Preparation method and application of lignin-carbohydrate complex based gel system

Abstract

The invention discloses a preparation method and application of a lignin-carbohydrate complex based gel system. Comprising the following steps: carrying out enzyme modification on a lignin-carbohydrate complex; after enzyme modification is finished, centrifugally collecting precipitate, washing the precipitate, and freeze-drying; dissolving the freeze-dried precipitate in a dichloromethane-ethanol mixed solution, stirring, centrifugally collecting the precipitate, and freeze-drying the precipitate to obtain modified LCC; dispersing the modified LCC in an oil phase; adding water into the oil phase to obtain an LCC emulsion; and adding a gelling agent into the LCC emulsion to obtain the lignin-carbohydrate complex based gel system. The lignin-carbohydrate complex based gel system disclosed by the invention has wide application prospects in the aspects of preparing health foods and medicines for improving the bioavailability of nutrients and realizing targeted release of intestinal tracts, developing healthy grease products, improving the functional activity of bioactive substances and the like.

Description

Technical Field

[0001] This invention relates to the field of materials technology, specifically to a method for preparing and applying a lignin-carbohydrate complex-based gel system. Background Technology

[0002] Traditional emulsions are thermodynamically unstable systems, typically relying on small-molecule surfactants to maintain their apparent stability. However, these surfactants exhibit kinetic instability at the interface, resulting in weak adhesion at the oil-water interface and a tendency for desorption and migration. Therefore, relatively high addition levels are generally required to achieve effective emulsification. Furthermore, traditional surfactants can cause biotoxicity and environmental residues. This study aims to improve the physical state and stability of traditional water-in-oil emulsions by modifying LCCs, adding oil gelling agents, and optimizing process conditions, thereby enhancing their effective applications.

[0003] Lignin-carbohydrate complexes (LCCs) are three-dimensional network structures formed by lignin and carbohydrates linked by chemical bonds. They possess advantages such as biodegradability, eco-friendliness, abundance, and low cost. Existing research indicates that LCCs, as natural amphiphilic polymers, can be adsorbed at the oil-water interface as emulsifiers, showing great potential to replace traditional surfactants. Compared to traditional surfactants, LCCs exhibit a more robust interfacial adsorption layer, resulting in stronger emulsifying stability, and are naturally non-toxic and environmentally friendly. LCCs are fundamentally different from traditional surfactants.

[0004] Peanut oil contains over 80% unsaturated fatty acids (including 37.6% linoleic acid) and is rich in sterols, wheat germ phenols, phospholipids, vitamin E, choline, and other bioactive components, which are beneficial for improving cardiovascular health and preventing diseases such as atherosclerosis. High-oleic rapeseed oil has an oleic acid content greater than 78%, is rich in nutrients, and is a novel and healthy oil with great development potential. It can regulate human physiological functions and plays a crucial role in human metabolism. Therefore, using peanut oil or high-oleic rapeseed oil as the oil phase and LCC as a stabilizer, researching an emulsion system based on natural LCC, and enhancing its stability after gelation, is bound to have good development value in the food, pharmaceutical, cosmetic, and environmental fields. However, no such reports have yet appeared. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a method for preparing and applying a lignin-carbohydrate complex-based gel system. The lignin-carbohydrate complex-based gel system of this invention has lower emulsifier addition requirements, a more stable interfacial network structure, and superior long-term storage and high-temperature stability compared to traditional emulsions.

[0006] To achieve the above objectives, the technical solution designed by the present invention is as follows: This invention provides a method for preparing a lignin-carbohydrate complex-based gel system, comprising the following steps: (1) Enzymatic modification of lignin-carbohydrate complex; (2) After enzyme modification, the precipitate is collected by centrifugation, washed, and then freeze-dried. (3) Dissolve the freeze-dried precipitate in a dichloromethane-ethanol mixture, stir and centrifuge to collect the precipitate, freeze-dry the precipitate to obtain modified LCC; (4) The modified LCC is dispersed in the oil phase to obtain a dispersion; (5) Add water to the dispersion from step (4) to obtain an LCC emulsion; (6) Add a gelling agent to the LCC emulsion to obtain a lignin-carbohydrate complex-based gel system.

[0007] Furthermore, in step (1), the enzyme modification step is as follows: the acetic acid-sodium acetate buffer is mixed with anhydrous ethanol, and lignin-carbohydrate complex, isoeugenol and laccase are added to carry out the enzyme modification reaction.

[0008] Furthermore, the volume ratio of the acetate-sodium acetate buffer to anhydrous ethanol is 1:1~1.5; the mass-volume ratio of the lignin-carbohydrate complex to the acetate-sodium acetate buffer is 1:25~30 g / mL; the mass ratio of the lignin-carbohydrate complex to isoeugenol is 1:3~5; the mass-volume ratio of the lignin-carbohydrate complex to laccase is 1:4~5 g / mL; and the enzyme activity of the laccase is 50~500 U / mL. The enzyme modification reaction is carried out at a temperature of 30-32°C for 1-5 days.

[0009] Furthermore, in step (1), the enzyme modification step is as follows: the lignin-carbohydrate complex is added to the acetate-sodium acetate buffer, and cellulase is added to carry out the enzyme modification reaction.

[0010] Furthermore, the mass-to-volume ratio of the lignin-carbohydrate complex to the acetate-sodium acetate buffer is 1:50~55 g / mL; the mass ratio of the lignin-carbohydrate complex to the cellulase is 1:0.5~1; The activity of the cellulase is 50~300 U / g; The enzyme modification reaction is carried out at a temperature of 50-55°C for 12-72 hours.

[0011] Furthermore, the preparation method of the lignin-carbohydrate complex is as follows: a. Take 40-80 mesh wood powder / grass powder, extract it with benzene-ethanol solution as solvent, and then vacuum dry it; b. Ball mill the dried sample to obtain 400-800 mesh wood flour / grass flour; c. Using dioxane-water solution as solvent, extract 400-800 mesh wood flour / grass flour and centrifuge to obtain precipitate; d. The precipitate was vacuum dried, and the precipitate was extracted with acetic acid-water solution. The supernatant was collected by centrifugation. e. The supernatant was concentrated by rotary evaporation and then freeze-dried. The resulting solid was extracted with N,N-dimethylformamide as a solvent. The extracted substance was added dropwise to a mixture of dichloroethane and ethanol, and the precipitated solid was collected. f. Wash and vacuum dry the precipitated solid sequentially, dissolve the dried substance in an acetic acid-water solution, add the dissolved substance dropwise to acetone, collect the precipitated solid and freeze dry it. g. The freeze-dried solid was washed and vacuum dried sequentially to obtain a lignin-carbohydrate complex.

[0012] Furthermore, in step (3), the volume ratio of dichloromethane to anhydrous ethanol in the dichloromethane-ethanol mixture is 2:1 to 1.5; the stirring time is 10 to 14 hours. In step (4), the concentration of modified LCC dispersed in the oil phase is 0.1~1.0 g / L; the oil phase is either peanut oil or rapeseed oil. In step (5), the volume ratio of water to oil phase is 1:10~100; In step (6), the gelling agent is either ethyl cellulose or hydroxyethyl cellulose; the concentration of the gelling agent added to the LCC emulsion is 50~200 g / L.

[0013] Furthermore, the volume ratio of dichloromethane to anhydrous ethanol is 2:1; the stirring time is 12 hours; The modified LCC is dispersed in the oil phase at a concentration of 0.1 g / L; the oil phase is rapeseed oil. The volume ratio of the water to the oil phase is 1:10; The gelling agent is ethyl cellulose; the concentration of the gelling agent added to the LCC emulsion is 200 g / L.

[0014] The present invention also provides a lignin-carbohydrate complex-based gel system prepared by the preparation method described above.

[0015] The present invention also provides the application of the lignin-carbohydrate complex-based gel system described above in the preparation of functional foods or pharmaceuticals for encapsulating, protecting or delivering sensitive bioactive factors.

[0016] The present invention also provides a functional gel system loaded with functional active substances, the functional gel system comprising the lignin-carbohydrate complex-based gel system and the functional active substances; The mass ratio of the functional active substance to the lignin-carbohydrate complex-based gel system is 50~100:1; The functional active substance is any one of curcumin, anthocyanin, β-carotene and vitamin E.

[0017] The beneficial effects of this invention are: The lignin-carbohydrate complex-based gel system of this invention exhibits lower emulsifier addition requirements, a more robust interfacial network structure, and superior long-term storage and high-temperature stability compared to traditional emulsions. This invention gels the outer oil phase of the W / O emulsion, resulting in a smaller particle size and higher encapsulation rate of bioactive substances, while better protecting the inner aqueous phase against temperature and pH changes. The lignin-carbohydrate complex-based gel system shows broad application prospects in developing healthy oil products and improving the functional activity of bioactive substances. Attached Figure Description

[0018] Figure 1 Figure showing the results of LCC emulsion stability analysis; Figure 2 The figure shows the results of stability analysis of the lignin-carbohydrate complex-based gel system. Figure 3 The image shows the results of the embedding efficiency measurement of the functional gel system. Figure 4 The figure shows the results of the delivery efficiency measurement of the functional gel system. Detailed Implementation

[0019] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can understand it.

[0020] Example 1 Preparation of lignin-carbohydrate complex (LCC) (1) Take 100g of 40-80 mesh poplar wood powder and extract it for 12 hours using benzene-ethanol (the volume ratio of benzene and anhydrous ethanol is 2:1) solution as solvent.

[0021] (2) The poplar powder extracted in step (1) is vacuum dried for 7 days.

[0022] (3) The dried sample from step (2) is ball-milled for 96 hours to obtain 400-800 mesh poplar powder.

[0023] (4) Using dioxane-water solution (dioxane and water volume ratio of 24:1) as solvent, extract the 400-800 mesh poplar powder in step (3) for 24 hours, and retain the precipitate after centrifugation.

[0024] (5) The precipitate from step (4) was vacuum dried for 7 days, extracted for 24 hours with acetic acid-water solution (the volume ratio of acetic acid and water is 1:1), and the supernatant was taken after centrifugation.

[0025] (6) The supernatant from step (5) was concentrated by rotary evaporation and then freeze-dried. The conditions for rotary evaporation concentration were 40°C and 150 rpm. The resulting solid was extracted with 300 mL of DMF for 24 hours. The extracted substance was added dropwise to a 3000 mL mixture of dichloroethane and ethanol (the volume ratio of dichloroethane to anhydrous ethanol was 2:1) and the precipitated solid was collected.

[0026] (7) The solid precipitated in step (6) was washed once with dichloroethane-ethanol (the volume ratio of dichloroethane to anhydrous ethanol is 2:1) and three times with anhydrous diethyl ether. After vacuum drying, it was dissolved in 300 mL of acetic acid-water solution (the volume ratio of acetic acid to water is 1:1) for 12 hours. After dissolution, it was added dropwise to 3000 mL of acetone. The precipitated solid was collected and freeze-dried.

[0027] (8) The freeze-dried solid in step (7) was washed once with acetone-acetic acid (the volume ratio of acetone and acetic acid was 99:1), three times with anhydrous diethyl ether, and once with petroleum ether. After vacuum drying, lignin-carbohydrate complex (LCC) was obtained.

[0028] Example 2 Preparation method of lignin-carbohydrate complex-based gel system 1 (1) Prepare a 0.1 M acetate-sodium acetate buffer solution with pH=4.8. Take 25 ml of acetate-sodium acetate buffer solution and mix it with an equal volume of anhydrous ethanol (anhydrous ethanol promotes dissolution). Add 1 g of LCC prepared in Example 1, 3 g of isoeugenol and 4 mL of laccase (50~500 U / mL, preferably 250 U / mL), and place it in a 30℃ water bath for reaction.

[0029] (2) The laccase modification reaction was carried out for 1 day. After the reaction was completed, the precipitate was centrifuged at 8000 rpm for 10 minutes, and the precipitate was collected. The precipitate was washed repeatedly with distilled water until the supernatant was clear. The solid was collected and freeze-dried.

[0030] (3) Dissolve the freeze-dried solid in step (2) with a mixture of dichloromethane and ethanol (volume ratio of dichloromethane and anhydrous ethanol is 2:1), stir magnetically for 12 hours, centrifuge again, freeze-dry the collected precipitate to obtain laccase-modified LCC.

[0031] (4) Disperse the laccase-modified LCC from step (3) in peanut oil at a concentration of 0.5 g / L to obtain a dispersion.

[0032] (5) Add water to the dispersion in step (4), with a volume ratio of water to peanut oil of 1:20, to prepare a water-in-oil emulsion, namely LCC emulsion 1, and evaluate its emulsification behavior and physicochemical properties.

[0033] (6) Ethyl cellulose was added to the LCC emulsion 1 in step (5) at a concentration of 50 g / L as a gelling agent to prepare lignin-carbohydrate complex-based gel system 1, and the stability was evaluated.

[0034] Example 3 Preparation method of lignin-carbohydrate complex-based gel system 2 (1) Prepare a 0.1 M acetate-sodium acetate buffer solution with pH=4.8. Mix 1 g LCC sample with 50 ml acetate-sodium acetate buffer solution, add 0.618 g cellulase (50~300 U / g, preferably 150 U / g), and react in a shaker at 50℃.

[0035] (2) The cellulose endopeptidase modification reaction was carried out for 48 hours. After the reaction was completed, the precipitate was centrifuged at 8000 rpm for 10 minutes, the precipitate was collected, and the precipitate was repeatedly washed with distilled water until the supernatant was clear. The solid was collected and freeze-dried.

[0036] (3) Dissolve the freeze-dried solid in step (2) with a mixture of dichloromethane and ethanol (the volume ratio of dichloromethane and anhydrous ethanol is 2:1), stir magnetically for 12 hours, centrifuge again, collect the precipitate, freeze-dry it to obtain cellulase-modified LCC.

[0037] (4) Disperse the cellulase-modified LCC from step (3) in peanut oil at a concentration of 0.1 g / L to obtain a dispersion.

[0038] (5) Add water to the dispersion in step (4), with a volume ratio of water to peanut oil of 1:100, to prepare a water-in-oil emulsion, namely LCC emulsion 2, and evaluate its emulsification behavior and physicochemical properties.

[0039] (6) Ethyl cellulose was added to the LCC emulsion 2 in step (5) at a concentration of 100 g / L as a gelling agent to prepare lignin-carbohydrate complex-based gel system 2, and the stability was evaluated.

[0040] Example 4 Preparation method of lignin-carbohydrate complex-based gel system 3 (1) Same as step (1) in Example 2.

[0041] (2) The laccase modification reaction was carried out for 5 days. After the reaction was completed, the precipitate was centrifuged at 8000 rpm for 10 minutes, and the precipitate was collected. The precipitate was repeatedly washed with distilled water until the supernatant was clear. The solid was collected and freeze-dried.

[0042] (3) Dissolve the freeze-dried solid in step (2) with a mixture of dichloromethane and ethanol (volume ratio of dichloromethane and anhydrous ethanol is 2:1), stir magnetically for 12 hours, centrifuge again, collect the precipitate, freeze-dry it to obtain laccase-modified LCC2.

[0043] (4) Disperse the laccase-modified LCC from step (3) in rapeseed oil at a concentration of 0.1 g / L to obtain a dispersion.

[0044] (5) Add water to the dispersion in step (4), with a volume ratio of water to rapeseed oil of 1:10, to prepare a water-in-oil emulsion, namely LCC emulsion 3, and evaluate its emulsification behavior and physicochemical properties.

[0045] (6) Ethyl cellulose was added to the LCC emulsion 3 in step (5) at a concentration of 200 g / L as a gelling agent to prepare lignin-carbohydrate complex-based gel system 3, and the stability was evaluated.

[0046] Example 5 Preparation method of lignin-carbohydrate complex-based gel system 4 (1) The steps are the same as steps (1) in Example 3.

[0047] (2) The cellulase modification reaction was carried out for 12 hours. After the reaction was completed, the precipitate was centrifuged at 8000 rpm for 10 minutes, the precipitate was collected, and the precipitate was repeatedly washed with distilled water until the supernatant was clear. The solid was collected and freeze-dried.

[0048] (3) Dissolve the freeze-dried solid in step (2) with a mixture of dichloromethane and ethanol (the volume ratio of dichloromethane and anhydrous ethanol is 2:1), stir magnetically for 12 hours, centrifuge again, collect the precipitate, freeze-dry it to obtain cellulase-modified LCC.

[0049] (4) Disperse the cellulase-modified LCC from step (3) in rapeseed oil at a concentration of 0.5 g / L to obtain a dispersion.

[0050] (5) Add water to the dispersion in step (4), with a volume ratio of water to rapeseed oil of 1:20, to prepare a water-in-oil emulsion, namely LCC emulsion 4, and evaluate its emulsification behavior and physicochemical properties.

[0051] (6) Ethyl cellulose was added to the LCC emulsion 4 in step (5) at a concentration of 50 g / L as a gelling agent to prepare lignin-carbohydrate complex-based gel system 4, and the stability was evaluated.

[0052] Example 6 Performance testing of lignin-carbohydrate complex-based gel systems 1. The stability of LCC emulsions 1-4 prepared in Examples 2-5 were determined, and the results are as follows: Figure 1 As shown, with Span-80 emulsifier as a blank control, the results showed that LCC emulsions 1-4 significantly improved the stability of water-in-oil emulsions compared with traditional emulsifiers (control group).

[0053] 2. The stability of the lignin-carbohydrate complex-based gel systems 1-4 prepared in Examples 2-5 was determined, and the results are as follows: Figure 2 As shown, with the emulsion without ethyl cellulose as the control group, the oleogel formed by ethyl cellulose can significantly improve the stability of the emulsion, and LCC and gelling agent have a certain synergistic effect.

[0054] Example 7 Functional gel system loaded with curcumin Curcumin was added to the lignin-carbohydrate complex-based gel system 1 of Example 2, with a mass ratio of curcumin to lignin-carbohydrate complex-based gel system 1 of 80:1. After mixing evenly, a functional gel system loaded with curcumin was obtained, and the encapsulation rate and its release behavior during storage were measured.

[0055] Example 8 Functional gel system loaded with anthocyanins Anthocyanins were added to the lignin-carbohydrate complex-based gel system 2 of Example 3. The mass ratio of anthocyanins to lignin-carbohydrate complex-based gel system 2 was 75:1. After mixing evenly, a functional gel system loaded with anthocyanins was obtained. The encapsulation rate and its release behavior during storage were measured.

[0056] Example 9 Functional gel system loaded with β-carotene β-carotene was added to the lignin-carbohydrate complex-based gel system 3 of Example 4. The mass ratio of β-carotene to lignin-carbohydrate complex-based gel system 3 was 100:1. After mixing evenly, a functional gel system loaded with β-carotene was obtained. The encapsulation rate and its release behavior during storage were measured.

[0057] Example 10 Vitamin E-loaded functional gel system Vitamin E was added to the lignin-carbohydrate complex-based gel system 4 of Example 5, with a mass ratio of vitamin E to lignin-carbohydrate complex-based gel system 4 of 50:1. After mixing evenly, a functional gel system loaded with vitamin E was obtained, and the encapsulation rate and its release behavior during storage were measured.

[0058] Experimental results 1. The encapsulation efficiency of the functional gel systems prepared in Examples 7 to 10 was determined, with Span 20 emulsion used as a control group (the corresponding functional active substances were added to the Span 20 emulsion in the same proportion). The results are as follows: Figure 3 As shown, the lignin-carbohydrate complex-based gel system can significantly improve the encapsulation rate of functional active substances (curcumin, anthocyanins, β-carotene, and vitamin E).

[0059] 2. The delivery efficiency of the functional gel systems prepared in Examples 7 to 10 was determined, with Span 20 emulsion used as a control group (the corresponding functional active substances were added to the Span 20 emulsion in the same proportion). The results are as follows: Figure 4 As shown, the lignin-carbohydrate complex-based gel system can significantly improve the delivery efficiency of functional active substances (curcumin, anthocyanins, β-carotene, and vitamin E).

[0060] All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for the preparation of a lignin-carbohydrate complex based gel system, characterized by: The method comprises the following steps: (1) enzyme modification of the lignin-carbohydrate complex; (2) after the enzyme modification, the precipitate is collected by centrifugation, and the precipitate is washed and then freeze-dried; (3) the freeze-dried precipitate is dissolved in a dichloromethane-ethanol mixture, stirred, and then centrifuged to collect the precipitate, which is freeze-dried to obtain modified LCC; (4) the modified LCC is dispersed in an oil phase to obtain a dispersion liquid; (5) water is added to the dispersion liquid of step (4) to obtain an LCC emulsion; (6) a gelling agent is added to the LCC emulsion to obtain a lignin-carbohydrate complex-based gel system.

2. The method of claim 1, wherein: In step (1), the enzyme modification is performed as follows: acetic acid-sodium acetate buffer solution is mixed with anhydrous ethanol, and then lignin-carbohydrate complex, isoeugenol and laccase are added for enzyme modification reaction.

3. The method of claim 2, wherein: The volume ratio of the acetic acid-sodium acetate buffer solution to the anhydrous ethanol is 1:1-1.5; the mass-volume ratio of the lignin-carbohydrate complex to the acetic acid-sodium acetate buffer solution is 1:25-30 g / mL; the mass ratio of the lignin-carbohydrate complex to the isoeugenol is 1:3-5; the mass-volume ratio of the lignin-carbohydrate complex to the laccase is 1:4-5 g / mL; and the enzyme activity of the laccase is 50-500 U / mL. The temperature of the enzyme modification reaction is 30-32℃, and the time is 1-5 days.

4. The method of claim 1, wherein: In step (1), the enzyme modification is performed as follows: the lignin-carbohydrate complex is added to acetic acid-sodium acetate buffer solution, and then endo-cellulase is added for enzyme modification reaction.

5. The method of claim 4, wherein: The mass-volume ratio of the lignin-carbohydrate complex to the acetic acid-sodium acetate buffer solution is 1:50-55 g / mL; and the mass ratio of the lignin-carbohydrate complex to the endo-cellulase is 1:0.5-1. The enzyme activity of the endo-cellulase is 50-300 U / g. The temperature of the enzyme modification reaction is 50-55℃, and the time is 12-72 hours.

6. The method of claim 1 or 2 or 4, wherein: The preparation method of the lignin-carbohydrate complex is as follows: a. 40-80 mesh wood powder / grass powder is extracted with a benzene-ethanol solution, and then vacuum dried; b. the dried sample is ball milled to obtain 400-800 mesh wood powder / grass powder; c. the 400-800 mesh wood powder / grass powder is extracted with a dioxane-water solution, and then centrifuged to obtain a precipitate; d. the precipitate is vacuum dried, and then extracted with an acetic acid-water solution, and then centrifuged to obtain a supernatant; e. the supernatant is concentrated by rotary evaporation and then freeze-dried, and then the obtained solid is extracted with N,N-dimethylformamide, and then the extracted material is added dropwise into a dichloroethane-ethanol mixture to collect the precipitated solid; f. the precipitated solid is washed and then vacuum dried, and then the dried material is dissolved in an acetic acid-water solution, and then the dissolved material is added dropwise into acetone to collect the precipitated solid and then freeze-dried; g. the freeze-dried solid is washed and then vacuum dried to obtain the lignin-carbohydrate complex.

7. The method of claim 1, wherein: In the step (3), the volume ratio of dichloromethane to anhydrous ethanol in the dichloromethane-ethanol mixture is 2:1-1.5, and the stirring time is 10-14 hours; In the step (4), the concentration of the modified LCC dispersed in the oil phase is 0.1-1.0 g / L, and the oil phase is any one of peanut oil and rapeseed oil; In the step (5), the volume ratio of water to the oil phase is 1:10-100; In the step (6), the gelling agent is any one of ethyl cellulose and hydroxyethyl cellulose, and the concentration of the gelling agent added to the LCC emulsion is 50-200 g / L.

8. A lignin-carbohydrate complex-based gel system prepared by the preparation method of claim 1.

9. Use of the lignin-carbohydrate complex-based gel system of claim 8 in the preparation of a functional food or medicine for embedding, protecting or delivering a sensitive bioactive factor.

10. A functional gel system loaded with a functionally active substance, characterized by: The functional gel system comprises the lignin-carbohydrate complex-based gel system of claim 8 and a functional active substance; The mass ratio of the functional active substance to the lignin-carbohydrate complex-based gel system is 50-100:1; The functional active substance is any one of curcumin, anthocyanin, beta-carotene and vitamin E.

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