A high-purity momordica grosvenori effective component gradient coupling extraction method and a powder obtained by the method

Abstract

The application discloses a high-purity mogroside effective component gradient coupling extraction process, and creatively deeply couples three technologies of gradient low-temperature plasma wall breaking, double-medium synergistic phase change extraction and membrane coupling dynamic purification in view of the problems of low extraction rate, poor purity, easy damage of heat-sensitive components and bitter taste residue in the prior art. Through the gradient plasma treatment of 'first weak and then strong', the mild and efficient wall breaking of the cell wall is realized at <=25 DEG C. The synergistic effect of the composite enzyme solution and the dimethyl carbonate / ethanol compound extraction agent is utilized to realize the efficient enrichment of the mogroside V and the synchronous separation of the bitter impurities. Then, the double-stage membrane coupling technology of ultrafiltration-nanofiltration is used for dynamic purification at low temperature, and the traditional resin process is replaced. The process produces the technical effect of '1+1+1>3' through the cooperation of parameters and steps, the extraction rate of the mogroside V of the momordica grosvenori is >=90%, the purity of the product is >=98%, the polysaccharide retention rate is >=90%, and the bitter impurities are completely removed.

Description

Technical Field

[0001] This invention belongs to the field of natural product extraction technology, specifically relating to a gradient coupling extraction method for extracting mogroside V, polysaccharides and flavonoids from monk fruit with high purity and high efficiency, as well as the high-purity powder prepared by this method. Background Technology

[0002] Monk fruit, a plant unique to my country and used both as food and medicine, has attracted much attention for its glycoside V component due to its high sweetness, low calories, and various physiological activities. However, the industrial extraction of the effective components of monk fruit has long faced three major bottlenecks: First, the extraction rate is low, as traditional water extraction and alcohol precipitation methods struggle to overcome the dense cell wall barrier, resulting in glycoside V extraction rates often below 60%; second, the purity is low and the taste is poor, as conventional solvents co-dissolve bitter impurities such as terpenes and flavonol glycosides during glycoside extraction, which are difficult to completely remove with traditional resin purification, and there is a risk of solvent residue; third, the active ingredients are severely lost, as high-temperature extraction and concentration processes easily lead to the degradation of heat-sensitive glycoside V and polysaccharides, affecting product efficacy.

[0003] To address these issues, existing technologies have explored methods such as ultrasound-assisted extraction, microwave extraction, enzymatic extraction, and membrane separation. However, these improvements are mostly applications of single technologies. For instance, while enzymatic hydrolysis alone can increase yield, it cannot solve the problem of co-dissolution of bitter substances; while membrane separation alone can purify, it often leads to severe membrane fouling and decreased flux due to excessive impurities in the extract. Therefore, developing an integrated process that can synergistically solve the three major challenges of "cell wall disruption, extraction, and purification" has been a long-desired but unfulfilled goal for professionals in this industry. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, a gradient extraction method is provided that deeply couples physical cell wall disruption, biological enzymatic hydrolysis, green solvent extraction, and membrane separation technology. This process achieves efficient, high-purity, and high-activity extraction of the effective components of monk fruit through the synergistic effect of the steps, and completely solves the problem of residual bitterness.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A gradient coupling extraction method for high-purity monk fruit active ingredients includes the following steps: Step (1) Gradient low-temperature plasma cell breaking pretreatment: After crushing the monk fruit pulp, place it in a low-temperature plasma reactor and control the temperature ≤25℃. First, treat it with 80-100W power for 3-5 minutes to loosen the cuticle of the cell wall and "loosen" its dense structure without generating high heat; then treat it with 150-180W power for 5-8 minutes to break through the cell wall. Using this 150-180W strong plasma to bombard the cell wall with instantaneous high energy, directional micropores are formed on the weakened cell wall. This avoids the local overheating and destruction of effective ingredients that may be caused by single high-power treatment. It is the key to achieving gentle and efficient cell breaking and finally obtaining broken monk fruit powder. Step (2) Dual-media synergistic phase change extraction: a. Mix the broken-cell wall monk fruit powder obtained in step (1) with the compound enzymatic hydrolysate at a material-to-liquid ratio of 1:12-1:15, and enzymatically hydrolyze for 60-90 min at pH 4.5-5.0 and 35-40℃; the compound enzymatic hydrolysate is composed of 0.3% cellulase, 0.2% pectinase and 0.1% neutral protease dissolved in deionized water; b. After enzymatic hydrolysis, add 20%-25% of the total volume of the hydrolysate of the green extractant to the system. The green extractant is a mixture of dimethyl carbonate and ethanol in a volume ratio of 7:3. Perform phase change extraction at 45-50℃ and a vacuum of -0.06MPa for 40-60 minutes. After extraction, cool to 20℃ to allow the green extractant to automatically undergo phase change and separate into layers, and then recover it to obtain the crude extract. The key to this step lies in the synergy of "dual media" and "phase change." First, the active ingredients encapsulated in the complex enzymatic hydrolysate of cellulase, pectinase, and neutral protease are fully released into the aqueous phase after hydrolysis. Then, a green extractant, a 7:3 mixture of dimethyl carbonate (DMC) and ethanol, is added. DMC is a low-boiling-point, environmentally friendly polar solvent with a good affinity for betaine V; ethanol acts as a co-solvent to adjust the polarity of the mixed solvent. Under vacuum conditions at 45-50°C, the extractant permeates into the material in gaseous form, efficiently extracting the target components. After cooling, the DMC / ethanol mixed solvent transitions to a liquid state and, due to its hydrophobicity, separates from water, allowing for automatic separation and recovery. This simplifies the process and avoids solvent residue.

[0006] Step (3) Membrane-coupled dynamic purification: The crude extract obtained in step (2) is sequentially separated by an ultrafiltration membrane with a molecular weight cutoff of 5000 Da and a nanofiltration membrane with a molecular weight cutoff of 800 Da, respectively removing macromolecular impurities and small molecule impurities, resulting in a concentrated solution enriched with effective components. This method uses a 5000 Da ultrafiltration membrane to precisely remove macromolecular impurities such as proteins and pectin, preventing them from contaminating the subsequent nanofiltration membrane; the 800 Da nanofiltration membrane removes mogroside V with a molecular weight of approximately 1287 Da, while allowing smaller monosaccharides, inorganic salts, and potentially residual trace bitter molecules to pass through. This physical fractionation purification method does not require elution with organic solvents, thus preserving the natural activity of the components to the greatest extent.

[0007] Step (4) Low-temperature drying: The concentrated liquid obtained in step (3) is subjected to vacuum freeze drying, with the temperature controlled at -45℃ and the vacuum degree below 10Pa, and dried for 4-6 hours to obtain high-purity monk fruit active ingredient powder.

[0008] By employing the aforementioned technical solutions, compared to existing technologies, this application's highly efficient cell wall disruption provides a mass transfer channel for enzymatic hydrolysis and extraction; dual-media extraction not only extracts the target compound but also removes most of the lipid-soluble impurities through phase change separation, significantly reducing the load on subsequent membrane separation; membrane separation utilizes physical retention to thoroughly remove trace impurities remaining in the extract. This results in a cell wall disruption rate of over 95% for monk fruit, an extraction rate of up to 90% for betaine V, a product purity consistently above 98%, and a polysaccharide retention rate exceeding 90%. Through the selective enrichment of dual-media extraction and the precise molecular weight retention of nanofiltration membranes, bitter substances that have long been difficult to remove from monk fruit extracts can be completely removed, resulting in a product with a pure taste that can be directly used in high-end food and beverage formulations. The use of recyclable green solvent DMC and physical membrane separation technology completely replaces the traditional macroporous resin adsorption-desorption process, resulting in no organic solvent emissions or residues, meeting green manufacturing standards.

[0009] To better achieve the purpose of the invention, the present invention also has the following superior technical solutions: In some embodiments, step (3) further includes a step of dehydrating and concentrating the nanofiltration concentrate via a reverse osmosis membrane after nanofiltration, with a concentration temperature ≤45°C.

[0010] The powder of effective components of Luo Han Guo purified by the above method has a content of ≥98% of glycoside V and a polysaccharide retention rate of ≥90%. Moreover, no bitter impurities were detected by high performance liquid chromatography. Attached Figure Description

[0011] Figure 1 This is a schematic diagram of the process flow of the gradient coupling extraction method of the present invention. Detailed Implementation

[0012] The present invention will be further described in detail below with reference to the embodiments. The embodiments are only intended to provide a clearer understanding of the technical features, objectives and effects of the present invention.

[0013] This invention provides a gradient coupling extraction method for high-purity monk fruit active ingredients, comprising the following steps: Step 1, Gradient low-temperature plasma cell disruption pretreatment: After crushing the monk fruit pulp, place it in a low-temperature plasma reactor, control the temperature ≤25℃, first treat with 80-100W power for 3-5 minutes to loosen the cell wall cuticle; then treat with 150-180W power for 5-8 minutes to break down the cell wall and obtain broken monk fruit powder. Step 2, Dual-media synergistic phase change extraction: a. Mix the broken-cell wall monk fruit powder obtained in step 1 with the compound enzymatic hydrolysate at a material-to-liquid ratio of 1:12-1:15, and enzymatically hydrolyze for 60-90 minutes at pH 4.5-5.0 and 35-40℃; the compound enzymatic hydrolysate is composed of 0.3% cellulase, 0.2% pectinase and 0.1% neutral protease dissolved in deionized water by mass fraction. b. After enzymatic hydrolysis, add 20%-25% of the total volume of the hydrolysate of the green extractant to the system. The green extractant is a mixture of dimethyl carbonate and ethanol in a volume ratio of 7:3. Perform phase change extraction at 45-50℃ and a vacuum of -0.06MPa for 40-60 minutes. After extraction, cool to 20℃ to allow the green extractant to automatically undergo phase change and separate into layers, and then recover it to obtain the crude extract. Step 3, membrane coupling dynamic purification: The crude extract obtained in step 2 is sequentially passed through an ultrafiltration membrane with a molecular weight cutoff of 5000 Da and a nanofiltration membrane with a molecular weight cutoff of 800 Da to separate macromolecular impurities and small molecule impurities, respectively, to obtain a concentrated solution enriched with effective components. Step 4 Low-temperature drying: The concentrate obtained in step 3 is subjected to vacuum freeze drying, with the temperature controlled at -45℃ and the vacuum degree below 10Pa, for 4-6 hours to obtain high-purity monk fruit active ingredient powder.

[0014] Step 3, following nanofiltration, also includes a step of further dehydrating and concentrating the nanofiltration concentrate using a reverse osmosis membrane, with a concentration temperature ≤45℃.

[0015] The powder of the effective components of monk fruit purified by the above method has a content of ≥98% of glycoside V and a polysaccharide retention rate of ≥90%, and no bitter impurities are detected by high performance liquid chromatography.

[0016] Raw materials and equipment used in the examples: Luo Han Guo: Select mature, mold-free, and insect-free dried fruit; Cellulase (enzyme activity ≥10000U / g), pectinase (enzyme activity ≥50000U / g), neutral protease (enzyme activity ≥100000U / g), dimethyl carbonate, ethanol; Low-temperature plasma blender with adjustable power and temperature control system, and membrane separation equipment equipped with 5000Da spiral wound ultrafiltration membrane, 800Da spiral wound nanofiltration membrane and reverse osmosis membrane modules.

[0017] Example 1

[0018] (1) Gradient cell wall breaking: Take dried monk fruit, remove the shell and take the pulp, and crush it through a 50-mesh sieve. Put the coarse powder into a low-temperature plasma cell wall breaking machine, control the temperature ≤25℃, first process it with 80W power for 4min, and then increase the pressure to 160W for 6min to obtain cell wall broken monk fruit powder.

[0019] (2) Dual-media extraction: Add a compound enzymatic hydrolysate (containing 0.3% cellulase, 0.2% pectinase, and 0.1% neutral protease, with deionized water as the solvent) to the cell wall-breaking powder at a material-to-liquid ratio of 1:13 (g / mL). Adjust the pH to 4.8 with citric acid or sodium hydroxide solution and enzymatically hydrolyze at 38℃ for 50 min. After enzymatic hydrolysis, add 22% of the total volume of the enzymatic hydrolysis system of green extractant (dimethyl carbonate: ethanol = 7:3, v / v), raise the temperature to 48℃, and extract under a vacuum of -0.06 MPa for 50 min. After extraction, cool to 20℃, separate and recover the upper extractant, and the lower layer is the crude extract.

[0020] (3) Membrane purification: The crude extract is first passed through a 5000 Da ultrafiltration membrane and the permeate is collected; the permeate is then concentrated through an 800 Da nanofiltration membrane and the retentate is collected.

[0021] (4) Freeze-drying: Place the nanofiltration concentrate in a vacuum freeze dryer and dry it at -45℃ and 5Pa vacuum for 5 hours to obtain high-purity monk fruit active ingredient powder.

[0022] Test results: Cell wall breakage rate 96.2%, mogroside V extraction rate 89.5%, finished product purity 98.3%, polysaccharide retention rate 92.1%, and no bitter impurities were detected in the sensory evaluation.

[0023] Example 2

[0024] (1) Gradient cell wall breaking: Take dried monk fruit, remove the shell and take the pulp, and crush it through a 40-mesh sieve. Put the coarse powder into a low-temperature plasma cell wall breaking machine, control the temperature ≤25℃, first process it with 90W power for 3min, then increase the pressure to 150W for 8min to obtain cell wall broken monk fruit powder.

[0025] (2) Dual-media extraction: Add the compound enzymatic hydrolysate (formulation same as in Example 1) to the cell wall-breaking powder at a material-to-liquid ratio of 1:12 (g / mL), adjust the pH to 4.5, and enzymatically hydrolyze at 35℃ for 60 min. After enzymatic hydrolysis, add 20% of the total volume of the enzymatic hydrolysis system of green extractant (same as in Example 1), raise the temperature to 45℃, and extract under a vacuum of -0.06MPa for 60 min. After extraction, cool to 20℃, separate and recover the extractant to obtain the crude extract.

[0026] (3) Membrane purification: Same as in Example 1.

[0027] (4) Freeze-drying: Dry at -45℃ and 8Pa vacuum for 4 hours.

[0028] Test results: Cell wall breakage rate 95.7%, mogroside V extraction rate 87.8%, finished product purity 98.1%, polysaccharide retention rate 90.3%, and no bitter impurities detected.

[0029] Example 3

[0030] (1) Gradient cell wall breaking: Take dried monk fruit, remove the shell and take the pulp, and crush it through a 60-mesh sieve. Put the coarse powder into a low-temperature plasma cell wall breaking machine, control the temperature ≤25℃, first process it with 100W power for 5min, then increase the pressure to 180W for 5min to obtain cell wall broken monk fruit powder.

[0031] (2) Dual-media extraction: Add the compound enzymatic hydrolysate (formulation same as in Example 1) to the cell wall-breaking powder at a material-to-liquid ratio of 1:15 (g / mL), adjust the pH to 5.0, and enzymatically hydrolyze at 40℃ for 90 min. After enzymatic hydrolysis, add 25% of the total volume of the enzymatic hydrolysis system of green extractant (same as in Example 1), raise the temperature to 50℃, and extract under a vacuum of -0.06MPa for 40 min. After extraction, cool to 20℃, separate and recover the extractant to obtain the crude extract.

[0032] (3) Membrane purification: Same as in Example 1.

[0033] (4) Freeze-drying: Dry at -45℃ and 3Pa vacuum for 6 hours.

[0034] Test results: Cell wall breakage rate 97.1%, mogroside V extraction rate 90.2%, finished product purity 98.7%, polysaccharide retention rate 93.4%, and no bitter impurities detected.

[0035] Comparative example (traditional process) Take 50-mesh monk fruit powder, add 12 times the amount of water, and extract by stirring at 80℃ for 2 hours, then filter. After concentrating the filtrate, add ethanol to a final concentration of 75%, precipitate overnight, and collect the precipitate by centrifugation. After redissolving the precipitate, pass it through a D101 macroporous adsorption resin, wash with water to remove impurities, then elute with 70% ethanol, collect the eluent, concentrate it, and dry it under normal pressure.

[0036] Test results: Cell wall breakage rate 68.3%, mogroside V extraction rate 52.4%, finished product purity 82.6%, polysaccharide retention rate 61.7%, sensory evaluation showed significant bitterness and astringency.

[0037] Comparison table of technical effects of each embodiment and comparative example

[0038] The comparison of the above embodiments and comparative examples shows that the gradient coupling extraction process for high-purity monk fruit active ingredients described in this application has achieved far superior results compared to traditional processes in terms of cell wall breakage rate, target component extraction rate, product purity, heat-sensitive component retention rate, and removal of bitter impurities.

[0039] The above descriptions are merely some embodiments of the present invention. Those skilled in the art can make various modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. A gradient coupling extraction method for high-purity monk fruit active ingredients, characterized in that, Includes the following steps: Step (1) Gradient low-temperature plasma cell disruption pretreatment: After crushing the monk fruit pulp, it is placed in a low-temperature plasma reactor with the temperature controlled at ≤25℃. First, it is treated with 80-100W power for 3-5 minutes to loosen the cuticle of the cell wall; then it is treated with 150-180W power for 5-8 minutes to break down the cell wall and obtain broken-cell-wall monk fruit powder. Step (2) Dual-media synergistic phase change extraction: a. Mix the broken-cell wall monk fruit powder obtained in step (1) with the compound enzymatic hydrolysate at a material-to-liquid ratio of 1:12-1:15, and enzymatically hydrolyze for 60-90 min at pH 4.5-5.0 and 35-40℃; the compound enzymatic hydrolysate is composed of 0.3% cellulase, 0.2% pectinase and 0.1% neutral protease dissolved in deionized water by mass fraction. b. After enzymatic hydrolysis, add 20%-25% of the total volume of the hydrolysate of the green extractant to the system. The green extractant is a mixture of dimethyl carbonate and ethanol in a volume ratio of 7:

3. Perform phase change extraction at 45-50℃ and a vacuum of -0.06MPa for 40-60 minutes. After extraction, cool to 20℃ to allow the green extractant to automatically undergo phase change and separate into layers, and then recover it to obtain the crude extract. Step (3) Membrane-coupled dynamic purification: The crude extract obtained in step (2) is separated by passing it through an ultrafiltration membrane with a molecular weight cutoff of 5000 Da and a nanofiltration membrane with a molecular weight cutoff of 800 Da in sequence to remove macromolecular impurities and small molecule impurities respectively, and a concentrated solution enriched with effective components is obtained. Step (4) Low-temperature drying: The concentrated liquid obtained in step (3) was subjected to vacuum freeze drying, with the temperature controlled at -45℃ and the vacuum degree below 10Pa, and dried for 4-6 hours to obtain high-purity monk fruit active ingredient powder.

2. The high-purity monk fruit effective component gradient coupling extraction method according to claim 1, characterized in that, In step (3), after nanofiltration, there is also a step of further dehydrating and concentrating the nanofiltration concentrate through a reverse osmosis membrane, with a concentration temperature ≤45℃.

3. A high-purity powder containing the effective components of monk fruit, characterized in that, The powder is prepared by the method according to claim 1 or 2, wherein the content of mogroside V in the powder is ≥98%, the polysaccharide retention rate is ≥90%, and no bitter impurities are detected by high performance liquid chromatography.

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