.89 (m, 1H),3.86-3.76 (m, 2H), 3.75-3.64 (m, 2H), 3.63-3.53 (m, 2H), 3.53-3.44 (m, 1H), 2.40-2.32 (m, 2H), 1.62 (td, J = 10.6, 9.0, 5.7 Hz, 2H), 1.29 (s, 28H), 0.90(t, J=6.9 Hz, 3H). 13 C NMR (125 MHz, Methanol- d 4) δ 173.81, 173.53, 101.68, 101.65, 96.80, 85.15, 80.85, 77.92, 76.19, 75.88, 75.60, 73.52, 73.28, 71.11, 48.11, 47.94, 47.77, 47.60, 47.43, 47.26, 47.09, 33.55, 31.68, 29.39, 29.36, 29.35, 29.24, 29.08, 29.04, 28.85, 24.56, 22.34, 13.04. MS (ESI, +ve): m / z 631.37 [M+Na + ]. This invention synthesized a series of lactulose-derived fatty acid monoesters (named compounds E-C8 to E-C18) with acyl side chain lengths of C8 to C18 using Novozym 435 as a catalyst. The structures of the products were confirmed by ¹H NMR, ¹³C{¹H} NMR, and HMBC spectroscopy, and the molecular weights were determined by mass spectrometry (MS). The results showed that the acylation reaction on the lactulose backbone preferentially occurred at the three primary hydroxyl groups, generating a mixture of three regiomeric monoesters with side chains at the C1, C6, and C6' positions. Figure 1 The preferential acylation of the primary hydroxyl sites is attributed to the smaller steric hindrance at these sites and the resulting greater accessibility. Careful analysis of the ¹H NMR spectra and corresponding HMBC data for each product mixture revealed that 1- O -Ayl lactulose is the major isomer, accounting for 75% to 90% of the product after chromatographic purification. Figure 4Key HMBC-related signals for lactulose monocaprylate (compound E-C8) are presented, and these signal features are representative throughout the series. A long-range correlation can be clearly observed between the ester carbonyl carbon (C=O) of the octanoyl group and the H-1a / 1b proton at the C1 position of the lactulose fructose unit, confirming the acylation site. The spectrum of lactulose fatty acid monoesters is shown below. Figures 2-15 As shown.

[0055] Experimental Example 1: Hydrophilic-lipophilic balance (HLB value), CMC value, and γ cmc Determination of value HLB determination method: 200 mg of commercial emulsifiers with known HLB values, namely Span 20 (HLB=8.6), Span 40 (HLB=6.7), Span 60 (HLB=4.7), Span 80 (HLB=4.3), Tween 20 (HLB=16.7), and Tween 80 (HLB=15.0), were dissolved in 25 ml of DMF / benzene (100:5, v / v) solvent mixture. The solutions were then titrated with distilled water until permanent turbidity appeared, and the volume of water consumed was recorded. A standard curve was obtained by plotting these water numbers against the corresponding HLB values. The same procedure was used for the synthesized series of lactulose esters, and the HLB values ​​were calculated based on their respective water numbers using this calibration curve.

[0056] CMC value determination method: First, prepare a series of aqueous solutions containing different concentrations of lactulose monoester and equilibrate at room temperature for 1 hour. Then, aspirate each solution into a 1 mL disposable syringe and fix it on the platform of an OCA-25 optical contact angle measuring instrument (Dataphysics, Germany). By gently pushing the syringe, a pendant droplet is formed at the tip of a 1.65 mm needle. The morphology of the droplet is recorded and analyzed using the instrument's built-in software, thereby determining its surface tension. The measured surface tension value is then calculated by dividing the logarithm of the solution concentration by the surface tension value. 10 Plot the curve; the concentration corresponding to the intersection of the two straight lines in the region of rapid decrease in surface tension and the plateau region is the CMC. This measurement was not performed because compound E-C18 has poor solubility in both water and oil.

[0057] γcmc is defined as the lowest surface tension that an aqueous solution of a surfactant can achieve at its CMC (Crystal-Cooled Surface Tension), and it is the core indicator of its maximum activity.

[0058] The test results are shown in Table 1 below. Figure 16 .

[0059] Table 1. HLB, CMC, and γcmc values ​​from E-C8 to E-C18

[0060] The HLB value is an important empirical indicator that can broadly define the emulsifying properties of a potential surfactant, thus predicting its optimal application in emulsion preparation. As shown in Table 1, the HLB values ​​of monoesters E-C8 to E-C18 decrease from 17.0 to 11.1 with increasing fatty acid side chain length. This trend is attributed to the stronger hydrophobicity conferred by the longer acyl chains, shifting the hydrophilic-lipophilic balance to a lower value. This behavior is consistent with the typical characteristics of such surfactants. Since all these esters have HLB values ​​above 9, this suggests that they may be best suited for stabilizing oil-in-water (O / W) emulsions.

[0061] CMC is a parameter of significant practical value, serving as a key indicator for measuring the micelle-forming efficiency of surfactants. (See Table 1 and...) Figure 16 As shown, with the growth of the relevant acyl side chains, the CMC values ​​of compounds E-C8 to E-C16 decreased sharply, from 61380.33 μmol / L to 159.47 μmol / L. From a thermodynamic perspective, this trend is attributed to the significant enhancement of intermolecular hydrophobic interactions by the growth of hydrophobic side chains, thus providing a stronger driving force for micelle formation. Notably, compound E-C16 exhibits an extremely low CMC value, indicating its superior micellization efficiency. In practical applications, this means that this compound requires only extremely low concentrations to achieve interfacial saturation adsorption and maximize surface activity. Compared to short-chain homologues, E-C16 has significant advantages in low addition amounts and high surface activity, making its application potential and economic value more prominent.

[0062] γcmc is defined as the lowest surface tension that an aqueous solution of a surfactant can achieve at its CMC (Cellular Magnitude Limit), and is a core indicator of its maximum activity. See Table 1 and... Figure 16 As shown, the γcmc values ​​of compounds E-C8 to E-C16 decrease significantly with increasing fatty acid side chain length. Molecular mechanism analysis suggests this trend is attributed to the strong hydrophobic effect of the long carbon chains, driving a more compact and ordered directional arrangement of surfactant molecules at the interface. Therefore, compound E-C16 exhibits the best surface activity among its homologues, indicating its significant technical advantages and broad development potential in applications requiring strong surface activity, such as emulsification and wetting.

[0063] Experimental Example 2: Interfacial Tension (IFT) and Diffusion Rate (K) diff Determination of ) The procedure for measuring interfacial tension is similar to that for surface tension. Prepare aqueous solutions of the ester at concentrations of 2.5 µM and 25 µM. Draw these solutions into the same syringe, then immerse the needle in a cuvette pre-filled with olive oil, the density of which is set to 0.9135 g / cm³. 3A droplet of approximately 5 µL was formed at the tip of the needle, and the appearance was recorded and analyzed in real time for 1 hour using software from an OCA-25 optical contact angle meter (Dataphysics GmbH, Germany) to obtain the IFT value.

[0064] Diffusion rate K diff This was derived by analyzing dynamic interfacial tension data using the Ward-Tordai model. Interfacial pressure (π) is used to quantify the degree of reduction in interfacial tension, defined as π = γ0. γ, where γ0 is the interfacial tension at the pure oil-water interface, and γ is the real-time interfacial tension of the sample. The change of π with adsorption time (t) can be determined by the modified Ward-Tordai equation (1):

[0065] Where C0 is the concentration of the additive in the continuous phase, K is the Boltzmann constant, T is the absolute temperature, D is the diffusion coefficient (in m² / s), and t is the adsorption time (in seconds). For diffusion-controlled adsorption processes, the Ward-Tordai model predicts a linear relationship between π and t¹ / ² in the initial stage of adsorption, and the slope of this linear relationship is defined as the diffusion rate constant K. diff .

[0066] The test results are shown in Table 2.

[0067] Table 2. Diffusion rates (K0) of E-C8 to E-C16 at concentrations of 2.5 µM and 25 µM diff ) and correlation coefficient (R) 2 )

[0068] The core effectiveness of emulsifiers depends on their adsorption capacity at the oil-water interface and their efficiency in reducing interfacial tension (IFT), which is the physicochemical basis for maintaining the stability of emulsion systems. Therefore, the dynamic IFT of compounds E-C8 to E-C16 was measured, and the results are as follows: Figure 17As shown, at low concentrations (2.5 μM), the interfacial activity of this series of monoesters exhibited a significant chain length dependence, with the IFT value decreasing significantly with increasing fatty acid side chain length (from 22 mN / m to 16-3 mN / m). Notably, compound E-C16 significantly reduced the IFT to approximately 3 mN / m, demonstrating its strongest interfacial activity among its homologues. In contrast, the short-chain compounds E-C8 and E-C10 showed weaker activity at this concentration. At high concentrations (25 μM), the activities of E-C10 and E-C12 were significantly enhanced, reducing the IFT to approximately 17 mN / m and 7 mN / m, respectively, but E-C8 still did not show significant interfacial activity. In summary, increasing the length of the hydrophobic side chain significantly enhances the interfacial activity of lactulose esters. This mechanism is attributed to the longer carbon chain providing a greater hydrophobic driving force, promoting directional migration and tight adsorption of molecules at the oil-water interface, thereby more effectively reducing the interfacial energy.

[0069] The dynamic behavior of surfactants at the liquid / liquid interface is a complex process involving multiple steps such as diffusion, adsorption, and interfacial rearrangement. To gain a deeper understanding of the adsorption behavior of these esters at the oil / water interface, their apparent diffusion rate (K0) was determined using the Ward-Tordai model. diff ), the result is as follows Figure 18 As shown, the corresponding K diff The values ​​are listed in Table 2. As shown in 18, π-t 1 / 2 The curve initially shows linear growth during the adsorption phase, but then deviates from linearity, tending towards an asymptotic plateau over time. The former (linear) phase indicates that the surfactant migrates from the bulk phase to the interface, leading to a rapid decrease in IFT and a subsequent increase in π. The subsequent plateau indicates that the interfacial adsorption process is approaching saturation, at which point the interfacial tension tends to stabilize. This marks the transition of the process to molecular unfolding and rearrangement events at the interface. Monoester compounds with relatively short side chains, E-C8 and E-C10, exhibit weaker interfacial activity, with their π-t... 1 / 2 The curves failed to show a clear transition, resulting in K at concentrations of 2.5 µM and 25 µM. diff The values ​​have poor correlation (R). 2 =0.5-0.75). In contrast, the interfacially active monoesters E-C12, E-C14, and E-C16 exhibited consistent behavior under the same conditions (R...). 2 >0.95), indicating that its initial adsorption is diffusion-controlled, therefore K diff The results are highly reliable. Overall, monoesters containing longer acyl chains exhibit higher K values. diff The value indicates that they can diffuse to the oil / water interface more quickly, thus creating more favorable conditions for reducing interfacial tension.

[0070] Experimental Example 3: Determination of Contact Angle Approximately 200 mg of lactulose monostearate sample was compressed into a flat tablet at 10 MPa for 1 minute using a mechanical tablet press. The tablet was placed at the bottom of a container filled with olive oil. Using a 1 mL syringe filled with deionized water, the needle was inserted into the oil phase, and a drop of 3 µL water was added to the tablet surface. The interaction between the water droplet and the tablet surface was immediately recorded for 10 seconds using an OCA-25 optical contact angle meter (Dataphysics, Germany), and the contact angle was calculated using the instrument's built-in software.

[0071] Given the limited water solubility of compound E-C18, its dispersion behavior and microstructure were systematically characterized. Solubility tests ( Figure 19 (Left) This indicates that the long-chain monoester exhibits extremely low water solubility. Its aqueous ester solution, after being dissolved by heating at 90°C and then cooled, forms a stable white emulsion suspension in the concentration range of 0.2–2.0 mg / mL. Furthermore, even at a high temperature of 150°C, the compound remains insoluble in olive oil (0.5 mg / mL). These observations suggest that in oil / water mixtures, E-C18 exists primarily as solid particles, rather than dissolved in either phase as molecules. To further characterize these particles, dynamic light scattering (DLS) analysis was employed. Figure 20 (Right) Further quantification of its particle characteristics revealed an average particle diameter of 1.437 μm, with a particle size distribution ranging from 300 nm to 4.5 μm. To further analyze its particle state and morphology, evaluation was performed using bright-field, polarized light, and transmission electron microscopy, with results as follows: Figure 20 As shown. Under a bright-field microscope, small, irregularly dispersed, round particles are clearly visible; however, under a polarizing microscope, no birefringence effect associated with the isotropic structure of liquid crystals was observed, indicating that the suspended particles of compound E-C18 are in an amorphous state. TEM image ( Figure 20 Further observation revealed nearly spherical particles with a diameter of approximately 2 μm, which is in high agreement with the DLS measurements. These results confirm that the compound exists in water as solid particles.

[0072] To elucidate the interfacial wetting properties of compound E-C18 particles and their behavior at the oil / water interface, their contact angles were measured, and the results are as follows: Figure 21As shown in the figure, tests indicate that within the initial 6 seconds of water droplet contact with the particle surface, the contact angle rapidly decreases from 146.8° to 80.8°, subsequently stabilizing at around 80°. Based on Young's equation and wetting theory, a contact angle close to 90° indicates that the solid particles possess a moderate affinity for both the oil and aqueous phases, exhibiting "near-neutral" wettability. This is a crucial prerequisite for the effective stabilization of Pickering emulsions by solid particles. Under this wetting state, the particles can partially penetrate into both phases, forming a robust mechanical barrier at the interface, thereby effectively inhibiting droplet coalescence. Therefore, based on the characteristic that the contact angle of E-C18 particles at the oil / water interface is less than 90°, it is expected that they can stably form oil-in-water (O / W) Pickering emulsions.

[0073] Experimental Example 4: Emulsion Preparation, Microstructure and Shelf Life Assessment First, 0.25 g of various lactulose esters (E-C8 to E-C18) were dissolved in 45 g of preheated deionized water. After cooling to room temperature, 5 g of olive oil was added to construct a mixture with an oil-to-water volume ratio of 1:9 and an emulsifier mass fraction of 0.5 wt.%. This mixture was subjected to high-speed shearing at 16,000 rpm for 2 minutes to prepare a crude emulsion, which was then transferred to a high-pressure homogenizer (AH-NANO type, Guangzhou Aolong Biotechnology Co., Ltd.) and homogenized 5 times at 1200 MPa pressure. The resulting emulsion was allowed to stand at room temperature for 2 hours, and its appearance was recorded. It was then stored at 4°C.

[0074] The emulsion was stored at 4°C, and the average particle size and particle size distribution were evaluated every 2 days. During testing, the sample was diluted approximately 100 times and then placed in a laser diffractometer (SALD-2300, Shimadzu, Japan) for analysis. The refractive index parameter was set to 1.35–1.00i, and the instability of the emulsion was assessed by monitoring changes in particle size.

[0075] 4.1 Transmission Electron Microscopy (TEM) Three drops (5-10 µL) of an aqueous suspension of compound E-C18 (2 mg / mL) were carefully added to a 200-mesh carbon-supported copper grid and allowed to stand for 1-2 minutes to facilitate sample adsorption. Excess liquid was then carefully blotted away from the edges of the grid using filter paper. The sample was allowed to air dry thoroughly at room temperature for 24 hours or until completely dry. The dried copper grid was then mounted on the sample holder of a transmission electron microscope (Tecnai G2 Spirit Twin, Thermo Fisher Scientific, USA) for morphological observation and image acquisition at an accelerating voltage of 120 kV.

[0076] 4.2 Laser confocal scanning electron microscope Place approximately 5 μL of the emulsion in the center of a glass slide, and add an equal volume of Nile Red staining solution (0.01 wt.%, solvent: 1,2-propanediol / water, volume ratio 50:1). Gently mix and stain for 2 minutes, then cover with a coverslip. Observe using a laser confocal scanning microscope (CLSM, LSM 880, Zeiss, Germany) equipped with a 40x oil immersion objective, with the excitation wavelength set to 543 nm to detect the fluorescence signal.

[0077] The emulsifying properties of lactulose monoesters were evaluated by combining macroscopic observation and particle size distribution determination. All monoesters formed uniform milky white emulsions after dispersion in an oil-water system, indicating that this series of compounds possesses good emulsifying ability and can construct uniform and stable dispersion systems. Except for E-C18, the remaining emulsions all exhibited a unimodal particle size distribution. Figure 24 (B) No coarse droplets or initial aggregation were observed, confirming the excellent emulsifying and dispersing properties of these monoesters. For the E-C18 system (Pickering emulsion), the overlapping bimodal distribution is attributed to the partial overlap in size between the solid particle aggregates and the emulsion droplets in the system.

[0078] Overall, the average droplet size of the emulsions exhibited a significant chain length dependence. As shown in Table 3 (Day 0), the average droplet diameter decreased with increasing hydrophobic side chain length; among them, the emulsion prepared by E-C18 had the smallest droplet size (approximately 1.3 μm). This trend is consistent with reports on classic sugar esters, and the mechanism lies in the superior interfacial activity of long-chain lactulose esters: they not only significantly reduce oil-water interfacial tension but also exhibit faster interfacial adsorption kinetics. These characteristics facilitate the rapid formation of a dense interfacial film during emulsification, thereby efficiently generating and stabilizing smaller oil droplets. Smaller initial droplet size not only reflects higher emulsification efficiency but also indicates better long-term stability of the system.

[0079] To assess the stability of the emulsion, the change in its average particle size over storage time was monitored, and the results are shown in Table 3 and 4. Figure 23 .

[0080] Table 3. Mean particle size of blank control group and E-C8 to E-C18 stable emulsion over time.

[0081] Note: Values ​​= mean ± standard deviation, n = 3. Oil separation: The oil phase appears at the top of the emulsion. Creaming: Droplets move upward under the influence of gravity, causing the lower layer to gradually clarify.

[0082] Stability monitoring results showed that the instability behavior of the emulsion was closely related to the hydrophobic chain length of the emulsifier. The system constructed from short-chain monoesters (E-C8) exhibited the worst stability, with its average particle size significantly increasing from 1694 nm to 1979 nm within 3 days, and macroscopic oil phase precipitation occurring on day 5. The E-C10 and E-C12 systems showed similar instability behavior, with particle size continuously increasing with storage time until oil-water separation occurred on day 7. Figure 23 The evolution of the particle size distribution curves visually reflects this phenomenon, with the characteristic region representing the internal droplets shifting significantly towards the larger droplet size, confirming significant droplet coalescence within the system. In contrast, emulsions derived from E-C14 and E-C16 did not exhibit instability until day 13, primarily in the form of emulsification rather than oil phase separation. A slight increase in average droplet diameter and size distribution was observed before emulsification, suggesting that droplet coalescence may have promoted or occurred simultaneously with the emulsification process. The emulsification phenomenon is attributed to gravitational migration caused by the density difference between the dispersed and continuous phases. It is worth noting that emulsification is not equivalent to the complete destruction of the emulsion structure; this process can be effectively suppressed by adding thickeners or adjusting the formulation. As expected, the Pickering emulsion formed from compound E-C18 exhibited the best storage stability in this series of systems, with no significant changes in its composition observed throughout the 15-day period, indicating a stable period of at least 15 days. This result is highly consistent with the view that Pickering emulsions with stable solid particles are more durable than conventional surfactant systems.

[0083] Experimental Example 5: In vitro digestion behavior of lactulose ester-stabilized emulsions The emulsion was diluted with 5 mM phosphate buffer (pH 7.0) to reduce the oil content from 10 wt.% to 2 wt.%. It was then mixed with an equal volume of simulated saliva (containing 3 mg / mL porcine gastric mucin type II). The pH was adjusted to 6.8, and the mixture was incubated for 10 minutes to simulate the oral phase. Subsequently, 25 mL of the digest was mixed with an equal volume of simulated gastric juice (SGF) containing 3.2 mg / mL pepsin. The pH was then adjusted to 2.5 using 0.1 M HCl, and the mixture was incubated for 2 hours to simulate gastric digestion. Finally, 30 mL of the gastric digest was transferred to a 100 mL beaker, the pH was adjusted to 7.0, and a small intestinal digestion program was simulated using a 902 Titrando automated titrator (Metrophon, Switzerland). In the first 100 seconds, 1.5 mL of simulated intestinal fluid (SIF) and 3.5 mL of bile extract were added, and the pH was then finely adjusted to 6.995–6.999. Immediately afterwards, 2.5 mL of lipase solution (24 mg / mL) was added, and the system was monitored and maintained at pH 7.0 by controlling the addition of 0.1 M NaOH. All procedures were performed at 37 °C, and the release rate of free fatty acids (FFAs) was calculated using equation (2).

[0084]

[0085] V NaOH and C NaOH These represent the volume and concentration of NaOH solution consumed, respectively. M lipid (879.67 g·mol) - ¹) and W lipid These represent the molar mass and mass (g) of digestible lipids, respectively.

[0086] Food-grade emulsions undergo complex physicochemical changes as they pass through various segments of the gastrointestinal tract. A thorough understanding of this process is crucial for developing bioactive delivery systems aimed at improving human health. The digestive process typically comprises three stages: the oral cavity, stomach, and intestines. During this process, changes in emulsion particle size are key indicators of its structural transformation and digestive fate. Therefore, the mean particle size and size distribution characteristics of emulsions stabilized by target monoesters were systematically evaluated at each digestive stage. Figure 24 As shown in Figure A, after simulated oral digestion, the average droplet size of the emulsion stabilized by lactulose monoester increased slightly, but remained below 3 μm overall. The particle size distribution curve after oral digestion (compared to the initial emulsion) is shown in Figure A. Figure 24 The shift of the B-type droplets towards larger particle sizes is attributed to the interaction between emulsion droplets and components such as mucins in saliva, which may induce the aggregation of some droplets through bridging or evacuation flocculation mechanisms. Despite these changes, the CLSM images ( Figure 25 (A in the middle, the red area represents oil droplets) shows that the internal droplet structure remains basically intact, indicating that although the oral environment induces a certain degree of flocculation, it does not cause significant structural damage or droplet aggregation.

[0087] After entering the gastric digestion stage, all emulsion systems underwent significant structural reorganization. Particle size distribution diagram ( Figure 24(Figure B) more clearly reveals this trend, showing that the emulsions after gastric digestion exhibit a wider and larger size distribution compared to the initial system and oral samples. Emulsions stabilized by short-chain lactulose esters (i.e., compounds E-C8 to E-C12) show bimodal or multimodal distributions, indicating the presence of heterogeneous aggregates or partial coalescence in the system. The strong acidity and high ionic strength of the gastric environment are the main drivers of these changes. Studies show that sugar ester-stabilized emulsions are prone to instability under these conditions, mainly manifested as emulsification and coalescence, rather than complete phase separation. Although the electrostatic shielding effect and low pH conditions of the gastric environment usually lead to the instability of emulsions stabilized by ionic surfactants or protein-based emulsifiers (near their isoelectric points), sugar esters are nonionic surfactants. Although significant particle coalescence was observed, the resulting interfacial layer effectively prevented complete emulsion disintegration and macroscopic oil phase separation. CLSM microscopy characterization of these systems ( Figure 25 Image A) further confirms the structural changes after gastric digestion. The enlarged oil droplets in the image indicate that although the system underwent redispersion and partial aggregation, the overall structure largely maintained its integrity.

[0088] Upon entering the small intestine for digestion, the emulsion system undergoes dramatic morphological remodeling. CLSM image ( Figure 25 A) shows that the emulsion exhibits a highly dispersed and irregular morphology, attributed to the extensive cleavage of triglycerides by the added pancreatic lipase, leading to the generation of fatty acids and monoglycerides. During this process, the lipase penetrates the interfacial barrier and promotes the hydrolysis of lipids within the olive oil, resulting in the release of free fatty acids (FFA). Simultaneously, lactulose monoester, acting as an emulsifier, may also undergo hydrolysis, dissociating to generate parent sugar molecules and corresponding fatty acids. In summary, in the third digestion stage, the structure of the original emulsion was completely destroyed, and particle size measurements clearly revealed the presence of colloidal aggregates. These aggregates are mainly composed of mixed micelles assembled from digestion products, bile salts, and phospholipids, while the intact oil droplets in the original emulsion no longer exist. Figure 24 The particle size distribution curve shown in Figure B is significantly broadened, further confirming that the material exhibits a complex and diverse aggregation state at this stage.

[0089] Although different lactulose monoesters exhibit varying emulsifying abilities, their stable emulsions demonstrate highly similar behavioral patterns during simulated in vitro digestion. Specifically, all systems showed minimal resistance to structural damage during both oral and gastric digestion. While droplet aggregation occurred in the stomach due to partial instability, the oil phase emulsion structure remained intact until reaching the intestine. This behavior is highly consistent with the digestive properties of common glycosyl fatty acid ester emulsions, suggesting that the lactulose ester emulsions in this study hold promise as carriers for the selective delivery of nutrients or pharmaceutically active ingredients to the small intestine. Crucially, given that lactulose monoesters may hydrolyze in the intestinal environment, the released prebiotic precursor glycosides will provide additional health benefits to the host.

[0090] During intestinal digestion, the hydrolysis of triglycerides releases free fatty acids (FFA) and monoacylglycerols. Therefore, the release rate curve of FFA was monitored to assess the extent and kinetic characteristics of lipid digestion. Figure 25 Figure B shows that all emulsions exhibited rapid FFA release within the first 20 minutes of digestion. This is attributed to the rapid contact of pancreatic lipase with the oil-water interface, thereby efficiently initiating the ester hydrolysis reaction. Subsequently, the FFA release rate gradually slowed down over time due to the decrease in substrate concentration and the reaction approaching equilibrium. Among them, the emulsion stabilized by compound E-C18 showed the highest degree of lipid digestion, with a total FFA release of 90%. The emulsions prepared from the other homologues (E-C8 to E-C16) had final FFA release rates ranging from 60% to 80%, and did not show a significant chain length dependence. The high FFA release rate exhibited by the E-C18 emulsion may be attributed to its unique interfacial environment, which is more conducive to the activity of lipase, or the change in its interfacial steric hindrance effect, which makes the core triacylglycerol (TAG) easier to digest. Nevertheless, the 60-80% FFA release levels of the other homologue emulsions still have significant application value, confirming that all lactulose monoester-stabilized emulsions in this study can achieve effective lipid digestion under gastrointestinal conditions.

[0091] The present invention has been described in detail above through specific embodiments, but the content thereon should not be construed as limiting the present invention. Those skilled in the art can make various equivalent substitutions, modifications, or improvements to the technical solutions and embodiments without departing from the spirit and scope of the present invention, and all such changes fall within the protection scope of the present invention, which is defined by the appended claims.

Claims

1. A method for preparing lactulose fatty acid monoesters using an enzymatic process, characterized in that: Includes the following steps: Lactulose and ethylene fatty acid esters were mixed, and then a mixed solvent of anhydrous pyridine and tert-butanol was added. The mixture was stirred until completely dissolved to obtain a mixed solution. An enzyme catalyst was added to the mixed solution and the reaction was continued. The reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was completed, the enzyme catalyst was removed while hot, the filtrates were combined, and the solution was concentrated under reduced pressure to obtain a yellow oily crude product. Then, rapid column chromatography was performed using a dichloromethane / methanol gradient elution to separate the target fraction. The target fraction was collected and concentrated to obtain purified lactulose monoester. The enzyme catalyst is Novozym 435.

2. The method according to claim 1, characterized in that: The molar ratio of lactulose to ethylene fatty acid ester is 1:

3.

3. The method according to claim 1, characterized in that: The volume ratio of anhydrous pyridine to tert-butanol is 1:

1.

4. The method according to claim 1, characterized in that: The volume-to-mass ratio of the mixed solvent to lactulose is 20 mL:1 g.

5. The method according to claim 1, characterized in that: The amount of enzyme catalyst added is 30% of the mass of lactulose.

6. The method according to claim 1, characterized in that: The reaction temperature of the reaction system is 50℃.

7. The method according to claim 1, characterized in that: The specific steps for rapid column chromatography separation using dichloromethane / methanol gradient elution are as follows: The reaction mixture was filtered and concentrated under reduced pressure to obtain an oily substance, which was then transferred to a chromatography column packed with 200-300 mesh silica gel. Gradient elution was performed using mixed solvents with dichloromethane and methanol volume ratios of 100:1, 47:3, 22:3, and 41:9, respectively. The collected samples were analyzed by thin-layer chromatography to detect R. f The fraction with a value of 0.3 was concentrated under reduced pressure to obtain the target product.

8. The method according to claim 1, characterized in that: The aforementioned fatty acid vinyl ester is selected from one of vinyl octanoate, vinyl decanoate, vinyl laurate, vinyl myristate, vinyl palmitate, and vinyl stearate.

9. The method according to any one of claims 1-8, characterized in that: Includes the following steps: 8.8 mmol of lactulose and 26.4 mmol of ethylene fatty acid ester were placed in a 200 mL round-bottom flask. 60 mL of a 1:1 mixture of anhydrous pyridine and tert-butanol was added to the flask, and the mixture was magnetically stirred at 50 °C until the starting materials were completely dissolved. Then, 0.9 g of Novozym 435 was added to the reaction system to initiate the reaction. The resulting mixture was continuously stirred at 50 °C. The reaction progress was monitored by thin-layer chromatography. After 24 hours, the reaction was terminated, and Novozym 435 was removed by hot filtration. The filter cake was washed with methanol, and the filtrate and washings were combined and concentrated under reduced pressure to obtain a yellow, oily crude product. This product was then separated by rapid column chromatography using a dichloromethane / methanol gradient elution at volume ratios of 47:3, 22:3, and 41:

9. R0 was collected. f The target fraction of 0.3 was extracted and concentrated to obtain purified lactulose monoester.

10. The use of lactulose fatty acid monoester prepared by the method of any one of claims 1-8 in food, medicine or cosmetics.

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