Composite process for green and efficient extraction of inonotus obliquus polysaccharide

Abstract

The invention discloses a green and efficient compound process for extracting inonotus obliquus polysaccharide, which comprises the following steps: pretreatment of raw materials: pretreating dried inonotus obliquus sporocarp, removing impurities and lipid, and preparation of a deep eutectic solvent (DES): mixing choline chloride as a hydrogen bond acceptor and lactic acid as a hydrogen bond donor according to a specific molar ratio, and stirring to obtain a mixture; adding a fixed proportion of water to adjust the viscosity. The deep eutectic solvent (DES) and ultrahigh pressure synergistic extraction system is adopted, 3-5 min short-time efficient extraction is achieved by precisely regulating and controlling key parameters such as the DES ratio, ultrahigh pressure and pressure maintaining time, compared with a traditional process, the extraction period is greatly shortened, energy consumption is reduced, choline chloride-lactic acid DES is used as an extraction solvent, an ethanol closed-loop recovery management mode is matched, and the extraction efficiency is greatly improved. The problems of pollution and residues of a traditional acid-base and organic solvent extraction method are avoided, the DES recycling rate reaches 91.5%-93.2%, the recycling loss is low, and the method has both environmental friendliness and cost controllability.

Description

Technical Field

[0001] This invention relates to the field of Inonotus obliquus polysaccharide extraction technology, specifically a green and efficient composite process for extracting Inonotus obliquus polysaccharides. Background Technology

[0002] Currently, the mainstream extraction methods for Inonotus obliquus polysaccharides include:

[0003] Hot water extraction: This method uses water as a solvent and high-temperature heating to dissolve polysaccharides. While simple to operate, it is time-consuming and yields low results. For example, using this method, with a material-to-liquid ratio of 1:40 g:mL and no auxiliary extraction, the extraction rate was only 2.81%. After optimization, with a material-to-liquid ratio of 1:50 g:mL, ultrasonic pretreatment for 20 min, and two extractions in a 60℃ constant-temperature water bath, the extraction rate increased to 3.17%.

[0004] Alkaline extraction: Using alkaline solutions as solvents, this method easily leads to the destruction of polysaccharide structures and causes wastewater pollution. While it achieves a relatively high extraction rate, the conditions are demanding. The optimal process, optimized through single-factor and orthogonal experiments, is: sodium hydroxide solution concentration of 0.5 mol·L⁻¹, material-to-liquid ratio of 1:30 g:mL, and extraction temperature of 80℃, achieving an extraction rate of 3.83%. However, this method carries significant operational risks and is difficult to implement.

[0005] Enzymatic extraction: This method uses cellulase and other enzymes to break down cell walls. The extraction rate is 5.86%, but enzyme preparations are expensive, the reaction conditions require strict control, and the production cost is high.

[0006] Ultrasonic extraction: This method utilizes the cavitation effect of ultrasound to disrupt cell structure and shorten extraction time. However, localized high temperatures can easily lead to polysaccharide degradation, resulting in limited improvement in extraction rate and significant variations in process parameters. Ultrasonic extraction at 50℃ for 2 hours with a material-to-liquid ratio of 1:40 g:mL yielded only 3.02%, presumably due to polysaccharide degradation caused by excessive extraction time. After optimization, ultrasonic pretreatment for 20 minutes followed by extraction in a 60℃ water bath increased the extraction rate to 5.03%.

[0007] High temperature and high pressure extraction method: This method can achieve an extraction rate of up to 5.88%, which is the highest among existing traditional methods. However, high temperature may affect the activity of polysaccharides.

[0008] In related studies, DES has been proven to achieve efficient extraction of polysaccharides, resulting in polysaccharide products with more complete structures, which is in line with the concepts of green chemistry and sustainable development. However, there are no reports on the synergistic application of ultra-high pressure and DES in the extraction of polysaccharides from Inonotus obliquus, and there is also a lack of systematic optimization studies on the process parameters of this synergistic system.

[0009] Existing polysaccharide extraction technologies from Inonotus obliquus have significant shortcomings:

[0010] The extraction efficiency is relatively low: the extraction rate of traditional hot water extraction is generally between 3.02% and 3.81%, and although the alkaline extraction method reaches 3.83%, it is limited by harsh conditions; even the high-temperature and high-pressure method with an extraction rate of 5.88% and the enzymatic extraction method with an extraction rate of 5.86% still have room for improvement, and there are problems such as activity destruction or excessive cost.

[0011] Imbalance between process rationality and cost: Ultrasonic extraction is prone to polysaccharide degradation due to improper parameter control, enzymatic extraction is costly, and alkaline extraction has high operational risks, making it difficult to apply on a large scale.

[0012] Insufficient greenness and environmental friendliness: Alkali extraction has wastewater pollution problems, and traditional solvent extraction methods partially rely on organic solvents, which do not conform to the concept of green production;

[0013] Mass transfer efficiency and applicability are limited: The DES extraction method alone has insufficient mass transfer efficiency due to the high viscosity of the solvent, and there is currently no research on its compatibility with Inonotus obliquus polysaccharides, so the extraction process has not yet been optimized.

[0014] The activity retention effect needs to be improved: High temperature, strong alkali, and long-term ultrasound conditions can easily lead to the degradation of polysaccharide structure, affecting its antioxidant, blood sugar regulation and other biological activities. For example, the hydroxyl radical scavenging rate of traditional water-extracted polysaccharides is only 56.53% (2.0 mg / mL), which is significantly lower than that of products from optimized processes. Summary of the Invention

[0015] The purpose of this invention is to provide a green and efficient composite process for extracting polysaccharides from Inonotus obliquus, so as to solve the problems in the background art mentioned above.

[0016] To achieve the above objectives, the present invention provides the following technical solution: a green and efficient composite process for extracting polysaccharides from Inonotus obliquus, comprising the following steps:

[0017] (1) Raw material pretreatment: The dried Inonotus obliquus fruiting bodies are pretreated to remove impurities and lipids;

[0018] (2) Preparation of eutectic solvent (DES): Choline chloride is used as hydrogen bond acceptor and lactic acid is used as hydrogen bond donor. They are mixed in a specific molar ratio, and a fixed proportion of water is added to adjust the viscosity. The mixture is heated and stirred to form a uniform and transparent DES.

[0019] (3) Ultra-high pressure assisted extraction: The pretreated Inonotus obliquus powder is mixed with the DES according to the set material-liquid ratio and transferred into an ultra-high pressure device for extraction under specific pressure and holding time;

[0020] (4) Separation and purification: The extract was filtered, concentrated, precipitated with alcohol, centrifuged, deproteinized, dialyzed and freeze-dried in sequence to obtain purified Inonotus obliquus polysaccharide;

[0021] (5) Solvent recovery: The DES used in the extraction process is recovered by vacuum distillation, and the recovered DES can be recycled for the next batch of extraction.

[0022] As a preferred embodiment of the present invention, the pretreatment of raw materials in step (1) specifically involves: crushing the dried Inonotus obliquus fruiting bodies to 60 mesh, soaking them in 95% ethanol overnight, filtering and drying them, then drying them at 90°C for 24 hours, and sealing and storing them for later use.

[0023] As a preferred embodiment of the present invention, the molar ratio of choline chloride to lactic acid in step (2) is 1:2, the water content accounts for 30% of the total mass of DES, the heating and stirring conditions are stirring at 80°C for 30 min, and the prepared DES has a viscosity of 35.1 mPa·s at 25°C.

[0024] As a preferred embodiment of the present invention, the material-to-liquid ratio in step (3) is 1:30 g / mL, the ultra-high pressure extraction pressure is 350-450 MPa, and the pressure holding time is 3-5 min.

[0025] As a preferred embodiment of the present invention, the ultra-high pressure extraction pressure in step (3) is 400 MPa and the pressure holding time is 4 min.

[0026] As a preferred embodiment of the present invention, the alcohol precipitation conditions in step (4) are as follows: add 3 times the volume of 95% ethanol to the concentrated supernatant to make the alcohol content of the system reach more than 70%, and let it stand overnight at 4°C.

[0027] As a preferred embodiment of the present invention, the centrifugation parameters in step (4) are: rotation speed 5000 r / min, centrifugation time 15 min; the protein removal is performed using a chloroform-n-butanol mixture, wherein the volume ratio of chloroform to n-butanol is 4:1, and the protein is removed by shaking until no precipitate is formed.

[0028] As a preferred embodiment of the present invention, the dialysis in step (4) uses a dialysis bag with a molecular weight cutoff of 3500 Da, uses ultrapure water as the dialysis medium, and the dialysis time is 48 hours.

[0029] As a preferred embodiment of the present invention, the vacuum distillation conditions in step (5) are: vacuum degree 0.085-0.09MPa, distillation temperature 55-65℃, DES recovery rate 91.5%-93.2%, and the polysaccharide yield decreases by ≤1.8% when the recovered DES is recycled.

[0030] As a preferred embodiment of the present invention, the refined Inonotus obliquus polysaccharide obtained has an extraction rate of ≥10% and a purity of ≥53%; when the polysaccharide concentration is 10 mg / mL, the DPPH free radical scavenging rate is ≥78%; and when the polysaccharide concentration is 2.0 mg / mL, the ABTS antioxidant capacity is ≥83%.

[0031] Compared with the prior art, the beneficial effects of the present invention are:

[0032] 1. This invention employs a eutectic solvent (DES) and ultra-high pressure synergistic extraction system. By precisely controlling key parameters such as the DES ratio, ultra-high pressure, and holding time, it achieves efficient extraction in a short time of 3-5 minutes, significantly shortening the extraction cycle and reducing energy consumption compared to traditional processes. Simultaneously, it addresses the pain points of large parameter variations and poor repeatability in traditional methods, resulting in strong process stability and a polysaccharide extraction rate of ≥10%, far exceeding the maximum of 5.88% achieved by traditional processes. This significantly improves raw material utilization and fills the technological gap in the synergistic application of DES and ultra-high pressure in the extraction of Inonotus obliquus polysaccharides, laying a solid foundation for industrial-scale production.

[0033] 2. This invention uses choline chloride-lactic acid DES as the extraction solvent, combined with a closed-loop ethanol recovery management mode, avoiding the pollution and residue problems of traditional acid-base and organic solvent extraction methods. The DES recovery rate reaches 91.5%-93.2% with low recycling losses, combining environmental friendliness and cost control. Ultra-high pressure short-time extraction is used to replace the high-temperature strong alkali process, avoiding polysaccharide structure degradation. The final purified polysaccharide has a purity of ≥53%, and its antioxidant activity is significantly better than that of traditional water-extracted products, expanding its application prospects in food, health products and other fields. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0035] In the description of this invention, it should be understood that the terms "length," "width," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," and "outer," etc., indicating orientation or positional relationships, are based on the indicated orientation or positional relationships and are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this invention. Furthermore, in the description of this invention, "first," "second," "third," and "fourth" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0036] In the description of this invention, it should also be noted that, unless otherwise explicitly specified and limited, the terms "set," "install," "connect," and "link" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or a connection through an intermediate medium; and they can refer to the internal communication between two components. For those skilled in the art, the specific meaning of the above terms in this invention can be understood according to the specific circumstances. "Multiple" means two or more, and unless otherwise explicitly limited, all such meanings fall within the scope of protection of this invention.

[0037] Example 1

[0038] This invention provides a technical solution:

[0039] 1. Raw material pretreatment: Take dried Inonotus obliquus fruiting bodies, crush them to 60 mesh, soak them in 95% ethanol overnight, filter and air dry, then dry them at 90℃ for 24 hours, and seal and store for later use;

[0040] 2. Preparation of eutectic solvent (DES): Choline chloride was used as a hydrogen bond acceptor and lactic acid as a hydrogen bond donor. They were mixed in a molar ratio of 1:2. Deionized water was added at 30% of the total mass of DES to adjust the viscosity. The mixture was stirred at 80°C for 30 min to form a uniform and transparent DES (viscosity of 35.1 mPa·s at 25°C).

[0041] 3. Ultra-high pressure assisted extraction: The pretreated Inonotus obliquus powder was mixed with the above DES at a material-to-liquid ratio of 1:30 g / mL, and then transferred into an ultra-high pressure device. The mixture was held at 400 MPa for 4 min to complete the extraction.

[0042] 4. Separation and Purification: The extract was filtered, and the supernatant was concentrated. Three volumes of 95% ethanol were added to bring the alcohol content to over 70%. The mixture was then allowed to stand overnight at 4°C. Subsequently, it was centrifuged at 5000 rpm for 15 min, and the precipitate was collected. The precipitate was shaken with a chloroform-n-butanol mixture (volume ratio 4:1) to remove proteins until no more precipitate formed. The protein-free solution was transferred to a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed with ultrapure water for 48 h. Finally, the dialysate was freeze-dried to obtain purified Inonotus obliquus polysaccharide.

[0043] 5. Solvent recovery: The filtered DES mother liquor is transferred to a vacuum distillation apparatus and distilled under vacuum conditions of 0.09 MPa and 60°C to recover DES.

[0044] This embodiment uses the preferred parameters (400 MPa pressure, 4 min holding time) as described in the claims, ultimately achieving a refined Inonotus obliquus polysaccharide extraction rate of 10.8% and a purity of 55.2%. At a polysaccharide concentration of 10 mg / mL, the DPPH free radical scavenging rate was 81.3%; and at a concentration of 2.0 mg / mL, the ABTS antioxidant capacity was 85.7%. Simultaneously, the DES recovery rate reached 93.2%, and when recycled for the next batch extraction, the polysaccharide yield decreased by only 1.2%. This embodiment fully leverages the advantages of synergistic extraction with DES and ultra-high pressure, achieving highly efficient and highly active polysaccharide extraction with optimal solvent recovery efficiency, demonstrating outstanding economic and environmental benefits.

[0045] Example 2

[0046] This invention provides a technical solution:

[0047] 1. Raw material pretreatment: Same as in Example 1, take dried Inonotus obliquus fruiting bodies, crush them to 60 mesh, soak them in 95% ethanol overnight, filter and air dry them, and then dry them at 90℃ for 24 hours for later use;

[0048] 2. Preparation of eutectic solvent (DES): Same as in Example 1, with a choline chloride-lactic acid molar ratio of 1:2, 30% water added, and stirred at 80°C for 30 min to prepare homogeneous and transparent DES;

[0049] 3. Ultra-high pressure assisted extraction: The pretreated Inonotus obliquus powder was mixed with DES at a material-to-liquid ratio of 1:30 g / mL, and then transferred into an ultra-high pressure device. The mixture was held at 350 MPa for 3 minutes to complete the extraction.

[0050] 4. Separation and purification: Filter the extract, concentrate the supernatant and add 3 times the volume of 95% ethanol, let stand overnight at 4℃; centrifuge at 5000 r / min for 15 min to collect the precipitate; remove protein by shaking with a chloroform-n-butanol mixture (4:1) until no precipitate remains; dialyze with ultrapure water in a 3500 Da dialysis bag for 48 h; freeze dry to obtain purified polysaccharide;

[0051] 5. Solvent recovery: DES is recovered by vacuum distillation of the DES mother liquor under vacuum conditions of 0.085 MPa and distillation temperature of 55℃.

[0052] This embodiment uses the lower limit of the ultra-high pressure parameters (350 MPa, 3 min) and the lower limit of the solvent recovery parameters (0.085 MPa, 55 °C) in the claims. The final refined Inonotus obliquus polysaccharide extraction rate is 10.1%, and the purity is 53.5%. The DPPH free radical scavenging rate is 78.2% at a concentration of 10 mg / mL, and the ABTS antioxidant capacity is 83.1% at a concentration of 2.0 mg / mL, both meeting the minimum requirements for product performance in the claims. The DES recovery rate reaches 91.5%, and the polysaccharide yield decreases by 1.8% when recycled after recovery. This embodiment demonstrates that the process parameter range of the present invention has good stability and adaptability. Even under the lower limit of the parameter conditions, efficient extraction and green recovery can still be achieved, reducing the stringent requirements for equipment parameters in industrial production and further improving the practicality of the process.

[0053] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No markings in the claims should be construed as limiting the scope of the claims.

Claims

1. A green and efficient process for extracting polysaccharides from Inonotus obliquus, characterized by, The method comprises the following steps: (1) raw material pretreatment: pretreat the dried Inonotus obliquus fruiting body, remove impurities and lipids; (2) deep eutectic solvent (DES) preparation: mix choline chloride as a hydrogen bond acceptor and lactic acid as a hydrogen bond donor according to a specific molar ratio, add a fixed proportion of water to adjust the viscosity, and heat and stir to form a uniform and transparent DES; (3) ultrahigh pressure assisted extraction: mix the pretreated Inonotus obliquus powder with the DES according to a set solid-liquid ratio, transfer into an ultrahigh pressure device, and extract under a specific pressure and pressure holding time; (4) separation and purification: sequentially filter, concentrate, alcohol precipitate, centrifuge, remove protein, dialyze and freeze-dry the extract to obtain refined Inonotus obliquus polysaccharide; (5) solvent recovery: recover the DES used in the extraction process by vacuum distillation, and the recovered DES can be recycled for the next batch of extraction.

2. The composite process of claim 1, wherein, In step (1), the raw material pretreatment is as follows: the dried Inonotus obliquus fruiting body is crushed to 60 mesh, soaked in 95% ethanol overnight, filtered and dried, then dried at 90°C for 24h, and sealed for storage.

3. The composite process of claim 1, wherein, In step (2), the molar ratio of choline chloride to lactic acid is 1:2, the water accounts for 30% of the total mass of the DES, the heating and stirring condition is 80°C for 30min, and the prepared DES has a viscosity of 35.1mPa・s at 25°C.

4. The composite process of claim 1, wherein, In step (3), the solid-liquid ratio is 1:30g / mL, the ultrahigh pressure extraction pressure is 350-450MPa, and the pressure holding time is 3-5min.

5. The composite process of claim 4, wherein, In step (3), the ultrahigh pressure extraction pressure is 400MPa, and the pressure holding time is 4min.

6. The composite process of claim 1, wherein, In step (4), the alcohol precipitation condition is as follows: 3 times the volume of 95% ethanol is added to the concentrated supernatant to make the alcohol content of the system more than 70%, and the system is placed at 4°C overnight.

7. The composite process of claim 1, wherein, In step (4), the centrifugation parameters are as follows: the rotation speed is 5000r / min, and the centrifugation time is 15min; the protein removal is performed using a chloroform-n-butanol mixture with a volume ratio of chloroform to n-butanol of 4:1, and the protein is removed by oscillation until no precipitate is precipitated.

8. The composite process of claim 1, wherein, In step (4), the dialysis is performed using a dialysis bag with a molecular weight cut-off of 3500Da, and ultrapure water is used as the dialysis medium, and the dialysis time is 48h.

9. The composite process of claim 1, wherein, In step (5), the vacuum distillation condition is as follows: the vacuum degree is 0.085-0.09MPa, and the distillation temperature is 55-65°C; the DES recovery rate is 91.5%-93.2%, and the polysaccharide yield decreases by ≤1.8% when the recycled DES is used.

10. The composite process according to any one of claims 1-9, wherein, The refined Inonotus obliquus polysaccharide has an extraction rate of ≥10% and a purity of ≥53%; when the polysaccharide concentration is 10mg / mL, the DPPH free radical scavenging rate is ≥78%; and when the polysaccharide concentration is 2.0mg / mL, the ABTS antioxidant capacity is ≥83%.