Method for enzyme-assisted water extraction of orange peel polysaccharide

Abstract

The invention discloses a method for enzyme-assisted water extraction of orange peel polysaccharide. The method comprises the following steps: S1, enzyme pretreatment: dissolving orange peel powder with water, adding a certain amount of enzyme, adjusting the pH value with a citrate buffer solution, and heating to obtain an enzyme-pretreated orange peel solution; s2, water extraction: performing water extraction on the orange peel liquid subjected to enzyme pretreatment in the step S1, and performing suction filtration and concentration after water extraction to obtain an orange peel polysaccharide concentrated solution; s3, performing alcohol precipitation: performing alcohol precipitation on the orange peel polysaccharide concentrated solution in the step S2, performing suction filtration, and performing freeze drying to obtain an orange peel polysaccharide crude product; s4, protein and impurity removal: removing impure protein and small molecular impurities from the crude orange peel polysaccharide product in the step S3 to obtain purified orange peel polysaccharide. According to the extraction method, the high-purity orange peel polysaccharide can be obtained, the prepared orange peel polysaccharide has certain antioxidant activity, the product purity is high, the energy consumption is low, and the high-purity polysaccharide has huge application value in the fields of medicine, health care products, food and the like.

Description

Technical Field

[0001] The invention relates to the field of extraction of medicinal and edible substances, and particularly relates to a method for enzyme-assisted water extraction of orange peel polysaccharide. Background Art

[0002] Tangerine peel, the outer rind of the common citrus fruit, is rich in volatile oils, flavonoids, pectin, vitamins, and minerals. While often discarded as leftover waste, tangerine peel holds significant value for both food and medicinal purposes. Tangerine peel can be used for drinking, seasoning, candied fruit, and extracting essential oils. Medicinally, dried tangerine peel, made from dried tangerine peel, is a common Chinese herbal medicine, known for its benefits of regulating qi and strengthening the spleen, clearing dampness, and resolving phlegm. It is commonly used to treat symptoms such as abdominal distension, loss of appetite, vomiting, diarrhea, and cough with excessive phlegm. Tangerine peel polysaccharides, one of the main active ingredients in tangerine peel, possess antioxidant, anti-inflammatory, antibacterial, and anti-tumor biological and pharmacological properties.

[0003] Currently, the industry mostly uses simple water extraction, acid-base extraction, and ultrasonic methods to extract orange peel polysaccharides from citrus peel. The extraction efficiency is low, the energy consumption is high, and purification and decolorization are difficult. There are few research reports on enzymatic hydrolysis-assisted water extraction of orange peel polysaccharides. Summary of the Invention

[0004] Under these background conditions, the present invention proposes a method for enzyme-assisted water extraction of tangerine peel polysaccharide, which can obtain high-purity tangerine peel polysaccharide. The obtained tangerine peel polysaccharide has certain antioxidant activity, and the product is high in purity and low in energy consumption. The high-purity polysaccharide has great application value in the fields of medicine, health products, food, etc.

[0005] In order to overcome at least one of the above disadvantages of the prior art, the present invention provides a method for enzyme-assisted water extraction of orange peel polysaccharide, comprising the following steps: S1 Enzyme pretreatment: Dissolve orange peel powder in water, add a certain amount of enzyme, adjust the pH with citrate buffer, and heat to obtain enzyme-pretreated orange peel liquid; S2 Water extraction: extracting the tangerine peel liquid pretreated with enzymes in step S1, filtering and concentrating the liquid to obtain a tangerine peel polysaccharide concentrate; S3: Alcohol precipitation: The orange peel polysaccharide concentrate in step S2 is subjected to alcohol precipitation, filtered, and freeze-dried to obtain a crude orange peel polysaccharide; S4: removing proteins and impurities: removing impurities such as proteins and small molecule impurities from the crude tangerine peel polysaccharide in step S3 to obtain purified tangerine peel polysaccharide.

[0006] Furthermore, in step S1, the ratio of orange peel powder to water is 1:90-1:95 (g / mL).

[0007] Furthermore, in step S1, the enzyme is cellulase, the added amount is 10000U / 1g orange peel powder, and the end point of pH adjustment is 5.

[0008] Furthermore, in step S2, the conditions for water extraction are: solid-liquid ratio 1:90-1:95 (g / mL), temperature 50-55°C, and time 4-5h.

[0009] Furthermore, in step S2, the concentration is concentrated under reduced pressure, and the pressure is lower than 0.01 MPa.

[0010] Furthermore, in step S3, the alcohol concentration is reduced to 80%, the temperature is 3-4° C., and the time is 20-24 hours.

[0011] Furthermore, in step S4, the protein removal is performed by the Sevage method, specifically, using distilled water to dissolve the crude orange peel polysaccharide, adding Sevage reagent, and fully oscillating at room temperature on a constant temperature oscillator, and then centrifuging. The centrifugation conditions are: 8500~9000rpm, 10~15min, after separating the organic phase and the aqueous phase, Sevage reagent is added to the aqueous phase, and the above process is repeated until there is no white precipitate at the junction of the organic layer and the aqueous layer after centrifugation.

[0012] Furthermore, in step S4, the volume ratio of Sevage reagent is chloroform: n-butanol = 4:1.

[0013] Furthermore, in step S4, the impurity removal is specifically as follows: placing the orange peel polysaccharide solution after protein removal into a dialysis bag, adding deionized water into the dialysis bag, changing the water every half a day, collecting the solution in the bag after 24 hours, and freeze-drying to obtain the finished orange peel polysaccharide.

[0014] Furthermore, in step S4, the dialysis bag model is MW: 3500Da.

[0015] The beneficial effects of the present invention are: (1) This method uses cellulase to specifically destroy plant cell walls before extraction, which can release polysaccharides under mild conditions, combining high efficiency with structural protection. After the orange peel powder is pretreated with enzymes and then extracted with water, the yield of orange peel polysaccharides is high.

[0016] (2) The tangerine peel polysaccharide obtained by enzyme pretreatment and water extraction of tangerine peel powder has less protein impurities and higher purity, which is more conducive to subsequent purification. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The present invention will be further described below with reference to the accompanying drawings and examples.

[0018] Figure 1 This is the ultraviolet spectrum of the orange peel polysaccharide obtained in Example 1 of the present invention; Figure 2 This is a bar chart comparing the yields of crude orange peel polysaccharide of Examples 1 to 5 of the present invention and Comparative Examples 1 to 3. DETAILED DESCRIPTION

[0019] Before discussing the exemplary embodiments in more detail, it should be mentioned that some exemplary embodiments are described as processes or methods depicted as flow charts. Although the flow charts describe the various operations as sequential processes, many of the operations therein can be implemented in parallel, concurrently, or simultaneously. In addition, the order of the various operations can be rearranged. The process can be terminated when its operation is completed, but can also have additional steps not included in the accompanying drawings. The process can correspond to a method, function, procedure, subroutine, subprogram, etc.

[0020] It should be understood that although the terms "first," "second," and the like may be used herein to describe various elements, these elements should not be limited by these terms. These terms are used solely to distinguish one element from another. For example, a first element may be referred to as a second element, and similarly, a second element may be referred to as a first element, without departing from the scope of the exemplary embodiments. The term "and / or" as used herein includes any and all combinations of one or more of the listed associated items.

[0021] The present invention provides a method for enzyme-assisted water extraction of orange peel polysaccharide, characterized in that the method comprises the following steps: S1 Enzyme pretreatment: Dissolve orange peel powder in water, add a certain amount of enzyme, adjust the pH with citrate buffer, and heat to obtain enzyme-pretreated orange peel liquid; The present invention investigates different enzymes (pectinase, cellulase, hemicellulase) and mixed enzymes in different proportions to study the extraction effect.

[0022] S2 Water extraction: extracting the tangerine peel liquid pretreated with enzymes in step S1, filtering and concentrating the liquid to obtain a tangerine peel polysaccharide concentrate; S3: Alcohol precipitation: The orange peel polysaccharide concentrate in step S2 is subjected to alcohol precipitation, filtered, and freeze-dried to obtain a crude orange peel polysaccharide; S4: removing proteins and impurities: removing impurities such as proteins and small molecule impurities from the crude tangerine peel polysaccharide in step S3 to obtain purified tangerine peel polysaccharide.

[0023] Example 1 S1 Enzyme pretreatment: Dissolve 1 g of orange peel powder in 94.6 mL of water, add 200 mg of cellulase, adjust the pH to 5 with citrate buffer, and heat at 30°C to obtain enzyme-pretreated orange peel solution; S2 Water Extraction: The enzyme-pretreated orange peel liquid in step S1 was subjected to water extraction under the following conditions (solid-to-liquid ratio of 1:94.6, where the solid-to-liquid ratio includes the water in step S1; temperature of 51.7°C; time of 4.3 h), followed by filtration and concentration under reduced pressure at 0.01 MPa to a volume of 1 / 3 of the original volume, to obtain an orange peel polysaccharide concentrate; S3: Alcohol precipitation: The orange peel polysaccharide concentrate in step S2 was subjected to alcohol precipitation (alcohol precipitation conditions: 80% ethanol concentration, temperature 4°C, time 24 h), and freeze-dried to obtain 0.42 g of crude orange peel polysaccharide; S4: Protein and impurity removal: Dissolve 0.2 g crude tangerine peel polysaccharide in 10 mL of distilled water, add 1 / 4 volume of Sevage reagent (chloroform: n-butanol = 4:1 / V:V), and shake thoroughly on a thermostatic oscillator at room temperature for 30 minutes (using vortexing). Then centrifuge at 9000 rpm for 15 minutes. Separate the organic and aqueous phases, then add 1 / 4 volume of Sevage reagent to the aqueous phase. Repeat the above process until no white precipitate is observed at the interface of the organic and aqueous layers after centrifugation, indicating that the protein has been completely removed. Place the tangerine peel polysaccharide solution after protein removal using the Sevage method in a dialysis bag (MW: 3500Da). Add deionized water to the dialysis bag and change the water every half a day. After 24 hours, collect the solution in the bag and freeze-dry to obtain tangerine peel polysaccharide, obtaining 0.17 g of purified tangerine peel polysaccharide.

[0024] Determination of orange peel polysaccharide content using the phenol-sulfuric acid method: Take a 1 mg / mL glucose standard solution, dilute to 10 mL with distilled water, and shake well to obtain a 0.1 mg / mL glucose reference stock solution. Pipette 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, and 1.6 mL of the glucose stock solution into test tubes. Add distilled water to each test tube to 2.0 mL. Add 1 mL of 6% phenol solution, mix thoroughly, and quickly add 5 mL of concentrated sulfuric acid. Heat in a boiling water bath for 10 min. After heating, cool to room temperature and measure the absorbance at 490 nm using a microplate reader. Repeat each experiment three times. Plot the glucose standard curve with concentration (x) as the horizontal axis and absorbance (y) as the vertical axis. To determine the content of tangerine peel polysaccharide: Dilute the tangerine peel polysaccharide solution to be tested by a specific dilution factor. Accurately pipette 2.0 mL of the test solution and perform the assay according to the aforementioned method.

[0025] The purity of the purified orange peel polysaccharide is 81.6%.

[0026] The above orange peel polysaccharide was characterized by UV, such as Figure 1 As shown, from Figure 1It can be seen that orange peel polysaccharide has a characteristic absorption peak at 190-200nm, which is a typical characteristic of polysaccharides; it has weak absorption at 260-280nm, indicating that it contains almost no protein.

[0027] Example 2 S1 Enzyme pretreatment: Dissolve 1 g of orange peel powder in 90 ml of water, add 200 mg of cellulase, adjust the pH to 5 with citrate buffer, and heat at 30°C to obtain enzyme-pretreated orange peel solution; S2 Water Extraction: The enzyme-pretreated orange peel liquid in step S1 was subjected to water extraction under the following conditions: a material-liquid ratio of 1:90, where the material-liquid ratio includes the water in step S1; a temperature of 51.7°C; and a time of 4.3 hours. After water extraction, the liquid was filtered and concentrated under reduced pressure at 0.01 MPa to a volume of 1 / 3 of the original volume to obtain an orange peel polysaccharide concentrate. S3: Alcohol precipitation: The orange peel polysaccharide concentrate in step S2 was subjected to alcohol precipitation (alcohol precipitation conditions: 80% ethanol concentration, temperature 4°C), filtered, and freeze-dried to obtain 0.39 g of crude orange peel polysaccharide; S4: Protein and impurity removal: Dissolve 0.2 g crude tangerine peel polysaccharide in 10 mL of distilled water, add 1 / 4 volume of Sevage reagent (chloroform: n-butanol = 4:1 / V:V), and shake thoroughly on a thermostatic shaker at room temperature for 30 minutes (using vortexing). Then centrifuge at 9000 rpm for 15 minutes. Separate the organic and aqueous phases, then add 1 / 4 volume of Sevage reagent to the aqueous phase. Repeat the above process until no white precipitate is observed at the interface of the organic and aqueous layers after centrifugation, indicating that the protein has been completely removed. The tangerine peel polysaccharide solution after protein removal using the Sevage method is placed in a dialysis bag (MW: 3500Da). Deionized water is added to the dialysis bag, and the water is changed every half a day. After 24 hours, the solution in the bag is collected and freeze-dried to obtain tangerine peel polysaccharide, resulting in 0.16 g of purified tangerine peel polysaccharide.

[0028] The purity of the purified orange peel polysaccharide measured using the method in Example 1 was 79.9%.

[0029] Example 3 S1 Enzyme pretreatment: Dissolve 1 g of orange peel powder in 95 ml of water, add 200 mg of cellulase, adjust the pH to 5 with citrate buffer, and heat at 30°C to obtain enzyme-pretreated orange peel liquid; S2 Water Extraction: The enzyme-pretreated orange peel liquid in step S1 was subjected to water extraction under the following conditions: a material-liquid ratio of 1:95, where the material-liquid ratio includes the water in step S1; a temperature of 50°C; and a time of 4.3 hours. After water extraction, the liquid was filtered and concentrated under reduced pressure at 0.01 MPa to a volume of 1 / 3 of the original volume to obtain an orange peel polysaccharide concentrate. S3: Alcohol precipitation: The orange peel polysaccharide concentrate in step S2 was subjected to alcohol precipitation (alcohol precipitation conditions: 80% ethanol concentration, temperature 4°C), filtered, and freeze-dried to obtain 0.41 g of crude orange peel polysaccharide; S4: Protein and impurity removal: Dissolve 0.2 g crude tangerine peel polysaccharide in 10 mL of distilled water, add 1 / 4 volume of Sevage reagent (chloroform: n-butanol = 4:1 / V:V), and shake thoroughly on a thermostatic shaker at room temperature for 30 minutes (using vortexing). Then centrifuge at 9000 rpm for 15 minutes. Separate the organic and aqueous phases, then add 1 / 4 volume of Sevage reagent to the aqueous phase. Repeat the above process until no white precipitate is observed at the interface of the organic and aqueous layers after centrifugation, indicating that the protein has been completely removed. The tangerine peel polysaccharide solution after protein removal using the Sevage method is placed in a dialysis bag (MW: 3500Da). Add deionized water to the dialysis bag and change the water every half a day. After 24 hours, collect the solution in the bag and freeze-dry to obtain tangerine peel polysaccharide, obtaining 0.18 g of purified tangerine peel polysaccharide.

[0030] The purity of the purified orange peel polysaccharide measured using the method in Example 1 was 80.3%.

[0031] Example 4 S1 Enzyme pretreatment: Dissolve 1 g of orange peel powder in 94.6 ml of water, add 200 mg of cellulase, adjust the pH to 5 with citrate buffer, and heat at 30°C to obtain enzyme-pretreated orange peel liquid; S2 Water Extraction: The enzyme-pretreated orange peel liquid in step S1 was subjected to water extraction under the following conditions: a material-liquid ratio of 1:94.6, where the material-liquid ratio includes the water in step S1; a temperature of 55°C; and a time of 4 hours. After water extraction, the liquid was filtered and concentrated under reduced pressure at 0.01 MPa to a volume of 1 / 3 of the original volume to obtain an orange peel polysaccharide concentrate. S3: Alcohol precipitation: The orange peel polysaccharide concentrate in step S2 was subjected to alcohol precipitation (alcohol precipitation conditions: 80% ethanol concentration, temperature 4°C), filtered, and freeze-dried to obtain 0.39 g of crude orange peel polysaccharide; S4: Protein and impurity removal: Dissolve 0.2 g crude tangerine peel polysaccharide in 10 mL of distilled water, add 1 / 4 volume of Sevage reagent (chloroform: n-butanol = 4:1 / V:V), and shake thoroughly on a thermostatic oscillator at room temperature for 30 minutes (using vortexing). Then centrifuge at 9000 rpm for 15 minutes. Separate the organic and aqueous phases, then add 1 / 4 volume of Sevage reagent to the aqueous phase. Repeat the above process until no white precipitate is observed at the interface of the organic and aqueous layers after centrifugation, indicating that the protein has been completely removed. Place the tangerine peel polysaccharide solution after protein removal using the Sevage method in a dialysis bag (MW: 3500Da). Add deionized water to the dialysis bag and change the water every half a day. After 24 hours, collect the solution in the bag and freeze-dry to obtain tangerine peel polysaccharide, obtaining 0.17 g of purified tangerine peel polysaccharide.

[0032] The purity of the purified orange peel polysaccharide measured using the method in Example 1 was 80.7%.

[0033] Example 5 S1 Enzyme pretreatment: Dissolve 1 g of orange peel powder in 94.6 ml of water, add 200 mg of cellulase, adjust the pH to 5 with citrate buffer, and heat at 30°C to obtain enzyme-pretreated orange peel liquid; S2 Water Extraction: The enzyme-pretreated orange peel liquid in step S1 was subjected to water extraction under the following conditions: a material-liquid ratio of 1:94.6, where the material-liquid ratio includes the water in step S1; a temperature of 51.7°C; and a time of 5 hours. After water extraction, the liquid was filtered and concentrated under reduced pressure at 0.01 MPa to a volume of 1 / 3 of the original volume to obtain an orange peel polysaccharide concentrate. S3: Alcohol precipitation: The orange peel polysaccharide concentrate in step S2 was subjected to alcohol precipitation (alcohol precipitation conditions: 80% ethanol concentration, temperature 4°C), filtered, and freeze-dried to obtain 0.40 g of crude orange peel polysaccharide; S4: Protein and impurity removal: Dissolve 0.2 g crude tangerine peel polysaccharide in 10 mL of distilled water, add 1 / 4 volume of Sevage reagent (chloroform: n-butanol = 4:1 / V:V), and shake thoroughly on a thermostatic shaker at room temperature for 30 minutes (using vortexing). Then centrifuge at 9000 rpm for 15 minutes. Separate the organic and aqueous phases, then add 1 / 4 volume of Sevage reagent to the aqueous phase. Repeat the above process until no white precipitate is observed at the interface of the organic and aqueous layers after centrifugation, indicating that the protein has been completely removed. The tangerine peel polysaccharide solution after protein removal using the Sevage method is placed in a dialysis bag (MW: 3500Da). Add deionized water to the dialysis bag and change the water every half a day. After 24 hours, collect the solution in the bag and freeze-dry to obtain tangerine peel polysaccharide, obtaining 0.18 g of purified tangerine peel polysaccharide.

[0034] The purity of the purified orange peel polysaccharide measured using the method in Example 1 was 81.1%.

[0035] Comparative Example 1 S1 Enzyme pretreatment: Dissolve 1 g of orange peel powder in 20 ml of water, add 200 mg of cellulase, adjust the pH to 5 with citrate buffer, and heat at 30°C to obtain enzyme-pretreated orange peel solution; S2 Water Extraction: The enzyme-pretreated orange peel liquid in step S1 was subjected to water extraction under the following conditions: a material-liquid ratio of 1:20, where the material-liquid ratio includes the water in step S1; a temperature of 70°C; and a time of 2 hours. After water extraction, the liquid was filtered and concentrated under reduced pressure at 0.01 MPa to a volume of 1 / 3 of the original volume to obtain an orange peel polysaccharide concentrate. S3: The orange peel polysaccharide concentrate in step S2 was precipitated with alcohol (ethanol concentration of 80% and temperature of 4°C), filtered, and freeze-dried to obtain 0.22 g of crude orange peel polysaccharide; S4: Protein and impurity removal: Dissolve 0.2 g crude tangerine peel polysaccharide in 10 mL of distilled water, add 1 / 4 volume of Sevage reagent (chloroform: n-butanol = 4:1 / V:V), and shake thoroughly on a thermostatic shaker at room temperature for 30 minutes (using vortexing). Then centrifuge at 9000 rpm for 15 minutes. Separate the organic and aqueous phases, then add 1 / 4 volume of Sevage reagent to the aqueous phase. Repeat the above process until no white precipitate is observed at the interface of the organic and aqueous layers after centrifugation, indicating that the protein has been completely removed. The tangerine peel polysaccharide solution after protein removal using the Sevage method is placed in a dialysis bag (MW: 3500Da). Add deionized water to the dialysis bag and change the water every half a day. After 24 hours, collect the solution in the bag and freeze-dry to obtain tangerine peel polysaccharide, obtaining 0.14 g of purified tangerine peel polysaccharide.

[0036] The purity of the purified orange peel polysaccharide measured using the method in Example 1 was 61.3%.

[0037] Comparative Example 2 S1 Enzyme pretreatment: Dissolve 1 g of orange peel powder in 50 ml of water, add 200 mg of cellulase, adjust the pH to 5 with citrate buffer, and heat at 30°C to obtain enzyme-pretreated orange peel liquid; S2 Water Extraction: The enzyme-pretreated orange peel liquid in step S1 was subjected to water extraction under the following conditions: a material-liquid ratio of 1:50, where the material-liquid ratio includes the water in step S1; a temperature of 40°C; and a time of 2 hours. After water extraction, the liquid was filtered and concentrated under reduced pressure at 0.01 MPa to a volume of 1 / 3 of the original volume to obtain an orange peel polysaccharide concentrate. S3: Alcohol precipitation: The orange peel polysaccharide concentrate in step S2 was subjected to alcohol precipitation (alcohol precipitation conditions: 80% ethanol concentration, temperature 4°C), filtered, and freeze-dried to obtain 0.24 g of crude orange peel polysaccharide; S4: Protein and impurity removal: Dissolve 0.2 g crude tangerine peel polysaccharide in 10 mL of distilled water, add 1 / 4 volume of Sevage reagent (chloroform: n-butanol = 4:1 / V:V), and shake thoroughly on a thermostatic shaker at room temperature for 30 minutes (using vortexing). Then centrifuge at 9000 rpm for 15 minutes. Separate the organic and aqueous phases, then add 1 / 4 volume of Sevage reagent to the aqueous phase. Repeat the above process until no white precipitate is observed at the interface of the organic and aqueous layers after centrifugation, indicating that the protein has been completely removed. The tangerine peel polysaccharide solution after protein removal using the Sevage method is placed in a dialysis bag (MW: 3500Da). Deionized water is added to the dialysis bag, and the water is changed every half a day. After 24 hours, the solution in the bag is collected and freeze-dried to obtain tangerine peel polysaccharide, resulting in 0.13 g of purified tangerine peel polysaccharide.

[0038] The purity of the purified orange peel polysaccharide measured using the method in Example 1 was 56.9%.

[0039] Comparative Example 3 S1 Enzyme pretreatment: Dissolve 1 g of orange peel powder in 50 ml of water, add 200 mg of cellulase, adjust the pH to 5 with citrate buffer, and heat at 30°C to obtain enzyme-pretreated orange peel liquid; S2 Water Extraction: The enzyme-pretreated orange peel liquid in step S1 was subjected to water extraction under the following conditions: a material-liquid ratio of 1:50, where the material-liquid ratio includes the water in step S1; a temperature of 70°C; and a time of 1 hour. After water extraction, the liquid was filtered and concentrated under reduced pressure at 0.01 MPa to a volume of 1 / 3 of the original volume to obtain an orange peel polysaccharide concentrate. S3: Alcohol precipitation: The orange peel polysaccharide concentrate in step S2 was subjected to alcohol precipitation (alcohol precipitation conditions: 80% ethanol concentration, temperature 4°C), filtered, and freeze-dried to obtain 0.15 g of crude orange peel polysaccharide; S4: Protein and impurity removal: Dissolve 0.1 g of crude tangerine peel polysaccharide in 5 mL of distilled water, add 1 / 4 volume of Sevage reagent (chloroform: n-butanol = 4:1 / V:V), and shake thoroughly on a thermostatic shaker at room temperature for 30 minutes (using vortexing). Then centrifuge at 9000 rpm for 15 minutes. Separate the organic and aqueous phases, then add 1 / 4 volume of Sevage reagent to the aqueous phase. Repeat the above process until no white precipitate is observed at the interface of the organic and aqueous layers after centrifugation, indicating that the protein has been completely removed. Place the tangerine peel polysaccharide solution after protein removal using the Sevage method in a dialysis bag (MW: 3500Da). Add deionized water to the dialysis bag and change the water every half a day. After 24 hours, collect the solution in the bag and freeze-dry to obtain tangerine peel polysaccharide, obtaining 0.06 g of purified tangerine peel polysaccharide.

[0040] The purity of the purified orange peel polysaccharide measured using the method in Example 1 was 58.5%.

[0041] The crude extraction rates of orange peel polysaccharides of Examples 1 to 5 and Comparative Examples 1 to 3 were plotted as a bar graph, as shown in FIG. Figure 1 As shown, the extraction rates of Examples 1 to 5 of the present invention are significantly higher than those of Comparative Examples 1 to 3.

[0042] Comparative Example 4 The method of Example 1 was used, except that pectinase, hemicellulase, hemicellulase, pectinase + cellulase, cellulase + hemicellulase, pectinase + hemicellulase, and pectinase + cellulase + hemicellulase were used to perform extraction experiments. The results are shown in the following table (the yield of crude tangerine peel polysaccharide is the mass percentage of the extracted crude tangerine peel polysaccharide to the tangerine peel powder).

[0043] As can be seen from the above table, the cellulase selected in the present invention has a higher extraction effect on orange peel polysaccharide than other enzymes.

[0044] The above is only an embodiment of the present invention. Common knowledge such as the known specific structures and characteristics in the scheme is not described in detail here. Ordinary technicians in the field are aware of all common technical knowledge in the technical field of the invention before the application date or priority date, can obtain all existing technologies in the field, and have the ability to apply conventional experimental means before that date. Ordinary technicians in the field can improve and implement this scheme in combination with their own abilities under the inspiration given by this application. Some typical known structures or known methods should not become obstacles for ordinary technicians in the field to implement this application. It should be pointed out that for those skilled in the art, without departing from the structure of the present invention, several variations and improvements can be made, which should also be regarded as the scope of protection of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A method for enzyme-assisted water extraction of orange peel polysaccharides, characterized by: The method comprises the following steps S1 Enzyme pretreatment: Dissolve orange peel powder in water, add a certain amount of enzyme, adjust the pH with citrate buffer, and heat to obtain enzyme-pretreated orange peel liquid; S2 Water extraction: extracting the tangerine peel liquid pretreated with enzymes in step S1, filtering and concentrating the liquid to obtain a tangerine peel polysaccharide concentrate; S3: Alcohol precipitation: The orange peel polysaccharide concentrate in step S2 is subjected to alcohol precipitation, filtered, and freeze-dried to obtain a crude orange peel polysaccharide; S4: removing proteins and impurities: removing impurities such as proteins and small molecule impurities from the crude tangerine peel polysaccharide in step S3 to obtain purified tangerine peel polysaccharide.

2. The method for enzyme-assisted water extraction of orange peel polysaccharide according to claim 1, characterized in that: In step S1, the ratio of orange peel powder to water is 1:90-1:95 (g / mL).

3. The method for enzyme-assisted water extraction of orange peel polysaccharide according to claim 1, characterized in that: In step S1, the enzyme is cellulase, the amount added is 10000 U / 1 g orange peel powder, and the end point of pH adjustment is 5.

4. The method for enzyme-assisted water extraction of orange peel polysaccharide according to claim 1, characterized in that: In step S2, the conditions for water extraction are: solid-liquid ratio 1:90-1:95 (g / mL), temperature 50-55° C., and time 4-5 h.

5. The method for enzyme-assisted water extraction of orange peel polysaccharide according to claim 1, characterized in that: In the step S2, the concentration is carried out under reduced pressure, and the pressure is lower than 0.01 MPa.

6. The method for enzyme-assisted water extraction of orange peel polysaccharide according to claim 1, characterized in that: In step S3, the alcohol concentration is reduced to 80%, the temperature is 3-4° C., and the time is 20-24 hours.

7. The method for enzyme-assisted water extraction of orange peel polysaccharide according to claim 1, characterized in that: In step S4, the protein is removed by the Sevage method, specifically, using distilled water to dissolve the crude orange peel polysaccharide, adding Sevage reagent, and fully oscillating on a constant temperature oscillator at room temperature, and then centrifuging. The centrifugation conditions are: 8500-9000 rpm, 10-15 min, after separating the organic phase and the aqueous phase, Sevage reagent is added to the aqueous phase, and the above process is repeated until there is no white precipitate at the junction of the organic layer and the aqueous layer after centrifugation.

8. The method for enzyme-assisted water extraction of orange peel polysaccharide according to claim 7, characterized in that: In step S4, the volume ratio of the Sevage reagent is chloroform: n-butanol = 4:

1.

9. The method for enzyme-assisted water extraction of orange peel polysaccharide according to claim 7, characterized in that: In step S4, the impurity removal is specifically as follows: placing the orange peel polysaccharide solution after protein removal into a dialysis bag, adding deionized water into the dialysis bag, changing the water every half a day, collecting the solution in the bag after 24 hours, and freeze-drying to obtain the finished orange peel polysaccharide.

10. The method for enzyme-assisted water extraction of orange peel polysaccharide according to claim 9, characterized in that: In step S4, the dialysis bag model is MW: 3500Da.

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