Preparation process of water-soluble paramoecium beta-glucan by enzymatic method

Abstract

The application provides a preparation process of water-soluble paramylon beta-glucan prepared by enzymatic hydrolysis, and the specific steps of the process include the pretreatment of paramylon and the enzymatic hydrolysis reaction of paramylon and the like. In the application, multiple enzymes are used to jointly perform the enzymatic hydrolysis reaction, and key conditions such as the material-liquid ratio, the enzymatic hydrolysis time and the enzymatic hydrolysis temperature in the enzymatic hydrolysis reaction are controlled, so that the prepared water-soluble paramylon beta-glucan has high purity and low protein content, and the total sugar content of the water-soluble paramylon beta-glucan can reach more than 60%. In the application, the paramylon beta-glucan is prepared by using the enzymatic hydrolysis method, the reaction condition is mild, a large amount of organic reagent is avoided, safety and environmental protection are achieved, the preparation process is simple, and the production cost is low.

Description

Technical Field

[0001] This invention relates to the field of polysaccharide extraction technology, specifically to a preparation process for water-soluble Euglena β-glucan by enzymatic hydrolysis. Background Technology

[0002] Euglena gracilis, also known as Euglena or Green Euglena, is a single-celled eukaryotic organism intermediate between animals and plants, capable of photoautotrophic, polytrophic, and heterotrophic cultivation. Euglena is rich in nutrients, especially Euglena polysaccharide, a polysaccharide composed of linear β-1,3-glucan, which is a highly efficient energy storage substance within Euglena cells. Due to its porous structure, Euglena polysaccharide has excellent adsorption properties, capable of adsorbing and expelling excess substances such as cholesterol, triglycerides, heavy metals, and alcohol from the body, while also promoting the activity of beneficial enzymes and regulating gastrointestinal function.

[0003] Euglena polysaccharides are composed of a triple helix structure formed by linear β-1,3-glucan. They are insoluble in water and weakly alkaline to acidic solutions, but soluble in strongly alkaline solutions. Once the pH is adjusted to weakly alkaline or acidic, a gel-like precipitate forms. The solubility of the polysaccharide is crucial to its usability in injectable solutions, skin lotions, etc. The inability to obtain pH-independent soluble Euglena β-glucan is a key factor limiting its application in cosmetics, pharmaceuticals, and other fields. In existing technologies, the preparation of water-soluble Euglena β-glucan mainly utilizes the ability of chemical reagents to dissolve Euglena polysaccharides. Continuous stirring at high temperatures followed by high-pressure microfluidics alters the original structure of the polysaccharide, significantly increasing its hydrolysis rate. However, high-pressure microfluidics are energy-intensive and have low flow rates, limiting the large-scale application of this method. Furthermore, the introduction of organic reagents may have toxic effects on the body, causing consumer concerns about adverse effects during selection. Therefore, a green and safe preparation method is urgently needed to improve production efficiency while alleviating consumer concerns. Summary of the Invention

[0004] To address the aforementioned needs, this invention provides a process for preparing water-soluble Euglena β-glucan via enzymatic hydrolysis. The process employs a composite enzyme hydrolysis technique to extract water-soluble Euglena β-glucan, thereby improving the extraction rate and purity, and laying the foundation for further separation, purification, and structural analysis.

[0005] This invention provides a process for preparing water-soluble Euglena β-glucan via enzymatic hydrolysis. The specific process steps of the enzymatic hydrolysis method for preparing water-soluble Euglena β-glucan are as follows:

[0006] Step S1: Pretreatment of Euglena polysaccharide: Euglena polysaccharide is subjected to SDS heating treatment, washed with water multiple times, dried, pulverized, passed through a 40-80 mesh sieve, and stored for later use.

[0007] Step S2: Enzymatic hydrolysis: The pretreated Euglena polysaccharide was placed in a reactor and treated with alkali at 20–40°C for 3 hours. After adjusting the pH to 5–7, the enzyme was added. After the enzymatic hydrolysis was completed, the supernatant was collected by centrifugation, and the hydrolysis rate was measured. The supernatant was frozen for 4 hours and then freeze-dried to obtain water-soluble Euglena β-1,3-glucan powder.

[0008] To effectively remove proteins from Euglena polysaccharide, the preferred step in step S1, which involves adding SDS for heating, is as follows: Euglena polysaccharide is added to an aqueous solution containing 1-3% SDS, heated at 60-90°C for 2-5 hours, filtered to separate the liquid and precipitate, and the precipitate is repeatedly washed with water until the Euglena polysaccharide turns white, then filtered and dried.

[0009] In the pretreatment step, the present invention involves heating followed by multiple rinsing with clean water, which effectively removes residual SDS, oils, and other water-soluble substances from Euglena polysaccharides, facilitating subsequent full enzymatic hydrolysis and improving extraction purity.

[0010] Preferably, in step S2, the amount of Euglena polysaccharide added to the water is 2-4%. In production, if the material-to-liquid ratio is too high, the subsequent concentration process will significantly increase in time and energy consumption, and losses will also increase further, leading to a decrease in yield.

[0011] Preferably, in step S2, the alkaline reagent is one or a mixture of sodium hydroxide, potassium hydroxide, and barium hydroxide.

[0012] To further improve the yield of water-soluble Euglena β-glucan, preferably, in step S2, the amount of alkaline reagent used is 10-30 g / L, as a strongly alkaline solution is beneficial for the swelling of Euglena polysaccharides.

[0013] Preferably, in step S2, the reaction enzyme is one or a mixture of several of β-glucanase, hemicellulase, and xylanase.

[0014] To further improve the yield of water-soluble Euglena β-glucan, preferably, in step S2, the proportion of β-glucanase, hemicellulase, and xylanase used is 2-8%: 1-3%: 2-7%.

[0015] Preferably, in step S2, the enzymatic hydrolysis reaction conditions are: hydrolysis time of 1-5 hours, hydrolysis temperature of 30-60°C, and hydrolysis pH of 4-7.

[0016] Preferably, in step S2, the freeze-drying is vacuum freeze-drying, and the specific drying conditions are: vacuum degree < 20 Pa, temperature -60℃, and drying time 12-26 h.

[0017] Unless otherwise specified, all raw materials and equipment used in this application are commonly used in the field and are commercially available products. Unless otherwise specified, all methods used in this application are conventional methods in the field.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] (1) This invention uses an enzymatic hydrolysis method to prepare water-soluble Euglena β-glucan. The reaction conditions are mild, avoiding the use of a large amount of organic reagents. It is safe and environmentally friendly, and the preparation process is simple and the production cost is low.

[0020] (2) This invention uses multiple enzymes to carry out enzymatic hydrolysis and controls key conditions such as the material-liquid ratio, reaction enzymes, hydrolysis time, and hydrolysis temperature in the enzymatic hydrolysis reaction. The resulting water-soluble Euglena β-glucan has high purity, low protein content, and a total sugar content of over 60%. Attached Figure Description

[0021] Figure 1 This is a glucose standard curve for testing polysaccharide content using the phenol-sulfuric acid method according to the present invention.

[0022] Figure 2 This is a protein standard curve for determining protein content using the BCA method according to the present invention. Detailed Implementation

[0023] To help those skilled in the art better grasp the technical details of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below. It should be understood that these described embodiments do not represent all possible implementations of the present invention. Based on these embodiments, any other implementations that can be conceived by those skilled in the art without innovative work should be considered within the scope of protection of the present invention.

[0024] The Euglena gracilis strain used in this embodiment is the mutant strain ZWang109, which was deposited on June 28, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCCNo.40684.

[0025] Example 1

[0026] This embodiment provides a process for preparing water-soluble Euglena β-glucan via enzymatic hydrolysis, the specific steps of which are as follows:

[0027] Step S1: Pretreatment of Euglena polysaccharide: Add Euglena polysaccharide to an aqueous solution containing 2% SDS, heat at 85°C for 3 hours, filter to separate the material and liquid, wash the precipitate repeatedly with water until the Euglena polysaccharide turns white, filter and dry.

[0028] Step S2: Enzymatic hydrolysis: The pretreated Euglena polysaccharide was placed in a reactor, and a 20 g / L sodium hydroxide alkaline solution was added. The mixture was treated at 40°C for 3 hours. After adjusting the pH to 5.3 with glacial acetic acid, the following enzymes were added: β-glucanase (5% of the solution), hemicellulase (1.5%), and xylanase (4.5%). The hydrolysis time was 3 hours, the hydrolysis temperature was 45°C, and the hydrolysis pH was 5.5. After hydrolysis, the supernatant was collected by centrifugation. The polysaccharide hydrolysis rate was determined using the phenol-sulfuric acid method. The supernatant was frozen for 4 hours and then subjected to vacuum freeze-drying. The specific conditions for vacuum freeze-drying were: vacuum degree < 20 Pa, temperature -60°C, and drying time 24 hours. Water-soluble Euglena β-glucan powder was obtained after freeze-drying.

[0029] Example 2

[0030] This embodiment provides a process for preparing water-soluble Euglena β-glucan by enzymatic hydrolysis. The specific steps are the same as in Example 1, except that the concentration of sodium hydroxide in the enzymatic hydrolysis reaction of step S2 is 15 g / L.

[0031] Example 3

[0032] This embodiment provides a process for preparing water-soluble Euglena β-glucan by enzymatic hydrolysis. The specific steps are the same as in Example 1, except that the sodium hydroxide treatment time in step S2 is 1 hour.

[0033] Example 4

[0034] This embodiment provides a process for preparing water-soluble Euglena β-glucan by enzymatic hydrolysis. The specific steps are the same as in Example 1. The difference from Example 1 is that in the enzymatic hydrolysis reaction of step S2, glacial acetic acid is used to adjust the pH to 6.5.

[0035] Example 5

[0036] This embodiment provides a preparation process for water-soluble Euglena β-glucan by enzymatic hydrolysis. The specific steps are the same as in Embodiment 1. The difference from Embodiment 1 is that in the enzymatic hydrolysis reaction of step S2, the ratio of β-glucanase to the liquid is 4%, the ratio of hemicellulase to the liquid is 1%, and the ratio of xylanase to the liquid is 3%.

[0037] Example 6

[0038] This embodiment provides a process for preparing water-soluble Euglena β-glucan by enzymatic hydrolysis. The specific steps are the same as in Example 1, except that the enzymatic hydrolysis temperature in step S2 is 35°C.

[0039] Comparative Example 1

[0040] This comparative example provides a process for preparing water-soluble Euglena β-glucan by enzymatic hydrolysis. The specific steps are the same as in Example 1. The difference from Example 1 is that in the enzymatic hydrolysis reaction in step S2, the reaction enzymes are β-glucanase and hemicellulase. The material-to-liquid ratio of β-glucanase is 5%, and the material-to-liquid ratio of hemicellulase is 1.5%.

[0041] Comparative Example 2

[0042] This comparative example provides a process for preparing water-soluble Euglena β-glucan by enzymatic hydrolysis. The specific steps are the same as in Example 1. The difference from Example 1 is that in the enzymatic hydrolysis reaction of step S2, the reaction enzymes are hemicellulase and xylanase, and the ratio of hemicellulase to liquid is 1.5% and the ratio of xylanase to liquid is 4.5%.

[0043] Comparative Example 3

[0044] This comparative example provides a process for preparing water-soluble Euglena β-glucan by enzymatic hydrolysis. The specific steps are the same as in Example 1. The difference from Example 1 is that in the enzymatic hydrolysis reaction in step S2, the reaction enzymes are β-glucanase and xylanase, and the material-to-liquid ratio of β-glucanase is 5% and the material-to-liquid ratio of xylanase is 4.5%.

[0045] Comparative Example 4

[0046] This comparative example provides a process for preparing water-soluble Euglena β-glucan by enzymatic hydrolysis. The specific steps are the same as in Example 1. The difference from Example 1 is that in the enzymatic hydrolysis reaction of step S2, the reaction enzyme is β-glucanase, and the material-to-liquid ratio of β-glucanase is 11%.

[0047] Performance testing

[0048] 1. Determination of the yield of water-soluble Euglena β-glucan

[0049] The formula for calculating the water-soluble Euglena β-glucan (Y%) is as follows:

[0050] Y% = M1 / M0 × 100% (1)

[0051] (M1: Mass of water-soluble Euglena β-glucan; M0: Mass of Euglena polysaccharide after pretreatment)

[0052] 2. Determination of total sugar content

[0053] The total sugar content was determined using the phenol-sulfuric acid method.

[0054] The determination method is as follows:

[0055] Take 2.00 mL of the sample to be tested into a test tube, add 6.00 g of colorimetric solution, shake well, let stand at room temperature for 10 min, then incubate in a 40°C water bath for 20 min, followed by an ice bath for 3 min, and cool to room temperature. Measure the absorbance at 490 nm.

[0056] Glucose standard curve determination (see curve graph) Figure 2 Take 0, 20, 40, 60, 80, and 100 mg / L glucose solutions respectively and place them in test tubes. Add 2 mL of each solution to 6 mL of colorimetric reagent, shake well, let stand at room temperature for 10 min, then incubate in a 40℃ water bath for 20 min, followed by an ice bath for 3 min, and cool to room temperature. Measure the absorbance at 490 nm.

[0057] 3. Protein content determination

[0058] The BCA method was used to detect the amount of residual protein in the sample.

[0059] (1) Preparation of protein standards:

[0060] Add 0.8 mL of protein standard preparation solution to one tube of protein standard (20 mg BSA), dissolve thoroughly to prepare a 25 mg / mL protein standard solution, and set aside. Take 20 mL of the 25 mg / mL protein standard and dilute with PBS buffer to a final concentration of 0.5 mg / mL.

[0061] (2) Preparation of BCA working solution:

[0062] Prepare an appropriate amount of BCA working solution by mixing 50 mL of BCA reagent A with 1 mL of BCA reagent B (50:1) and mixing thoroughly. Take 20 mL of BCA reagent A and add 0.4 mL of BCA reagent B, mix well, and prepare the BCA working solution. The BCA working solution is stable at room temperature for 24 hours.

[0063] (3) Plotting the protein standard curve:

[0064] Add standards in increments of 0, 10, 20, 40, 80, 120, 160, and 200 μL to test tubes, and then add PBS solution to bring the volume to 200 μL. Add an appropriate volume of sample to the test tube. If the sample volume is less than 200 μL, add PBS solution to bring the volume to 200 μL. Record the sample volume. Add 2 mL of BCA working solution to each well and incubate at 37°C for 30 min. Measure the absorbance at wavelength A562 using a spectrophotometer; plot the protein standard curve based on the absorbance and protein concentration.

[0065] (4) Sample testing:

[0066] Take an appropriate amount of sample, use PBS as a buffer, repeat the above standard curve plotting process to detect the protein content in the sample, and input the absorbance value into the standard curve to calculate the protein concentration.

[0067] The total sugar content and protein content of water-soluble Euglena β-glucan from Examples 1-6 and Comparative Examples 1-4 were determined as shown in Table 1 below:

[0068] Table 1

[0069]

[0070]

[0071] Based on the polysaccharide yield and total sugar content data in Table 1, it can be seen that the water-soluble Euglena β-glucan prepared in Example 1 has the characteristics of high polysaccharide yield and high total sugar content. In Comparative Examples 2-6, the pH, time, enzymatic hydrolysis time, pH, and temperature of alkali swelling were adjusted respectively. This shows that changes in experimental conditions all affect the final polysaccharide yield. Among them, the pH and time of alkali swelling have a relatively small impact on the polysaccharide yield, while the enzymatic hydrolysis time, pH, and temperature have a significant impact. In Comparative Examples 1-4, the enzyme dosage was adjusted respectively. The experimental results showed that changes in enzyme dosage greatly affect the polysaccharide yield, and only when all three enzymes are used in combination does the best effect be achieved.

[0072] In this invention, the polysaccharides obtained in Example 1 were analyzed for their in vitro moisturizing and antioxidant properties. Water-soluble Euglena β-glucan exhibited good moisturizing properties. In vitro antioxidant experiments showed that water-soluble Euglena β-glucan had a good free radical scavenging rate; according to the above experimental methods, water-soluble Euglena β-glucan showed a free radical scavenging rate of approximately 94.76%.

[0073] Finally, it should be noted that although the present invention has been described in detail by way of preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, those skilled in the art can make various equivalent modifications and substitutions to the embodiments of the present invention, and such modifications and substitutions should all be within the scope of the present invention. Any variations and substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should also be covered within the protection scope of the present invention.

Claims

1. A method for preparing water-soluble Euglena β-glucan, characterized in that, The specific steps of the preparation method are as follows: Step S1: Add Euglena polysaccharide to an aqueous solution containing 2% SDS, heat at 85°C for 3 hours, filter to separate the material and liquid, wash the precipitate repeatedly with water until the Euglena polysaccharide turns white, filter and dry. Step S2: Place the pretreated Euglena polysaccharide from Step S1 into a reactor, add a 20 g / L sodium hydroxide alkaline solution, treat at 40°C for 3 h, adjust the pH to 5.3 with glacial acetic acid, and then add the reaction enzymes: β-glucanase at a feed-to-liquid ratio of 5%, hemicellulase at 1.5%, and xylanase at 4.5%; the enzymatic hydrolysis time is 3 h, the hydrolysis temperature is 45°C, and the hydrolysis pH is 5.5; after the enzymatic hydrolysis is completed, centrifuge and collect the supernatant, use the phenol-sulfuric acid method to detect the polysaccharide hydrolysis rate, freeze the supernatant for 4 h, and then perform vacuum freeze-drying to obtain water-soluble Euglena β-glucan powder.

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