Preparation method of rhizoma bletillae polysaccharide

Abstract

The invention relates to the technical field of deep processing of plants, in particular to a preparation method of rhizoma bletillae polysaccharide. The method comprises the following steps: (1) washing rhizoma bletillae tubers, slicing, drying and crushing, carrying out steam explosion treatment, and drying and crushing after treatment; (2) performing hot water extraction on the rhizoma bletillae powder subjected to steam explosion, retaining supernate, sequentially adding high-temperature-resistant alpha-amylase, saccharifying enzyme and protease, and taking supernate after enzyme deactivation; and (3) carrying out alcohol precipitation on the supernate, keeping the precipitate, washing with absolute ethyl alcohol, adding water into the precipitate, redissolving, and carrying out freeze drying to obtain the rhizoma bletillae polysaccharide. According to the method, the rhizoma bletillae is subjected to hot water extraction after being treated by the steam explosion technology, so that the extraction rate of the rhizoma bletillae polysaccharide is increased compared with that of only hot water extraction, and the prepared rhizoma bletillae polysaccharide is relatively high in sugar content.

Description

Technical Field

[0001] This invention relates to the field of plant deep processing technology, and in particular to a method for preparing Bletilla striata polysaccharide. Background Technology

[0002] Bletilla striata is a terrestrial herb belonging to the Orchidaceae family and the Bletilla genus. Harvesting typically occurs in September. In my country, it is a traditional Chinese medicine with a long history and high medicinal value. Among its components, Bletilla striata polysaccharide is the most important active substance found in its tuber. Due to its high viscosity, Bletilla striata polysaccharide can be used as a humectant, rheology modifier, and thickener in the fields of daily chemicals and food additives. The structure of Bletilla striata polysaccharide is mainly composed of D-mannose and D-glucose linked by β-(1→4)-glycosidic bonds, with an average relative molecular mass of 6.5 × 10⁻⁶. 4 -1.5×10 5 The molecular weight of Bletilla striata polysaccharides is closely related to the extraction and purification methods. Crude polysaccharides and purified polysaccharides differ in molecular weight and physicochemical properties.

[0003] Currently, common extraction methods for Bletilla striata polysaccharides include: (1) Enzymatic hydrolysis, which is highly selective and can efficiently break down cell walls to promote polysaccharide dissolution, but the enzyme cost is high and precise control of conditions is required; (2) Ultrasonic-assisted extraction, which is safe, has a short extraction time and high extraction efficiency, but the cavitation effect generated by high ultrasonic power will change the structure of polysaccharides, thereby affecting the bioactivity of polysaccharides, and the equipment cost is high, making industrial application difficult; (3) Microwave-assisted extraction, which has high extraction efficiency and low solvent consumption, but high temperature may affect the structure and activity of polysaccharides, and the equipment cost is high; Hot water extraction, which is simple to operate, low in cost and has no special equipment requirements, is suitable for large-scale industrial production, but it takes a long time and the extraction rate is low. Therefore, it is necessary to optimize the extraction process of Bletilla striata polysaccharides to improve its extraction rate. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies, meet practical needs, and provide a method for preparing Bletilla striata polysaccharides. This method can expand polysaccharide extraction pathways and improve the extraction rate of Bletilla striata polysaccharides, which is of great significance to the field of plant deep processing technology.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] One of the technical solutions of this invention is a method for preparing Bletilla striata polysaccharide, comprising the following steps:

[0007] (1) Wash and slice the tubers of Bletilla striata, dry and pulverize them, then steam-explode them, and dry and pulverize them after treatment.

[0008] (2) The powder of Bletilla striata after steam explosion was extracted with hot water, and the supernatant was retained. The heat-resistant α-amylase, saccharifying enzyme and protease were added in sequence to inactivate the enzymes and the supernatant was taken.

[0009] (3) The supernatant was precipitated with alcohol, and the precipitate was washed with anhydrous ethanol. The precipitate was redissolved with water and then freeze-dried to obtain Bletilla striata polysaccharide.

[0010] The second technical solution of the present invention is a polysaccharide of Bletilla striata prepared according to the above method.

[0011] The present invention discloses the following technical effects:

[0012] 1. This invention uses steam explosion technology to treat Bletilla striata and then performs hot water extraction, which improves the extraction rate of Bletilla striata polysaccharides compared to hot water extraction alone, and the prepared Bletilla striata polysaccharides have a higher sugar content.

[0013] 2. The method of the present invention has the advantages of high production efficiency, environmental protection and no chemical residue. Attached Figure Description

[0014] Figure 1 The yields of Bletilla striata polysaccharides obtained in Examples 1-3 and Comparative Example 1;

[0015] Figure 2 The sugar content of Bletilla striata polysaccharide obtained in Examples 1-3 and Comparative Example 1. Detailed Implementation

[0016] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0017] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0018] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0019] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0020] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0021] The first aspect of this invention provides a method for preparing Bletilla striata polysaccharide, comprising the following steps:

[0022] (1) Wash and slice the tubers of Bletilla striata, dry and pulverize them, then steam-explode them, and dry and pulverize them after treatment.

[0023] (2) The powder of Bletilla striata after steam explosion was extracted with hot water, and the supernatant was retained. The heat-resistant α-amylase, saccharifying enzyme and protease were added in sequence to inactivate the enzymes and the supernatant was taken.

[0024] (3) The supernatant was precipitated with alcohol, and the precipitate was washed with anhydrous ethanol. The precipitate was redissolved with water and then freeze-dried to obtain Bletilla striata polysaccharide.

[0025] In a preferred embodiment of the present invention, the pressure of the steam explosion treatment is 1.8 MPa, and the pressure holding time is 60 s.

[0026] In a preferred embodiment of the present invention, the material-to-liquid ratio of the hot water extraction is 1:30 g / mL, the extraction temperature is 80°C, and the stirring extraction time is 2 hours.

[0027] In a preferred embodiment of the present invention, the amount of the heat-resistant α-amylase added is 15 μL / g, the water bath temperature is 95°C, and the water bath time is 1 h; the amount of the saccharifying enzyme added is 1 μL / g, the water bath temperature is 60°C, and the water bath time is 0.5 h; and the amount of the protease added is 15 mg / g, the water bath temperature is 60°C, and the water bath time is 1 h.

[0028] In a preferred embodiment of the present invention, the enzyme inactivation temperature is 100°C and the enzyme inactivation time is 15 min.

[0029] In a preferred embodiment of the present invention, the alcohol precipitation time is 12 hours.

[0030] In a preferred embodiment of the present invention, the freeze-drying temperature is -50°C, the time is 72 hours, and the vacuum degree is 10-12 Pa.

[0031] A second aspect of the present invention provides a Bletilla striata polysaccharide prepared according to the above method.

[0032] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0033] The polysaccharide yield calculation formula in this invention is: weight of freeze-dried Bletilla striata polysaccharide / weight of Bletilla striata raw material × 100%;

[0034] Total sugar content determination method: Accurately weigh 0.01 g of D-glucose reference standard dried to constant weight, place it in a 100 mL volumetric flask, and prepare a 0.1 mg / mL standard solution, freshly prepared before use. Transfer 0, 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of glucose standard solution into test tubes, respectively, and add 1, 0.9, 0.8, 0.6, 0.4, 0.2, and 0 mL of distilled water to a final volume of 1 mL. Then add 1 mL of 3% phenol solution and 4 mL of concentrated sulfuric acid, shake well, and let stand for 30 min. Use a blank tube as a reference and measure the absorbance at a wavelength of 490 nm. Plot a standard curve with the glucose standard solution concentration on the x-axis and the absorbance value on the y-axis. Determination of soluble total sugar content in the sample: Accurately weigh 10 mg of crude polysaccharide sample, dilute to 10 mL with distilled water, and prepare a 1 mg / mL sample test solution. Follow the standard curve method described above and calculate the total sugar content in Bletilla striata polysaccharide based on the standard curve.

[0035] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0036] Example 1

[0037] A method for preparing Bletilla striata polysaccharide, comprising the following steps:

[0038] (1) Wash and slice the tubers of Bletilla striata, dry them, and then crush them;

[0039] (2) The pulverized Bletilla striata powder was subjected to steam explosion treatment, with the explosion pressure set at 1.8 MPa and the holding time at 60 s. The sample after explosion treatment was dried and pulverized.

[0040] (3) Weigh 10g of Bletilla striata powder after steam explosion, add 300mL of distilled water at a material-to-liquid ratio of 1:30g / mL, and extract by stirring at 80℃ for 2h. Centrifuge at 4500r / min for 15min to remove residue and take the supernatant.

[0041] (4) Add 15 μL / g thermostable α-amylase to the supernatant and keep it in a 95℃ water bath for 1 h; after cooling, add 1 μL / g saccharifying enzyme and keep it in a 60℃ water bath for 0.5 h; then add 15 mg / g protease and keep it in a 60℃ water bath for 1 h. After enzymatic hydrolysis, keep it in a 100℃ water bath for 15 min, cool, and centrifuge at 12000 r / min for 10 min to collect the supernatant for later use.

[0042] (5) Add 95% ethanol (V / V) to the supernatant until the ethanol concentration of the system reaches 80% (V / V), let stand for 12 h, and precipitate Bletilla striata polysaccharide. Centrifuge at 4500 r / min for 15 min to collect the precipitate, wash twice with anhydrous ethanol, centrifuge at 12000 r / min for 10 min to retain the precipitate, and redissolve it with water.

[0043] (6) The reconstituted liquid is placed in a -80°C freezer and then freeze-dried to obtain Bletilla striata polysaccharide. The freeze-drying temperature is -50°C, the time is 72 hours, and the vacuum degree is 10-12 Pa.

[0044] Example 2

[0045] A method for preparing Bletilla striata polysaccharide, comprising the following steps:

[0046] (1) Wash and slice the tubers of Bletilla striata, dry them, and then crush them;

[0047] (2) The pulverized Bletilla striata powder was subjected to steam explosion treatment, with the explosion pressure set at 1.2 MPa and the holding time at 60 s. The sample after explosion treatment was dried and pulverized.

[0048] (3) Weigh 10g of Bletilla striata powder after steam explosion, add 300mL of distilled water at a material-to-liquid ratio of 1:30g / mL, and extract by stirring at 80℃ for 2h. Centrifuge at 4500r / min for 15min to remove residue and take the supernatant.

[0049] (4) Add 15 μL / g thermostable α-amylase to the supernatant and keep it in a 95℃ water bath for 1 h; after cooling, add 1 μL / g saccharifying enzyme and keep it in a 60℃ water bath for 0.5 h; then add 15 mg / g protease and keep it in a 60℃ water bath for 1 h. After enzymatic hydrolysis, keep it in a 100℃ water bath for 15 min, cool, and centrifuge at 12000 r / min for 10 min to collect the supernatant for later use.

[0050] (5) Add 95% ethanol (V / V) to the supernatant until the ethanol concentration of the system reaches 80% (V / V), let stand for 12 h, and precipitate Bletilla striata polysaccharide. Centrifuge at 4500 r / min for 15 min to collect the precipitate, wash twice with anhydrous ethanol, centrifuge at 12000 r / min for 10 min to retain the precipitate, and redissolve it with water.

[0051] (6) The reconstituted liquid is placed in a -80°C freezer and then freeze-dried to obtain Bletilla striata polysaccharide. The freeze-drying temperature is -50°C, the time is 72 hours, and the vacuum degree is 10-12 Pa.

[0052] Example 3

[0053] A method for preparing Bletilla striata polysaccharide, comprising the following steps:

[0054] (1) Wash and slice the tubers of Bletilla striata, dry them, and then crush them;

[0055] (2) The pulverized Bletilla striata powder was subjected to steam explosion treatment, with the explosion pressure set at 0.6 MPa and the holding time at 60 s. The sample after explosion treatment was dried and pulverized.

[0056] (3) Weigh 10g of Bletilla striata powder after steam explosion, add 300mL of distilled water at a material-to-liquid ratio of 1:30g / mL, and extract by stirring at 80℃ for 2h. Centrifuge at 4500r / min for 15min to remove residue and take the supernatant.

[0057] (4) Add 15 μL / g thermostable α-amylase to the supernatant and keep it in a 95℃ water bath for 1 h; after cooling, add 1 μL / g saccharifying enzyme and keep it in a 60℃ water bath for 0.5 h; then add 15 mg / g protease and keep it in a 60℃ water bath for 1 h. After enzymatic hydrolysis, keep it in a 100℃ water bath for 15 min, cool, and centrifuge at 12000 r / min for 10 min to collect the supernatant for later use.

[0058] (5) Add 95% ethanol (V / V) to the supernatant until the ethanol concentration of the system reaches 80% (V / V), let stand for 12 h, and precipitate Bletilla striata polysaccharide. Centrifuge at 4500 r / min for 15 min to collect the precipitate, wash twice with anhydrous ethanol, centrifuge at 12000 r / min for 10 min to retain the precipitate, and redissolve it with water.

[0059] (6) The reconstituted liquid is placed in a -80°C freezer and then freeze-dried to obtain Bletilla striata polysaccharide. The freeze-drying temperature is -50°C, the time is 72 hours, and the vacuum degree is 10-12 Pa.

[0060] Comparative Example 1

[0061] A method for preparing Bletilla striata polysaccharide, comprising the following steps:

[0062] (1) Wash and slice the tubers of Bletilla striata, dry them, and then crush them;

[0063] (2) Weigh 10g of the pulverized Bletilla striata powder, add 300mL of distilled water at a material-to-liquid ratio of 1:30g / mL, and extract by stirring at 80℃ for 2h. Centrifuge at 4500r / min for 15min to remove the residue and take the supernatant.

[0064] (3) Add 15 μL / g thermostable α-amylase to the supernatant and keep it in a 95℃ water bath for 1 h; after cooling, add 1 μL / g saccharifying enzyme and keep it in a 60℃ water bath for 0.5 h; then add 15 mg / g protease and keep it in a 60℃ water bath for 1 h. After enzymatic hydrolysis, keep it in a 100℃ water bath for 15 min, cool, and centrifuge at 12000 r / min for 10 min to collect the supernatant for later use.

[0065] (4) Add 95% ethanol (V / V) to the supernatant until the ethanol concentration of the system reaches 80% (V / V), let stand for 12 h, and precipitate Bletilla striata polysaccharide. Centrifuge at 4500 r / min for 15 min to collect the precipitate, wash twice with anhydrous ethanol, centrifuge at 12000 r / min for 10 min to retain the precipitate, and redissolve it with water.

[0066] (5) The reconstituted liquid is placed in a -80°C freezer and then freeze-dried to obtain Bletilla striata polysaccharide. The freeze-drying temperature is -50°C, the time is 72 hours, and the vacuum degree is 10-12 Pa.

[0067] Experimental results:

[0068] like Figure 1As shown, the yield of Bletilla striata polysaccharide in the hot water extraction group was 22.56±2.47%, while the yield was 31.40±0.19% when the steam explosion pressure was 1.8 MPa, which was 1.39 times that of the hot water extraction group. Furthermore, among the three steam explosion treatments, the polysaccharide yield at an explosion pressure of 1.8 MPa was higher than that of the other two groups.

[0069] like Figure 2 As shown, the polysaccharide content of Bletilla striata obtained by the four methods is all around 80%, which is relatively high.

[0070] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing Bletilla striata polysaccharide, characterized in that, Includes the following steps: (1) Wash and slice the tubers of Bletilla striata, dry and pulverize them, then steam-explode them, and dry and pulverize them after treatment. (2) The powder of Bletilla striata after steam explosion was extracted with hot water, and the supernatant was retained. The heat-resistant α-amylase, saccharifying enzyme and protease were added in sequence to inactivate the enzymes and the supernatant was taken. (3) The supernatant was precipitated with alcohol, and the precipitate was washed with anhydrous ethanol. The precipitate was redissolved with water and then freeze-dried to obtain Bletilla striata polysaccharide.

2. The method for preparing Bletilla striata polysaccharide according to claim 1, characterized in that, The pressure of the steam explosion treatment in step (1) is 1.8 MPa, and the pressure holding time is 60 s.

3. The method for preparing Bletilla striata polysaccharide according to claim 1, characterized in that, In step (2), the material-to-liquid ratio for hot water extraction is 1:30 g / mL, the extraction temperature is 80℃, and the stirring extraction time is 2 hours.

4. The method for preparing Bletilla striata polysaccharide according to claim 1, characterized in that, In step (2), the amount of heat-resistant α-amylase added is 15 μL / g, the water bath temperature is 95℃, and the water bath time is 1h; the amount of saccharifying enzyme added is 1 μL / g, the water bath temperature is 60℃, and the water bath time is 0.5h; and the amount of protease added is 15 mg / g, the water bath temperature is 60℃, and the water bath time is 1h.

5. The method for preparing Bletilla striata polysaccharide according to claim 1, characterized in that, The enzyme inactivation temperature in step (2) is 100℃ and the enzyme inactivation time is 15min.

6. The method for preparing Bletilla striata polysaccharide according to claim 1, characterized in that, The alcohol precipitation time in step (3) is 12 hours.

7. The method for preparing Bletilla striata polysaccharide according to claim 1, characterized in that, The freeze-drying process in step (3) is carried out at a temperature of -50°C for 72 hours and a vacuum degree of 10-12 Pa.

8. The Bletilla striata polysaccharide prepared by the method according to any one of claims 1-7.

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