Method for extracting polysaccharide from horned melons

Abstract

The invention discloses a method for extracting polysaccharide from cucumis metuliferus, and relates to the field of polysaccharide preparation, the extraction method comprises the following steps: S1, adding cucumis metuliferus pulp into an aqueous solution containing an antioxidant, homogenizing, adding an electrolyte solution, and stirring to obtain a cucumis metuliferus homogenate; s2, adding a compound enzyme solution into the horned melon homogenate for enzymolysis to obtain an enzymolysis mixed solution; s3, adding an extraction solvent into the enzymolysis mixed solution for extraction, and performing heat preservation and stirring to obtain an extracting solution; s4, sequentially carrying out solid-liquid separation, adsorption decoloration and deproteinization, ethanol precipitation, washing and drying to obtain cucumis metuliferus polysaccharide powder. By adopting a composite enzymolysis process containing pectinase, cellulase and protease, a mucus colloidal network structure formed by pectin cellulose and protein in the cucumis metuliferus is effectively broken, and compared with the phenomena of polysaccharide structure damage and glucosidic bond breakage caused by extraction of cucumis metuliferus polysaccharide by a traditional extraction method, the extraction method has the advantages that the extraction efficiency is improved; the extraction efficiency and activity retention of the cucumis metuliferus polysaccharide can be effectively improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of polysaccharide preparation, and particularly relates to an extraction method of polysaccharide in Cucumis metuliferus. BACKGROUND

[0002] Cucumis metuliferus, also known as African horned melon or fire melon, is originally from Africa and is a fruit with unique appearance and rich nutritional value. The fruit pulp contains various bioactive components, especially polysaccharides, which have been confirmed by modern research to have multiple physiological functions such as enhancing immunity, regulating intestinal flora and antioxidant, and show broad application prospects in the fields of functional food, health products and pharmaceutical intermediates.

[0003] At present, the conventional extraction methods of plant polysaccharides include hot water extraction, acid and alkali extraction, ultrasonic-assisted extraction and enzyme-assisted extraction, etc. These methods have been widely used in the extraction of various plant polysaccharides. After the extraction of plant polysaccharides is completed, the crude extract needs to be purified to remove proteins, pigments, small molecule impurities, etc. However, due to the physicochemical properties of Cucumis metuliferus polysaccharides and the complexity of the crude extract, the traditional purification process needs to be operated in multiple steps, such as Sevag method for removing protein and macroporous resin for decolorization, etc. The process is complicated and time-consuming, and the polysaccharide recovery rate is generally less than 60%, which restricts the large-scale preparation and application of Cucumis metuliferus polysaccharides. SUMMARY

[0004] The present application overcomes the shortcomings of the prior art and provides an extraction method of polysaccharide in Cucumis metuliferus.

[0005] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows: an extraction method of polysaccharide in Cucumis metuliferus, comprising the following steps:

[0006] S1, homogenizing Cucumis metuliferus pulp in an aqueous solution containing an antioxidant, and then adding an electrolyte solution and stirring to obtain a Cucumis metuliferus homogenate;

[0007] S2, adding a complex enzyme solution to the Cucumis metuliferus homogenate, and carrying out enzymolysis at 38-42 ℃ and 150-200 r / min for 2-2.5 h to obtain an enzymolysis mixture; wherein the complex enzyme solution contains pectinase, cellulase and protease;

[0008] S3, adding an extraction solvent to the enzymolysis mixture, adjusting the solid-liquid ratio to 1:7-9, and carrying out ultrasonic extraction, and then stirring and incubating at 45-60 ℃ for 8-15 min to obtain an extract;

[0009] S4, sequentially performing solid-liquid separation, adsorption decolorization and deproteinization, ethanol precipitation, washing and drying to obtain Cucumis metuliferus polysaccharide powder.

[0010] In a preferred embodiment of the present invention, in step S1, the antioxidant is a vitamin C solution with a concentration of 0.05~0.1wt%, and the solid-liquid ratio of the horned melon pulp to the vitamin C solution is 1:2~4.

[0011] In a preferred embodiment of the present invention, in step S1, the electrolyte solution is a sodium chloride solution, accounting for 0.1-0.2% of the horned melon pulp, and the stirring time is 5-20 min.

[0012] In a preferred embodiment of the present invention, in step S1, the homogenization is performed using a colloid mill at a speed of 2500~3500 r / min for 5~10 min.

[0013] In a preferred embodiment of the present invention, in step S2, the mass ratio of pectinase, cellulase and protease in the composite enzyme solution is 2-5:1-3:1, the activity of the pectinase is 3000-5000 U / g, the activity of the cellulase is 4500-5500 U / g, the activity of the protease is 1700-2500 U / g, and the activity of the protease is 70-90% for endopeptidase and 10-30% for exopeptidase.

[0014] In a preferred embodiment of the present invention, in step S2, the enzyme concentration of the composite enzyme solution is 0.8~1.2wt%, the solvent is an acetate-sodium acetate buffer solution with a pH of 5.0, and the volume ratio of the homogenate to the composite enzyme solution is 4~5:1.

[0015] In a preferred embodiment of the present invention, in step S3, the extraction solvent is water containing 0.05~0.1% vitamin C.

[0016] In a preferred embodiment of the present invention, in step S3, the ultrasonic extraction adopts a pulse mode: ultrasonication for 3-8 minutes followed by an interval of 1-3 minutes, ultrasonic power of 180-220 W, ultrasonic frequency of 30-40 kHz, extraction temperature of 45-50℃, and ultrasonic time of 30-40 minutes.

[0017] In a preferred embodiment of the present invention, in step S4, the solid-liquid separation is centrifugation at 4000-5500 r / min for 10-30 min; the adsorption, decolorization, and deproteinization uses diatomaceous earth, with an addition amount of 1.0-1.5% of the supernatant by mass-volume ratio, an adsorption temperature of 20-40 ℃, a settling time of 10-30 min, and centrifugation at 3000-4000 r / min for 20-30 min after adsorption.

[0018] In a preferred embodiment of the present invention, in step S4, the ethanol precipitation uses ethanol with a volume fraction of 85-95%, the volume ratio of ethanol to clarified liquid is 1:2-3, and the precipitation conditions are refrigeration at 2-5 ℃ for 4-6 h; the washing uses an ethanol solution with a volume fraction of 60-80%, the washing is performed 2-3 times, and the amount used each time is 2-5 times the volume of the precipitate; the drying is performed under a vacuum of ≤-0.095 MPa at 45-60 ℃ for 6-8 h.

[0019] This invention addresses the shortcomings of the prior art and has the following beneficial effects:

[0020] (1) This invention provides a method for extracting polysaccharides from horned melon. By employing a complex enzymatic hydrolysis process containing pectinase, cellulase and protease, during the complex enzymatic hydrolysis process, pectinase can break the galacturonic acid bonds of pectin in the pulp, cellulase hydrolyzes the β-1,4 glycosidic bonds of cellulose, and protease cuts the peptide bonds of proteins, so as to effectively break the mucilaginous colloidal network structure formed by pectin, cellulose and proteins in horned melon. Under the condition of ultrasound assistance, the cavitation effect accelerates the diffusion of polysaccharide molecules from cell tissue to extraction solvent. At the same time, the low temperature is used to avoid the destruction of glycosidic bonds in polysaccharide molecules by high temperature, so that horned melon polysaccharides can be fully released and dissolved from the encapsulation barrier. Compared with the phenomenon of polysaccharide structure destruction and glycosidic bond breakage caused by the extraction of horned melon polysaccharides by traditional extraction methods, the extraction method of this invention can effectively improve the extraction efficiency and activity retention of horned melon polysaccharides.

[0021] (2) In the purification stage of this invention, a simplified process of diatomaceous earth adsorption combined with ethanol gradient precipitation is adopted. Diatomaceous earth has a porous structure and surface activity, which can capture pigment molecules and protein impurities through physical adsorption and ion interaction. Ethanol can act as a precipitant to reduce the hydration layer thickness of polysaccharide molecules through dehydration, so that polysaccharide molecular chains aggregate to form precipitates, while small molecule impurities remain in the ethanol solution. Decolorization and deproteinization can be completed in one step, directly shortening the purification process. Compared with the traditional Sevag method, which requires multiple extractions for deproteinization and has a long decolorization cycle with macroporous resin, this method can reduce equipment dependence and energy consumption, thereby further improving the polysaccharide recovery rate and reducing operating costs.

[0022] (3) In this invention, water is used as the main solvent in the extraction process, and vitamin C and low concentration of sodium chloride are added. Vitamin C, as an antioxidant, can provide a reducing environment, inhibit the oxidation of hydroxyl groups in polysaccharide molecules to carbonyl groups, and reduce the oxidative degradation of polysaccharides. Sodium ions in sodium chloride solution can neutralize the negative charge on the surface of mucus colloidal particles, destroy the colloidal double layer structure, and promote the dispersion of mucus aggregates. This can effectively reduce the oxidative loss of polysaccharides in the extraction process, and at the same time destroy the mucus structure to promote the release of polysaccharides, thereby improving the environmental friendliness of the extraction process. Compared with traditional methods that use strong acids or bases or large amounts of organic solvents, this method can eliminate the problems of harmful residues and waste liquid treatment, thereby reducing the overall production cost and environmental burden, and improving the safety of products and the utilization rate of raw materials.

[0023] (4) In the compound enzymatic hydrolysis of the present invention, a protease with a specific ratio of endopeptidase and exopeptidase activity is used. Endopeptidase can specifically cleave peptide bonds from inside the protein molecule and hydrolyze large protein molecules into small polypeptides. Exopeptidase hydrolyzes amino acid residues from the end of the polypeptide chain and further decomposes the polypeptide into free amino acids and small peptide fragments. This can avoid the formation of complexes between incompletely degraded polypeptides and polysaccharides through hydrophobic interactions, effectively exert the role of protease, and make the protein encapsulating polysaccharides completely degraded. This reduces the adsorption of protein impurities on polysaccharides, thereby not only enhancing the enzymatic hydrolysis efficiency and improving the polysaccharide extraction rate, but also further improving the polysaccharide purity. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 This is a flowchart of a preferred embodiment of the present invention for extracting polysaccharides from horned melon. Detailed Implementation

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein. Therefore, the scope of protection of the invention is not limited to the specific embodiments disclosed below.

[0028] Application Overview:

[0029] During their in-depth research on the extraction process of polysaccharides from horned melon, the applicant discovered that the pulp of horned melon is not only rich in polysaccharides, but also contains a large amount of gel-like mucus formed by pectin, cellulose and protein. This mucus tightly encapsulates the polysaccharide components in a colloidal network structure, forming a mucus-polysaccharide encapsulation system, which makes it difficult for the polysaccharides to be released from the cell tissue.

[0030] When conventional methods for extracting plant polysaccharides are used to extract horned melon polysaccharides, the following problems exist: First, the extraction efficiency is low. Traditional hot water extraction requires extraction at 80-100 ℃ for 4-6 hours, but under high temperature conditions, the mucilage colloidal structure can only be partially destroyed, and the polysaccharide dissolution rate is only 45-55%, resulting in low raw material utilization. Second, the activity is greatly destroyed. Although acid-base extraction can destroy the mucilage structure through strong acid (pH<2) or strong alkali (pH>11) environments, strong acids and alkalis will cause the glycosidic bonds in the polysaccharide molecules to break, resulting in a loss of biological activity of more than 40%, and easily introducing metal ion impurities. Third, the environmental friendliness and safety are insufficient. Some methods use organic solvents such as ethanol-acetone mixtures to precipitate polysaccharides, with large solvent consumption (solid-liquid ratio of 1:20), which not only results in high recovery costs but also poses a risk of solvent residue. At the same time, the waste liquid has a high content of organic solvents, making treatment difficult and failing to meet the requirements of green production.

[0031] To address the aforementioned problems, this invention proposes a method for extracting polysaccharides from horned melon. The method utilizes a colloid mill homogenization process and pretreatment with sodium chloride solution to disrupt the mucilage structure. Then, a complex enzymatic hydrolysis simultaneously degrades pectin, cellulose, and protein to break down the polysaccharide encapsulation barrier. Pulsed ultrasound and low-temperature conditions synergistically promote polysaccharide dissolution, and diatomaceous earth adsorption combined with ethanol gradient precipitation achieves efficient purification. This method enables highly efficient extraction and purification of horned melon polysaccharides, thereby improving the extraction rate and activity retention rate, shortening purification time, reducing solvent consumption and energy consumption, meeting food safety and environmental standards, and is suitable for large-scale production.

[0032] It should be noted that the raw materials, equipment and reagents used in this invention can all be purchased from the market or obtained through existing preparation methods.

[0033] like Figure 1 As shown, a method for extracting polysaccharides from horned melon includes the following steps:

[0034] S1. Add the horned melon pulp to an aqueous solution containing antioxidants and homogenize it. Then add an electrolyte solution and stir to obtain a horned melon homogenate.

[0035] S2. Add the compound enzyme solution to the horned melon homogenate and enzymatically hydrolyze it for 2-2.5 h at 38-42 ℃ and 150-200 r / min to obtain the enzymatic hydrolysis mixture; wherein, the compound enzyme solution contains pectinase, cellulase and protease.

[0036] S3. Add extraction solvent to the enzymatic hydrolysis mixture, adjust the solid-liquid ratio to 1:7~9, perform ultrasonic extraction, and keep warm and stir at 45~60 ℃ for 8~15 min to obtain the extract;

[0037] S4. After solid-liquid separation, adsorption decolorization and deproteinization, ethanol precipitation, washing and drying, horned melon polysaccharide powder is obtained.

[0038] In some specific embodiments, in step S1, the antioxidant is a vitamin C solution with a concentration of 0.05~0.1 wt%, and the solid-liquid ratio of horned melon pulp to vitamin C solution is 1:2~4.

[0039] In some specific embodiments, in step S1, the electrolyte solution is a sodium chloride solution, accounting for 0.1-0.2% of the horned melon pulp, and the stirring time is 5-20 min.

[0040] In some specific embodiments, in step S1, the homogenization is performed using a colloid mill at a speed of 2500~3500 r / min for 5~10 min.

[0041] In some specific embodiments, in step S2, the mass ratio of pectinase, cellulase and protease in the complex enzyme solution is 2~5:1~3:1, the enzyme activity of pectinase is 3000~5000 U / g, the enzyme activity of cellulase is 4500~5500 U / g, the enzyme activity of protease is 1700~2500 U / g, and the activity of endopeptidase in protease accounts for 70~90%, and the activity of exopeptidase accounts for 10~30%.

[0042] In some specific embodiments, in step S2, the enzyme concentration of the complex enzyme solution is 0.8~1.2 wt%, the solvent is an acetate-sodium acetate buffer solution with a pH of 5.0, and the volume ratio of the homogenate to the complex enzyme solution is 4~5:1.

[0043] In some specific embodiments, in step S3, the extraction solvent is water containing 0.05~0.1% vitamin C.

[0044] In some specific implementations, in step S3, the ultrasonic extraction adopts a pulse mode: ultrasonication for 3~8 min followed by an interval of 1~3 min, ultrasonic power of 180~220 W, ultrasonic frequency of 30~40 kHz, extraction temperature of 45~50 ℃, and ultrasonic time of 30~40 min.

[0045] In some specific embodiments, in step S4, solid-liquid separation is performed by centrifugation at 4000-5500 r / min for 10-30 min; diatomaceous earth is used for adsorption, decolorization, and deproteinization, with an addition amount of 1.0-1.5% of the supernatant by mass-volume ratio, an adsorption temperature of 20-40 ℃, a settling time of 10-30 min, and centrifugation after adsorption at 3000-4000 r / min for 20-30 min.

[0046] In some specific embodiments, in step S4, ethanol precipitation uses 85-95% ethanol by volume, with a volume ratio of ethanol to clarified liquid of 1:2-3, and precipitation conditions include refrigeration at 2-5°C for 4-6 hours; washing uses a 60-80% ethanol solution by volume, and washing is performed 2-3 times, with each wash using 2-5 times the volume of the precipitate; drying is performed at 45-60°C for 6-8 hours under a vacuum of ≤-0.095MPa.

[0047] To further simplify and make the present invention achieve its objectives and effects, the present invention will be further illustrated in conjunction with the following specific embodiments and comparative examples, but the present invention is not limited to the scope of the embodiments described herein.

[0048] It should be noted that the raw materials used in the examples and comparative examples are as follows: Pectinase: purity ≥99.9%, enzyme activity 4000 U / g, purchased from Guangdong Mingcheng Biotechnology; Cellulase: purity ≥99.9%, enzyme activity 5000 U / g, purchased from Shandong Fangchang Biotechnology; Protease: enzyme activity 2000 U / g, including endopeptidase with purity ≥99.9%, purchased from Zhongshan Dixing Chemical, and exopeptidase with purity ≥99.9%, purchased from Nantong Yingruida Biotechnology; Diatomaceous earth: particle size 200 mesh, purchased from Shandong Xinjiucheng Chemical.

[0049] Example 1:

[0050] A method for extracting polysaccharides from horned melon includes the following steps:

[0051] S1. Select fresh horned melons with a maturity of 70-80%, remove the hard thorns, stems and rotten parts of the peel, rinse with water, drain the water, cut open the fruit, scoop out the pulp containing gel-like mucus, add a 0.08 wt% vitamin C solution, the solid-liquid ratio of horned melon pulp to vitamin C solution is 1:3, homogenize with a colloid mill at 3000 r / min for 5 min, then add a 0.15% sodium chloride solution of horned melon pulp and stir for 10 min to obtain horned melon homogenate;

[0052] S2. Pectinase, cellulase, and protease in a mass ratio of 3:2:1 were added to an acetate-sodium acetate buffer solution with a pH of 5.0 to prepare a 1.0 wt% compound enzyme solution. The compound enzyme solution was added to the horned melon homogenate and enzymatically hydrolyzed at 40 ℃ and 180 r / min for 2.2 h to obtain an enzymatic hydrolysate. The volume ratio of homogenate to compound enzyme solution was 5:1, and the activity of endopeptidase in the protease was 83%, while the activity of exopeptidase was 17%.

[0053] S3. Add water containing 0.05% vitamin C to the enzymatic hydrolysis mixture, adjust the solid-liquid ratio to 1:8, set the ultrasonic power to 200 W and the ultrasonic frequency to 35 kHz, and sonicate at 48 ℃ for 35 min. During this period, use a pulse mode with ultrasonication intervals of 5 min to 2 min to avoid local overheating. After extraction, keep warm and stir at 50 ℃ for 10 min to obtain the extract.

[0054] S4. Centrifuge the extract at 5000 r / min for 20 min, collect the supernatant, add 1.2% (w / v) diatomaceous earth to the supernatant, stir well, and let stand at 30 ℃ for 20 min to adsorb pigments and proteins. Centrifuge at 4000 r / min for 15 min, collect the clear liquid, slowly add 95% (v / v) ethanol to the clear liquid at a volume ratio of 1:2, stir well, and refrigerate at 4 ℃ for 5 hours to allow the polysaccharides to fully precipitate. Collect the precipitate, wash it three times with 70% (v / v) ethanol solution (each time using three times the volume of the precipitate) to remove residual impurities, and place it in a vacuum drying oven with a vacuum degree of -0.095 MPa and dry at 50 ℃ for 7 h to obtain horned melon polysaccharide powder.

[0055] Example 2:

[0056] This embodiment is basically the same as Example 1, except that the enzyme concentration of the compound enzyme solution is different. Specifically, step S2 involves adding pectinase, cellulase, and protease in a mass ratio of 3:2:1 to an acetate-sodium acetate buffer solution with a pH of 5.0 to prepare a compound enzyme solution with a concentration of 0.8 wt%. The compound enzyme solution is then added to the horned melon homogenate and enzymatically hydrolyzed at 40 ℃ and 180 r / min for 2.2 h to obtain an enzymatic hydrolysis mixture. The volume ratio of the homogenate to the compound enzyme solution is 5:1, and the activity percentage of endopeptidase in the protease is 83%, while the activity percentage of exopeptidase is 17%.

[0057] Example 3:

[0058] This embodiment is basically the same as Example 1, except that the enzyme concentration of the compound enzyme solution is different. Specifically, step S2 involves adding pectinase, cellulase, and protease in a mass ratio of 3:2:1 to an acetate-sodium acetate buffer solution with a pH of 5.0 to prepare a 1.2 wt% compound enzyme solution. This compound enzyme solution is then added to the horned melon homogenate and enzymatically hydrolyzed at 40 ℃ and 180 r / min for 2.2 h to obtain an enzymatic hydrolysis mixture. The volume ratio of the homogenate to the compound enzyme solution is 5:1, and the activity percentage of endopeptidase in the protease is 83%, while the activity percentage of exopeptidase is 17%.

[0059] Example 4:

[0060] This embodiment is basically the same as Example 1, except that the activity ratios of endopeptidase and exopeptidase in the protease are different. The specific steps of S2 are as follows: pectinase, cellulase and protease in a mass ratio of 3:2:1 are added to an acetate-sodium acetate buffer solution with a pH of 5.0 to prepare a 1.0 wt% compound enzyme solution. The compound enzyme solution is added to the horned melon homogenate and enzymatically hydrolyzed for 2.2 h at 40℃ and 180 r / min to obtain an enzymatic hydrolysate. The volume ratio of the homogenate to the compound enzyme solution is 5:1, and the activity ratio of endopeptidase in the protease is 70% and the activity ratio of exopeptidase is 30%.

[0061] Example 5:

[0062] This embodiment is basically the same as Example 1, except that the activity ratios of endopeptidase and exopeptidase in the protease are different. The specific steps of S2 are as follows: pectinase, cellulase and protease in a mass ratio of 3:2:1 are added to an acetate-sodium acetate buffer solution with a pH of 5.0 to prepare a 1.0 wt% compound enzyme solution. The compound enzyme solution is added to the horned melon homogenate and enzymatically hydrolyzed for 2.2 h at 40℃ and 180 r / min to obtain an enzymatic hydrolysate. The volume ratio of the homogenate to the compound enzyme solution is 5:1, and the activity ratio of endopeptidase in the protease is 90% and the activity ratio of exopeptidase is 10%.

[0063] Comparative Example 1:

[0064] The traditional hot water extraction method for polysaccharides from horned melon includes the following steps:

[0065] S1. Select fresh horned melons with a maturity of 70-80%, remove the hard thorns, stems and rotten parts of the peel, rinse with water, drain the water, cut open the fruit, scoop out the pulp containing gel-like mucus, add water, the solid-liquid ratio of horned melon pulp to water is 1:3, homogenize with a colloid mill at 3000 r / min for 5 min to obtain horned melon homogenate;

[0066] S2. Add deionized water directly to the homogenate, adjust the solid-liquid ratio to 1:8, and let it stand in a 90 ℃ water bath for 5 h to obtain the extract.

[0067] S3. Centrifuge the extract at 5000 r / min for 20 min, collect the supernatant, and deproteinize using the Sevag method (chloroform: n-butanol = 4:1, add 1 / 5 of the supernatant volume, shake for 30 min and centrifuge, repeat 3 times). Then decolorize using a D101 macroporous resin column (flow rate 1 BV / h, elute with distilled water) and collect the clear liquid.

[0068] S4. Slowly add 95% ethanol (volume ratio of ethanol to clear liquid 1:2) to the clear liquid, refrigerate at 4 ℃ and let stand for 5 h, collect the precipitate, wash it 3 times with 70% ethanol solution (volume ratio 3 times the volume of precipitate each time), and dry it in a vacuum drying oven at 50 ℃ for 7 h to obtain horned melon polysaccharide powder.

[0069] Comparative Example 2:

[0070] The traditional acid-base extraction method for polysaccharides from horned melon includes the following steps:

[0071] S1. Select fresh horned melons with a maturity of 70-80%, remove the hard thorns, stems and rotten parts of the peel, rinse with water, drain the water, cut open the fruit, scoop out the pulp containing gel-like mucus, add water, the solid-liquid ratio of horned melon pulp to water is 1:3, homogenize with a colloid mill at 3000 r / min for 5 min to obtain horned melon homogenate;

[0072] S2. Add 1 mol / L hydrochloric acid solution to the homogenate, adjust the pH of the system to 1.0, stir evenly, and extract at a constant temperature of 60 ℃ in a water bath for 3 h, stirring once every 30 min for 5 min each time to obtain an acidic extract.

[0073] S3. Add 1 mol / L sodium hydroxide solution to the acidic extract to neutralize to pH 7.0, centrifuge at 5000 r / min for 20 min, collect the supernatant, add 732 type cation exchange resin to the supernatant (the amount of resin is 1 / 5 of the volume of the supernatant), shake for 2 h to remove impurity ions, filter and collect the desalination solution.

[0074] S4. Deproteinize using the Sevag method (chloroform: n-butanol = 4:1, add 1 / 5 of the desalting liquid volume, shake for 30 min and centrifuge, repeat 3 times), then decolorize using a D101 macroporous resin column (flow rate 1 BV / h, elute with distilled water), and collect the clear liquid.

[0075] S5. Slowly add a 1:1 volume ratio of ethanol-acetone mixture (volume ratio of mixture to clear liquid 1:20) to the clear liquid, refrigerate at 4 ℃ and let stand for 5 h, collect the precipitate, wash it 3 times with a 70% volume fraction ethanol solution, each time using 3 times the volume of the precipitate, and dry it in a vacuum drying oven at 50 ℃ for 7 h to obtain horned melon polysaccharide powder.

[0076] Comparative Example 3:

[0077] This comparative example is basically the same as Example 1, except that: no compound enzyme is used, and only single enzymatic hydrolysis is performed. The specific steps of S2 are as follows: an acetate-sodium acetate buffer solution with a pH of 5.0 is added to the pectinase to prepare a compound enzyme solution with a concentration of 1.0 wt%. The compound enzyme solution is added to the horned melon homogenate and enzymatically hydrolyzed for 2.2 h at 40 ℃ and 180 r / min to obtain an enzymatic hydrolysate. The volume ratio of the homogenate to the compound enzyme solution is 5:1, and the activity of endopeptidase in the protease accounts for 83% and the activity of exopeptidase accounts for 17%.

[0078] Comparative Example 4:

[0079] This comparative example is basically the same as Example 1, except that the enzyme concentration of the compound enzyme solution is different. Specifically, step S2 involves adding pectinase, cellulase, and protease in a mass ratio of 3:2:1 to an acetate-sodium acetate buffer solution with a pH of 5.0 to prepare a compound enzyme solution with a concentration of 0.5 wt%. The compound enzyme solution is then added to the horned melon homogenate and enzymatically hydrolyzed at 40 ℃ and 180 r / min for 2.2 h to obtain an enzymatic hydrolysate. The volume ratio of the homogenate to the compound enzyme solution is 5:1, and the activity of endopeptidase in the protease is 83%, while the activity of exopeptidase is 17%.

[0080] Comparative Example 5:

[0081] This comparative example is basically the same as Example 1, except that the enzyme concentration of the compound enzyme solution is different. Specifically, step S2 involves adding pectinase, cellulase, and protease in a mass ratio of 3:2:1 to an acetate-sodium acetate buffer solution with a pH of 5.0 to prepare a 1.5 wt% compound enzyme solution. This compound enzyme solution is then added to the horned melon homogenate and enzymatically hydrolyzed for 2.2 h at 40 ℃ and 180 r / min to obtain an enzymatic hydrolysate. The volume ratio of the homogenate to the compound enzyme solution is 5:1, and the activity of endopeptidase in the protease is 83%, while the activity of exopeptidase is 17%.

[0082] Comparative Example 6:

[0083] This comparative example is basically the same as Example 1, except that the activity ratios of endopeptidase and exopeptidase in the protease are different. The specific steps in S2 are as follows: pectinase, cellulase and protease in a mass ratio of 3:2:1 are added to an acetate-sodium acetate buffer solution with a pH of 5.0 to prepare a 1.0 wt% compound enzyme solution. The compound enzyme solution is added to the horned melon homogenate and enzymatically hydrolyzed for 2.2 h at 40°C and 180 r / min to obtain an enzymatic hydrolysate. The volume ratio of the homogenate to the compound enzyme solution is 5:1, and the activity ratio of endopeptidase in the protease is 65%, while the activity ratio of exopeptidase is 35%.

[0084] Comparative Example 7:

[0085] This comparative example is basically the same as Example 1, except that the activity ratios of endopeptidase and exopeptidase in the protease are different. The specific steps of S2 are as follows: pectinase, cellulase and protease in a mass ratio of 3:2:1 are added to an acetate-sodium acetate buffer solution with a pH of 5.0 to prepare a 1.0 wt% compound enzyme solution. The compound enzyme solution is added to the horned melon homogenate and enzymatically hydrolyzed for 2.2 h at 40℃ and 180 r / min to obtain an enzymatic hydrolysate. The volume ratio of the homogenate to the compound enzyme solution is 5:1, and the activity ratio of endopeptidase in the protease is 95%, while the activity ratio of exopeptidase is 5%.

[0086] Comparative Example 8:

[0087] This comparative example is basically the same as Example 1, except that: when extracting the enzymatic hydrolysis mixture, ultrasonic-assisted extraction was not performed. The specific steps of S3 are: add water containing 0.05% vitamin C to the enzymatic hydrolysis mixture, adjust the solid-liquid ratio to 1:8, and let it stand in an 80 ℃ water bath for 5 h to obtain the extract.

[0088] Performance testing: The horned melon polysaccharide powders obtained in Examples 1-5 and Comparative Examples 1-8 were designated as test samples; at the same time, the same mass of horned melon pulp was taken, freeze-dried, and then pulverized through an 80-mesh sieve to obtain horned melon pulp dry powder, which was designated as raw material control sample. The polysaccharide extraction rate, activity retention rate, and polysaccharide purity were tested sequentially, and the results are shown in Table 1.

[0089] Polysaccharide extraction rate: Weigh 2 g of the raw material control sample and place it in a 250 mL round-bottom flask. Add 80 mL of water and reflux in a 95℃ water bath for 2 h (stirring once every 30 min during the process). After cooling, transfer to a centrifuge tube and centrifuge at 4000 r / min for 15 min. Collect the supernatant. Repeat the extraction of the residue with 50 mL of water once. Combine the two supernatants and concentrate them to 20 mL by rotary evaporation. Add 100 mL of anhydrous ethanol and let stand overnight at 4 ℃. Centrifuge at 4000 r / min for 20 min and collect the precipitate. Wash three times with 70% ethanol and vacuum dry to obtain the crude extract of total polysaccharides from the raw material. Dissolve the crude extract in water and dilute to a volumetric flask with water. After shaking well, take 1 mL of the solution and determine the total sugar content using the phenol-sulfuric acid method: Add 1 mL of 5% phenol solution to the solution, mix well, and then quickly add 5 mL of concentrated sulfuric acid. Heat in a boiling water bath for 15 min, cool to room temperature, and measure the absorbance at a wavelength of 490 nm. Plot a standard curve using glucose standard solution and calculate the total polysaccharide content of the raw material (denoted as Mtotal, in g). Weigh the mass of the sample to be tested (denoted as Msample, in g). Extraction rate (%) = (Msample / Mtotal) × 100%.

[0090] Activity retention rate: Take 100 mL of the crude total polysaccharide extract solution prepared in the polysaccharide extraction rate test, dilute it with 0.1 mol / L phosphate buffer (pH 6.8) to a polysaccharide concentration of 1 mg / mL, and use it as the raw material activity assay solution; take 2 mL of this assay solution, add 2 mL of 0.2 mmol / L DPPH ethanol solution, mix well, and react in the dark for 30 min, and measure the absorbance at a wavelength of 517 nm (recorded as A0); at the same time, use phosphate buffer instead of the assay solution as a blank control (absorbance recorded as A1), and use ethanol instead of DPPH solution as a sample control (absorbance recorded as A2), and calculate the original DPPH scavenging rate of the raw material polysaccharide according to the formula: R0 (%) = [1 - (A0 - A2) / A1] × 100%. Weigh 0.1 g of the sample to be tested, dissolve it in 0.1 mol / L phosphate buffer (pH 6.8) and bring the volume to 100 mL to obtain a sample solution with a concentration of 1 mg / mL. Determine the DPPH scavenging rate (denoted as R1) using the method described above. Activity retention rate (%) = (R1 / R0) × 100%.

[0091] Polysaccharide purity: Take 1 mL of the sample solution to be tested in the activity retention rate test, and determine the total sugar content (denoted as C sugar, unit mg / mL) according to the phenol-sulfuric acid method in the polysaccharide extraction rate test. Total sugar content (%) = (C sugar × solution volume × dilution factor) / sample mass × 100%. Take 1 mL of the sample solution to be tested, add 5 mL of Coomassie Brilliant Blue G-250 reagent, mix well, and let stand for 5 min. Measure the absorbance at a wavelength of 595 nm. Plot a standard curve using bovine serum albumin standard solution, and calculate the protein content (denoted as C protein, unit mg / mL). Protein content (%) = (C protein × solution volume × dilution factor) / sample mass × 100%. Weigh 2 g of the sample to be tested, place it in a pre-weighed weighing bottle, and dry it in an oven at 105 ℃ until constant weight. Calculate the moisture content (denoted as C water, %). Polysaccharide purity (%) = total sugar content - protein content - moisture content.

[0092] Table 1: Performance test results of the horned melon polysaccharide powders obtained in Examples 1-5 and Comparative Examples 1-8

[0093] Item Extraction rate of polysaccharide (%) Activity retention rate (%) Purity of polysaccharide (%) Example 1 87.2 92.3 86.7 Example 2 82.5 91.1 85.3 Example 3 84.7 90.6 84.1 Example 4 83.6 91.2 83.8 Example 5 85.1 90.9 84.5 Comparative Example 1 52.3 68.2 65.8 Comparative Example 2 68.5 70.1 59.7 Comparative Example 3 65.3 78.5 72.6 Comparative Example 4 58.7 82.3 70.2 Comparative Example 5 72.1 85.6 75.8 Comparative Example 6 76.4 88.2 79.3 Comparative Example 7 74.2 87.5 78.1 Comparative Example 8 68.9 81.7 73.5

[0094] As shown in Table 1, the polysaccharide extraction rate (82.5%~87.2%), activity retention rate (90.6%~92.3%), and purity (83.8%~86.7%) of Examples 1-5 of the method of the present invention are significantly better than those of the comparative examples. The core of this method lies in the compound enzymatic hydrolysis. In the compound enzymatic hydrolysis, pectinase specifically breaks the galacturonic acid glycosidic bonds in pectin, cellulase hydrolyzes the β-1,4 glycosidic bonds in cellulose, and protease cuts the peptide bonds of proteins. These processes work together to degrade the mucilaginous colloidal network structure formed by pectin, cellulose, and protein in the flesh of horned melon, thereby breaking the polysaccharide encapsulation barrier and allowing the polysaccharides to be fully released.

[0095] In the subsequent extraction, the ultrasonic-assisted cavitation effect generates microjets and shear forces, accelerating the diffusion of polysaccharide molecules from cell tissues into the solvent. The low temperature conditions (45-50 ℃) prevent the high temperature from damaging the polysaccharide glycosidic bonds, ensuring the preservation of activity. In the purification stage, diatomaceous earth adsorption, with its porous structure, captures pigment and protein impurities through physical adsorption and ion interactions. Ethanol gradient precipitation induces selective precipitation of polysaccharides by changing the polarity of the solution, avoiding the cumbersome operation of traditional multi-step purification.

[0096] A comparison of Example 1 and Comparative Examples 1-2 reveals the following: Comparative Example 1, using the traditional hot water extraction method, while achieving a high temperature of 90°C to partially destroy the mucilage colloid, prolonged extraction at high temperatures easily leads to the breakage of glycosidic bonds in the polysaccharide molecules due to thermal vibration. In particular, the α-1,4 and β-1,4 glycosidic bonds are heat-sensitive and prone to hydrolysis, causing polysaccharide chain degradation. Consequently, the extraction rate was only 52.3%, and the activity retention rate was 68.2%. Simultaneously, the high temperature caused protein denaturation and aggregation in the mucilage colloid, encapsulating the polysaccharide, hindering dissolution, and initiating Maillard reactions to produce pigment impurities, reducing the purity to 65.8%. Comparative Example 2, using the acid-base extraction method, experienced acid-catalyzed hydrolysis of the polysaccharide glycosidic bonds in a strongly acidic environment. The glycosidic bonds at the reduction ends were particularly prone to breakage, resulting in a high rate of bioactivity loss. Furthermore, the introduced metal ions formed complexes with the polysaccharide, increasing the impurity content, resulting in a purity of only 59.7%. In addition, the salts generated after acid-base neutralization required an additional desalting step, further reducing the recovery rate. In contrast, the mild enzymatic hydrolysis and low-temperature ultrasound in Example 1 avoids the destruction of chemical bonds, achieving an extraction rate of 87.2% and an activity retention rate of 92.3%. This highlights the advantages of the present invention in protecting the integrity of polysaccharide molecules and avoiding the introduction of impurities, while significantly reducing solvent consumption and effectively solving the core pain points of traditional technologies.

[0097] A comparison between Example 1 and Comparative Example 3 reveals that: Comparative Example 3 used only a single pectinase. Although the pectinase could degrade the galacturonic acid chains of pectin, it could not effectively hydrolyze the β-1,4 glycosidic bonds of cellulose and the peptide bonds of proteins. As a result, the cellulose and protein barriers in the mucus network were not broken down, and the polysaccharides remained encapsulated in the cell walls and protein matrix, hindering polysaccharide dissolution, resulting in an extraction rate of only 65.3%. At the same time, the undegraded proteins bound to the polysaccharides through hydrophobic interactions, forming complexes, increasing the impurity content, and reducing the purity to 72.6%. In addition, the low efficiency of single enzymatic hydrolysis required longer time or higher temperature compensation, indirectly affecting the activity retention rate.

[0098] A comparison of Examples 1-3 with Comparative Examples 4-5 shows that the concentration of the complex enzyme solution in Examples 1-3 is in the range of 0.8-1.2 wt%. This concentration ensures that the enzyme molecules and the substrates (such as pectin, cellulose, and proteins) are fully in contact and bound to form an enzyme-substrate complex, thereby efficiently catalyzing bond breaking.

[0099] In Comparative Example 4, the enzyme concentration was too low, resulting in insufficient enzyme molecules to completely cover the substrate binding sites, leading to a slow catalytic reaction rate, incomplete degradation of the encapsulation structure, and an extraction rate of only 58.7%. Furthermore, residual protein and pectin impurities reduced the purity to 70.2%. In Comparative Example 5, although the enzyme concentration was too high, it could rapidly degrade the substrate. However, excessive aggregation of enzyme molecules could cause substrate inhibition or non-specific binding. For example, proteases might excessively hydrolyze peptide bonds on polysaccharide chains, causing slight breaks in the polysaccharide chains and reducing the activity retention rate to 85.6%. At the same time, the enzyme itself, as an impurity, increased the purification burden, resulting in a purity of only 75.8%.

[0100] A comparison of Examples 1, 4-5 and Comparative Examples 6-7 shows that an appropriate ratio of endopeptidase activity can ensure that endopeptidase preferentially cleaves peptide bonds inside proteins, rapidly hydrolyzing large protein molecules into polypeptides, while exopeptidase gradually releases amino acids from the ends of polypeptides, avoiding the generation of too many small peptide fragments. This balance ensures that proteins are completely degraded, reducing hydrophobic interactions with polysaccharides and thus improving the extraction rate.

[0101] Comparative Example 6 showed an excessively low proportion of endopeptidase activity and an excessively high proportion of exopeptidase activity, leading to premature hydrolysis of the exopeptidase from its terminal end, resulting in a large number of small peptides and amino acids. These small molecules easily compete with polysaccharides for solvents or form complexes, hindering polysaccharide dissolution, resulting in an extraction rate of only 76.4%, and an increase in small peptide impurities, reducing the purity to 79.3%. Comparative Example 7 showed an excessively high proportion of endopeptidase activity and an insufficient proportion of exopeptidase activity. Although it could quickly cleave internal peptide bonds, the accumulated peptides were not fully degraded. These peptides bound to polysaccharides through hydrogen bonds or electrostatic interactions, forming insoluble complexes, reducing the purity to 78.1%, and affecting the activity retention rate.

[0102] A comparison between Example 1 and Comparative Example 8 reveals that Comparative Example 8 did not employ ultrasound, relying solely on thermal stirring. Ultrasound's cavitation effect generates microbubbles and localized high pressure, disrupting the physical structure of the cell wall and mucus network, and accelerating polysaccharide molecule diffusion. In contrast, thermal stirring relies only on thermal motion and convection, resulting in low efficiency, slow solvent diffusion rate, and prolonged dissolution time of polysaccharide molecules from intercellular spaces. Furthermore, high temperature (80 °C) may cause glycosidic bond pyrolysis, leading to an extraction rate of only 68.9% and an activity retention rate of 81.7%. Simultaneously, without ultrasound, the mucus colloid was not sufficiently dispersed, polysaccharide release was incomplete, residual impurities increased, and purity decreased to 73.5%.

[0103] The above description is based on the preferred embodiments of the present invention. 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 exemplary and non-limiting in all respects. The scope of the invention is defined by the appended claims rather than the foregoing description, and all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0104] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for extracting polysaccharides from horned melon, characterized in that, Includes the following steps: S1. Add the horned melon pulp to an aqueous solution containing antioxidants and homogenize it. Then add an electrolyte solution and stir to obtain a horned melon homogenate. S2. Add a compound enzyme solution to the horned melon homogenate and hydrolyze it at 38-42 ℃ and 150-200 r / min for 2-2.5 h to obtain an enzymatic hydrolysis mixture; wherein the compound enzyme solution contains pectinase, cellulase and protease. S3. Add extraction solvent to the enzymatic hydrolysis mixture, adjust the solid-liquid ratio to 1:7~9, perform ultrasonic extraction, and keep warm and stir at 45~60 ℃ for 8~15 min to obtain the extract; S4. After solid-liquid separation, adsorption decolorization and deproteinization, ethanol precipitation, washing and drying, horned melon polysaccharide powder is obtained.

2. The method for extracting polysaccharides from horned melon according to claim 1, characterized in that: In step S1, the antioxidant is a vitamin C solution with a concentration of 0.05~0.1 wt%, and the solid-liquid ratio of the horned melon pulp to the vitamin C solution is 1:2~4.

3. The method for extracting polysaccharides from horned melon according to claim 1, characterized in that: In step S1, the electrolyte solution is a sodium chloride solution, accounting for 0.1-0.2% of the horned melon pulp, and the stirring time is 5-20 minutes.

4. The method for extracting polysaccharides from horned melon according to claim 1, characterized in that: In step S1, the homogenization is performed using a colloid mill at a speed of 2500~3500 r / min for 5~10 min.

5. The method for extracting polysaccharides from horned melon according to claim 1, characterized in that: In step S2, the mass ratio of pectinase, cellulase and protease in the composite enzyme solution is 2-5:1-3:

1. The activity of the pectinase is 3000-5000 U / g, the activity of the cellulase is 4500-5500 U / g, and the activity of the protease is 1700-2500 U / g. Furthermore, the activity of endopeptidase in the protease accounts for 70-90%, and the activity of exopeptidase accounts for 10-30%.

6. The method for extracting polysaccharides from horned melon according to claim 1, characterized in that: In step S2, the enzyme concentration of the composite enzyme solution is 0.8~1.2 wt%, the solvent is an acetate-sodium acetate buffer solution with a pH of 5.0, and the volume ratio of the homogenate to the composite enzyme solution is 4~5:

1.

7. The method for extracting polysaccharides from horned melon according to claim 1, characterized in that: In step S3, the extraction solvent is water containing 0.05-0.1% vitamin C.

8. The method for extracting polysaccharides from horned melon according to claim 1, characterized in that: In step S3, the ultrasonic extraction adopts a pulse mode: ultrasonication for 3-8 minutes followed by an interval of 1-3 minutes, ultrasonic power of 180-220 W, ultrasonic frequency of 30-40 kHz, extraction temperature of 45-50 ℃, and ultrasonic time of 30-40 minutes.

9. The method for extracting polysaccharides from horned melon according to claim 1, characterized in that: In step S4, the solid-liquid separation is performed by centrifugation at 4000-5500 r / min for 10-30 min; the adsorption, decolorization, and deproteinization uses diatomaceous earth, with an addition amount of 1.0-1.5% of the supernatant by mass-volume ratio, an adsorption temperature of 20-40 ℃, a settling time of 10-30 min, and centrifugation at 3000-4000 r / min for 20-30 min after adsorption.

10. The method for extracting polysaccharides from horned melon according to claim 1, characterized in that: In step S4, the ethanol precipitation uses ethanol with a volume fraction of 85-95%, the volume ratio of ethanol to clarified liquid is 1:2-3, and the precipitation conditions are refrigeration at 2-5 ℃ for 4-6 h; the washing uses an ethanol solution with a volume fraction of 60-80%, and the washing is performed 2-3 times, with each washing using 2-5 times the volume of the precipitate; the drying is performed under a vacuum of ≤-0.095 MPa at 45-60 ℃ for 6-8 h.

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