CN115260334A - Compound extraction process of mulberry leaf polysaccharide - Google Patents
Compound extraction process of mulberry leaf polysaccharide Download PDFInfo
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- CN115260334A CN115260334A CN202211110147.7A CN202211110147A CN115260334A CN 115260334 A CN115260334 A CN 115260334A CN 202211110147 A CN202211110147 A CN 202211110147A CN 115260334 A CN115260334 A CN 115260334A
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- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
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Abstract
The invention relates to the field of mulberry leaf polysaccharide extraction, and particularly discloses a compound extraction process of mulberry leaf polysaccharide. Folium mori is taken as a raw material, after extraction and degreasing treatment by an organic solvent, an enzymolysis method is adopted to combine deep eutectic solvent and ultrasonic extraction to remove impurities, and the folium mori polysaccharide is obtained, wherein the purity of the prepared folium mori polysaccharide reaches more than 70.8%. The invention adopts degreasing, enzyme extraction and deep eutectic solvent ultrasonic extraction, greatly reduces the impurities contained in the mulberry leaf polysaccharide, shortens the extraction time, further improves the extraction rate of the mulberry leaf polysaccharide and simultaneously improves the purity of the mulberry leaf polysaccharide. The extraction process of the mulberry leaf polysaccharide provided by the invention has positive promotion effect on the deep processing of the mulberry leaf polysaccharide and the development of the mulberry leaf polysaccharide extraction industry, and has wide application prospect.
Description
Technical Field
The invention relates to the field of extraction of mulberry leaf polysaccharide, in particular to a compound extraction process of mulberry leaf polysaccharide.
Background
Mulberry leaf, also known as "Tie Fan", is the leaf of Morus alba L. The mulberry leaves are traditional Chinese medicines in China, the pharmacological activity of the mulberry leaves is recorded in Shen nong's herbal Jing in the east Han period at the earliest time, the mulberry leaves have the effects of dispelling wind and clearing heat, cooling blood and improving eyesight, tonifying and the like, and are mainly used for treating wind-heat type common cold, lung heat type dry cough, dizziness and headache, thirst, lung heat type cough and other diseases. The mulberry leaves not only contain rich substances such as protein, fat, carbohydrate, vitamin and the like, but also contain amino acid and various bioactive components which are necessary for human bodies, thereby becoming one of ideal sources of natural functional foods.
Polysaccharides are substances widely existing in organisms, are natural polymer polymers formed by connecting aldose or ketose through glycosidic chains, are important biological macromolecules in organisms, and are one of basic substances for maintaining normal operation of life activities. Plant polysaccharides, also known as plant polysaccharides, are polysaccharides produced by plant cell metabolism with a degree of polymerization of more than 10. The mulberry leaves are rich in polysaccharide and are one of the main functional components of the mulberry leaves. According to a large number of researches, the mulberry leaf polysaccharide has various biological activities of reducing blood sugar and blood fat, regulating immunity, resisting tumors, resisting aging, resisting coagulation, inhibiting bacteria and the like, and has no toxic or side effect on organisms.
At present, more production technologies and processes are available for extracting the mulberry leaf polysaccharide from the mulberry leaves, but no matter which extraction method is adopted, the extraction rate of the mulberry leaf polysaccharide is low, and the obtained product contains more impurities, so that the purity is not high, and the product can only be used as a low-end mixed pharmaceutical preparation and is not suitable for development and application of high-end preparations, so that the improvement of the extraction rate and the purity of the mulberry leaf polysaccharide is particularly important.
Disclosure of Invention
Aiming at the problems of low extraction rate and more impurities in the existing mulberry leaf polysaccharide extraction process, the invention provides a compound extraction process of mulberry leaf polysaccharide.
In order to achieve the purpose of the invention, the embodiment of the invention adopts the following technical scheme:
the invention provides a composite extraction process of mulberry leaf polysaccharide, which comprises an enzymolysis process and an ultrasonic extraction process, wherein an extraction solvent of the ultrasonic extraction process comprises a deep eutectic solvent and deionized water.
The deep eutectic solvent includes a hydrogen bond donor and a hydrogen bond acceptor.
The hydrogen bond donor is one or more of xylitol, trimethylolethane or urea; the hydrogen bond acceptor is one or two of benzyltriethylammonium chloride or tetrabutylammonium hydrogen sulfate.
The enzymolysis process adopts a complex enzyme of chitinase, thiol protease and carboxypeptidase to carry out enzymolysis.
Compared with the prior art, the composite extraction process of the mulberry leaf polysaccharide provided by the invention has the advantages that the mulberry leaves are used as raw materials, the mulberry leaf polysaccharide is obtained by combining an ultrasonic extraction process and an enzymolysis process, the fat and protein in the mulberry leaves are effectively removed, the impurities in the mulberry leaf polysaccharide are greatly reduced, the extraction time of the mulberry leaf polysaccharide is shortened, the extraction rate of the mulberry leaf polysaccharide is improved, and the mulberry leaf polysaccharide with higher purity can be obtained. According to the invention, the degreased mulberry leaf powder is treated by using chitinase, thiol protease and carboxypeptidase together, so that the mulberry leaf polysaccharide in the mulberry leaves can be fully dissolved out, and the extraction rate of the mulberry leaf polysaccharide is improved; the deep eutectic solvent in the ultrasonic extraction solvent uses one or more of xylitol, trimethylolethane or urea as a hydrogen bond donor, one or two of benzyltriethylammonium chloride or tetrabutylammonium hydrogen sulfate as a hydrogen bond acceptor, wherein the xylitol is green, safe and environment-friendly, is a five-carbon sugar alcohol, has five hydroxyl groups, is very soluble in water, and can interact with polysaccharide molecules through hydrogen bonds, the benzyl triethylammonium chloride has very strong dissolving capacity in a water phase, and positive ions and negative ions in quaternary ammonium salts form ion pairs in the water phase. Meanwhile, through ultrasonic extraction, by utilizing ultrasonic cavitation effect, thermal effect and mechanical effect, great pressure is generated to cause the cell walls and the whole tissues of the mulberry leaves to be cracked, and inclusion is released, the whole cracking process is completed instantly, and the extraction time of the mulberry leaf polysaccharide is greatly shortened.
Furthermore, the mass ratio of the deionized water to the deep eutectic solvent in the extraction solvent is 2-5.
Preferably, the mass ratio of the hydrogen bond donor to the hydrogen bond acceptor in the deep eutectic solvent is 1-4.
Further, the deep eutectic solvent is prepared by the following method: and (3) uniformly mixing the hydrogen bond donor and the hydrogen bond acceptor, heating to 80-90 ℃, and reacting for 2-3h to obtain the deep eutectic solvent.
Preferably, the total mass of the chitinase, the thiol protease and the carboxypeptidase accounts for 2-5% of the total mass of the mulberry leaf powder.
Preferably, the mass ratio of the chitinase to the thiol protease to the carboxypeptidase is 2-3.
Further, the mulberry leaf polysaccharide compound extraction process specifically comprises the following steps:
step a, washing, drying, crushing and sieving fresh mulberry leaves to obtain mulberry leaf powder.
And b, adding distilled water into the mulberry leaf powder, uniformly mixing, then adding an organic solvent, extracting, taking the degreased solution, and drying to obtain degreased powder.
The extraction is that mixed solution of mulberry leaf powder and distilled water is placed in a separating funnel, an organic reagent is added, after shaking vigorously up and down for 3-5min, the mixture is placed on an iron support and is kept stand for layering up and down, and lower layer solution is taken out.
The organic solvent is at least one of n-hexane, petroleum ether or ethanol.
And c, dissolving the degreased powder in distilled water, adding chitinase, thiol protease and carboxypeptidase, and stirring for enzymolysis to obtain an enzymolysis solution.
Step d, adding an extraction solvent into the enzymatic hydrolysate, performing ultrasonic extraction to obtain an extracting solution, filtering, and concentrating to obtain a concentrated solution; precipitating the concentrated solution with ethanol, and drying to obtain folium Mori polysaccharide powder.
Preferably, the mass ratio of the distilled water to the mulberry leaf powder in the step b is 5-10; the mass ratio of the organic solvent to the mulberry leaf powder is 5-10.
Preferably, the mass ratio of the distilled water to the mulberry leaf powder in the step c is 2-3; the stirring time is 25-35 min.
Preferably, the mass ratio of the extraction solvent to the mulberry leaf powder in the step d is 3-5; the ultrasonic power is 40000-50000Hz, and the ultrasonic time is 15-20 min.
Preferably, the concentration in the step d is vacuum concentration until the solid content is more than or equal to 20%, and the temperature of the vacuum concentration is 75-90 ℃.
Preferably, the alcohol precipitation in the step d is specifically: adding absolute ethyl alcohol with the mass 1-3 times of that of the mulberry leaf powder into the concentrated solution, and standing for 2-3 h.
Preferably, the drying in step d is a pressure spray drying method; the air inlet temperature of the pressure spray drying is 180-190 ℃, and the air outlet temperature is 80-100 ℃; the spraying pressure of the pressure spray drying is 0.7-1.0 MPa.
By the technical scheme, the invention at least has the following advantages and beneficial effects:
the invention adopts ultrasonic extraction, shortens the extraction time, improves the dissolution rate of the mulberry leaf polysaccharide and is beneficial to improving the extraction rate of the mulberry leaf polysaccharide. By adopting degreasing treatment and enzymatic extraction, the grease and protein components in the mulberry leaves can be effectively removed, and the impurities in the mulberry leaf polysaccharide are greatly reduced. The invention optimizes the extraction process of the mulberry leaf polysaccharide, has wide application prospect and has positive promotion effect on the deep processing of the mulberry leaf polysaccharide and the development of the mulberry leaf polysaccharide extraction industry.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The following examples are provided to better illustrate the embodiments of the present invention.
Example 1
The preparation method of the ultrasonic extracting solution comprises the following steps: mixing xylitol and benzyltriethylammonium chloride according to the mass ratio of 1.
Step a, washing fresh mulberry leaves with clear water, drying in a 60 ℃ oven for 12h, crushing, and sieving with a 60-mesh sieve.
And step b, adding 500mL of distilled water into 100g of mulberry leaf powder for dissolving, placing the mixture into a separating funnel, then adding 500mL of petroleum ether, shaking the mixture for 3min violently up and down, placing the mixture on an iron support for standing till layering up and down, taking the solution at the lower layer, and drying the solution in a 60 ℃ oven to constant weight to obtain degreased powder.
And c, dissolving the degreased powder in 200mL of distilled water, adding 1g of chitinase, 0.5g of thiol protease and 0.5g of carboxypeptidase, and stirring for 25min to obtain an enzymatic hydrolysate.
Step d, adding 300mL of prepared ultrasonic extraction solvent into the enzymatic hydrolysate, treating for 15min in 40000Hz ultrasonic waves to obtain an extracting solution, filtering, and concentrating in vacuum at 75 ℃ until the soluble solid is more than or equal to 20% to obtain a concentrated solution; adding 100mL of absolute ethyl alcohol into the concentrated solution, standing for 2h, filtering and separating, and washing a solid part with the absolute ethyl alcohol; through pressure spray drying, the air inlet temperature is adjusted to be 180 ℃, the air outlet temperature is adjusted to be 80 ℃, the spray pressure is 0.7MPa, 10.2g of mulberry leaf polysaccharide powder is obtained through drying, and according to GB/T15672-2009, the content of polysaccharide in the prepared mulberry leaf polysaccharide powder reaches 72.4% through determination of a phenol-sulfuric acid method.
Example 2
The preparation method of the ultrasonic extracting solution comprises the following steps: xylitol and benzyltriethylammonium chloride are mixed according to the mass ratio of 2.
Step a, washing fresh mulberry leaves with clear water, drying in a 60 ℃ oven for 12h, crushing, and sieving with a 60-mesh sieve.
And b, adding 100g of mulberry leaf powder into 800mL of distilled water for dissolving, placing the solution into a separating funnel, then adding 800mL of petroleum ether, shaking the solution vigorously up and down for 4min, placing the solution on an iron support for standing and layering up and down, taking the lower layer solution, and drying the solution in an oven at the temperature of 60 ℃ until the weight is constant to obtain degreased powder.
And c, dissolving the degreased powder in 250mL of distilled water, adding 2.5g of chitinase, 1g of thiol protease and 1g of carboxypeptidase, and stirring for 30min to obtain an enzymatic hydrolysate.
Step d, adding 400mL of prepared ultrasonic extraction solvent into the enzymatic hydrolysate, treating for 18min in 45000Hz ultrasonic waves to obtain an extracting solution, filtering, and concentrating in vacuum at 80 ℃ until the soluble solid is more than or equal to 20% to obtain a concentrated solution; adding 200mL of absolute ethyl alcohol into the concentrated solution, standing for 2.5h, filtering and separating, and washing a solid part with the absolute ethyl alcohol; through pressure spray drying, the air inlet temperature is adjusted to be 185 ℃, the air outlet temperature is adjusted to be 90 ℃, the spray pressure is 0.9MPa, 11.5g of mulberry leaf polysaccharide powder is obtained through drying, and according to GB/T15672-2009, the content of polysaccharide in the mulberry leaf polysaccharide powder is measured to reach 70.8% through a phenol-sulfuric acid method.
Example 3
The preparation method of the ultrasonic extracting solution comprises the following steps: xylitol and benzyltriethylammonium chloride are mixed according to the mass ratio of 4.
Washing fresh mulberry leaves with clear water, drying in a 60 ℃ oven for 12h, crushing, and sieving with a 60-mesh sieve.
And b, adding 1000mL of distilled water into 100g of mulberry leaf powder for dissolving, placing the mixture into a separating funnel, then adding 1000mL of petroleum ether, shaking up and down for 5min violently, placing the mixture on an iron support for standing until layering up and down, taking the lower layer solution, and drying the lower layer solution in an oven at 60 ℃ until the weight is constant to obtain degreased powder.
And step c, dissolving the degreased powder in 300mL of distilled water, adding 3g of chitinase, 1g of thiol protease and 1g of carboxypeptidase, and stirring for 35min to obtain an enzymatic hydrolysate.
And d, adding 500mL of prepared ultrasonic extraction solvent into the enzymolysis liquid, and treating for 20min in 50000Hz ultrasonic waves to obtain an extracting solution to obtain the extracting solution.
Filtering, and vacuum concentrating at 90 deg.C until soluble solid is not less than 20% to obtain concentrated solution; adding 300mL of absolute ethyl alcohol into the concentrated solution, standing for 3 hours, filtering and separating, and washing a solid part by using the absolute ethyl alcohol; the mulberry leaf polysaccharide powder is dried by adjusting the air inlet temperature to 190 ℃, the air outlet temperature to 100 ℃ and the spraying pressure to 1MPa to obtain 10.9g of mulberry leaf polysaccharide powder, and the content of the polysaccharide in the mulberry leaf polysaccharide powder is up to 71.3 percent according to GB/T15672-2009 by a phenol-sulfuric acid method.
Comparative example 1
This comparative example used the same starting materials and preparation as in example 1, except that the chitinase was replaced with an equal amount of cellulase. The prepared mulberry leaf polysaccharide powder is 18.2g, and the content of polysaccharide in the mulberry leaf polysaccharide powder is up to 35.7% according to GB/T15672-2009 by a phenol-sulfuric acid method.
Comparative example 2
This comparative example used the same starting materials and preparation method as example 1, except that the ultrasonic extraction solvent in step c was replaced with an equivalent amount of dodecyl pyridinium bromide ionic liquid. 15.9g of mulberry leaf polysaccharide powder is prepared, and the content of polysaccharide in the mulberry leaf polysaccharide powder is up to 42.6 percent according to GB/T15672-2009 through a phenol-sulfuric acid method.
Comparative example 3
This comparative example uses the same starting materials and preparation method as example 1, except that benzyltriethylammonium chloride in the ultrasonic extraction solvent of step c was replaced with an equal amount of choline chloride. 17.4g of mulberry leaf polysaccharide powder is prepared, and the content of polysaccharide in the mulberry leaf polysaccharide powder is up to 40.5 percent according to GB/T15672-2009 by a phenol-sulfuric acid method.
In conclusion, the purity of the mulberry leaf polysaccharide compound extraction process provided by the invention reaches more than 70.8%. Compared with comparative examples 1-3, the purity of the product is obviously improved. The mulberry leaf polysaccharide compound extraction process provided by the invention has wide application prospect and has positive promotion effect on the deep processing of the mulberry leaf polysaccharide and the development of the mulberry leaf polysaccharide extraction industry.
The above description is intended to be illustrative of the preferred embodiment of the present invention and should not be taken as limiting the invention, but rather, the invention is intended to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention.
Claims (10)
1. A compound extraction process of mulberry leaf polysaccharide is characterized in that: the method comprises an enzymolysis process and an ultrasonic extraction process, wherein an extraction solvent of the ultrasonic extraction process comprises a deep eutectic solvent and deionized water;
the deep eutectic solvent comprises a hydrogen bond donor and a hydrogen bond acceptor;
the hydrogen bond donor is one or more of xylitol, trimethylolethane or urea; the hydrogen bond acceptor is one or two of benzyltriethylammonium chloride or tetrabutylammonium hydrogen sulfate;
the enzymolysis process adopts a complex enzyme of chitinase, thiol protease and carboxypeptidase to carry out enzymolysis.
2. The mulberry leaf polysaccharide complex extraction process of claim 1, which is characterized in that: the mass ratio of the deionized water to the deep eutectic solvent in the extraction solvent is 2-5.
3. The complex extraction process of mulberry leaf polysaccharide as claimed in claim 1 or 2, wherein: the mass ratio of the hydrogen bond donor to the hydrogen bond acceptor in the deep eutectic solvent is 1-4.
4. The mulberry leaf polysaccharide complex extraction process of claim 3, which is characterized in that: the deep eutectic solvent is prepared by the following method: and (3) uniformly mixing the hydrogen bond donor and the hydrogen bond acceptor, heating to 80-90 ℃, and reacting for 2-3h to obtain the deep eutectic solvent.
5. The complex extraction process of mulberry leaf polysaccharide as claimed in claim 1, wherein: the total mass of the chitinase, the thiol protease and the carboxypeptidase accounts for 2 to 5 percent of the total mass of the mulberry leaf powder;
the mass ratio of the chitinase to the thiol protease to the carboxypeptidase is 2-3.
6. The mulberry leaf polysaccharide complex extraction process of claim 1, which is characterized in that: the method specifically comprises the following steps:
washing, drying, crushing and sieving fresh mulberry leaves to obtain mulberry leaf powder;
step b, adding distilled water into the mulberry leaf powder, uniformly mixing, then adding an organic solvent for extraction, taking the degreased raffinate, and drying to obtain degreased powder;
c, adding the degreased powder into distilled water, uniformly dispersing, adding chitinase, thiol protease and carboxypeptidase, and stirring for enzymolysis to obtain an enzymolysis solution;
d, adding an extraction solvent into the enzymatic hydrolysate, performing ultrasonic extraction to obtain an extracting solution, filtering, and concentrating to obtain a concentrated solution; precipitating the concentrated solution with ethanol, and drying to obtain folium Mori polysaccharide powder.
7. The mulberry leaf polysaccharide complex extraction process of claim 6, which is characterized in that: in the step b, the mass ratio of the distilled water to the mulberry leaf powder is 5-10; the mass ratio of the organic solvent to the mulberry leaf powder is 5-10;
the organic solvent is at least one of n-hexane, petroleum ether or ethanol.
8. The mulberry leaf polysaccharide complex extraction process of claim 6, which is characterized in that: in the step c, the mass ratio of the distilled water to the mulberry leaf powder is 2-3; the stirring time is 25-35 min.
9. The mulberry leaf polysaccharide complex extraction process of claim 6, which is characterized in that: in the step d, the mass ratio of the extraction solvent to the mulberry leaf powder is 3-5;
in the step d, the ultrasonic power is 40000-50000Hz, and the ultrasonic time is 15-20 min;
in the step d, the concentration is carried out in vacuum until the solid content is more than or equal to 20 percent, and the temperature of the vacuum concentration is 75-90 ℃;
the alcohol precipitation specifically comprises the following steps: adding absolute ethyl alcohol with the mass 1-3 times of that of the mulberry leaf powder into the concentrated solution, and standing for 2-3 h.
10. The mulberry leaf polysaccharide complex extraction process of claim 6, which is characterized in that: the drying in the step d is pressure spray drying; the air inlet temperature of the pressure spray drying is 180-190 ℃, and the air outlet temperature is 80-100 ℃; the spraying pressure of the pressure spray drying is 0.7-1.0 MPa.
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| CN116178577A (en) * | 2023-01-31 | 2023-05-30 | 杭州佳嘉乐生物技术有限公司 | High-purity microalgae polysaccharide and extraction method thereof |
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| 夏平;谢何青;: "复合酶法提取桑叶中多糖的工艺条件优化" * |
| 王芳;郑海雪;: "桑叶多糖的提取工艺研究" * |
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| CN115926013A (en) * | 2022-12-08 | 2023-04-07 | 天然(广州)新材料研究发展有限公司 | Nostoc sphaeroides polysaccharide and preparation method thereof |
| CN115926013B (en) * | 2022-12-08 | 2024-01-26 | 天然(广州)新材料研究发展有限公司 | Nostoc sphaeroids kutz polysaccharide and preparation method thereof |
| CN116178577A (en) * | 2023-01-31 | 2023-05-30 | 杭州佳嘉乐生物技术有限公司 | High-purity microalgae polysaccharide and extraction method thereof |
| CN116178577B (en) * | 2023-01-31 | 2024-04-19 | 杭州佳嘉乐生物技术有限公司 | High-purity microalgae polysaccharide and extraction method thereof |
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| Publication number | Publication date |
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| CN115260334B (en) | 2023-04-07 |
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