Efficient and green extraction method of plant marine polysaccharide based on superfine grinding-enzymolysis coupling ultrasound

Abstract

Belonging to the technical field of polysaccharide extraction, the invention discloses a botanical marine polysaccharide efficient green extraction method, which comprises: (1) carrying out superfine grinding on a seaweed raw material until the particle size is less than or equal to 50 [mu] m; (2) carrying out enzymolysis wall breaking by adopting a cellulase / xylanase composite system; (3) carrying out low-frequency ultrasonic-assisted extraction (40kHz, 300 to 500W); and (4) membrane separation and purification (the molecular weight cut-off is 5-100 kDa). According to the method, a three-stage physical-biological synergistic wall breaking technology is innovatively adopted, the dependence of an organic solvent adopted in a traditional process is broken through, no organic solvent and no ethanol addition are achieved in the whole process, the polysaccharide yield is increased to 38.7% + / -1.2% (kelp) and 32.5% + / -0.8% (asparagus), the extraction time is shortened to 45 min, and the energy consumption is reduced by 40%.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of marine bioactive ingredient extraction, and in particular to a high-efficiency green extraction process for plant marine polysaccharides using ultrafine grinding pretreatment, combined enzymatic hydrolysis and low-frequency ultrasonic synergy. BACKGROUND

[0002] Marine plant polysaccharides are a class of natural high-molecular compounds with various biological activities extracted from marine plants (such as kelp brown algal gum and dragon's whisker carrageenan). Due to their anti-tumor, hypoglycemic and other biological activities, they have wide application prospects in the fields of medicine, functional food and cosmetics.

[0003] The extraction and purification methods of marine polysaccharides mainly include hot water extraction, acid and alkali extraction, enzymatic hydrolysis, ultrasonic-assisted extraction, etc. However, these traditional extraction processes face three dilemmas. 1. Efficiency bottleneck: hot water extraction requires long-time high-temperature (80-100℃) boiling for 6-8 hours, which leads to the degradation of heat-sensitive polysaccharides. For example, the sulfuric acid group of kelp polysaccharides breaks at >60℃, resulting in a loss of activity of more than 40%. 2. Environmental protection defects: solvent precipitation method relies on a large amount of ethanol (solid-liquid ratio 1:3), which generates 5 tons of alcohol-containing wastewater per ton of raw material, with a COD value of more than 8000 mg / L, and the treatment cost accounts for 35% of the total production cost. 3. Technical limitations: single ultrasonic extraction has insufficient penetration for dense cell walls (such as kelp epidermis), with a yield of only 18-25%; although the enzymatic-hydrosonic synergistic process improves the efficiency, the enzymatic hydrolysis temperature of 55℃ still causes the denaturation of β-glucan; the subsequent purification relies on acid / alkali treatment, which destroys the integrity of the polysaccharide molecular structure. At the same time, the existing methods generally have the problems of complicated purification steps, high cost and difficulty in realizing industrial production.

[0004] There are many literatures and patents related to plant polysaccharides, most of which use traditional methods. For example: Chinese patent CN119930858A discloses a plant polysaccharide extract and its preparation method and application. The patent uses a composite acid solution composed of acetic acid and oxalic acid for pretreatment, and uses a low-melting solvent for efficient extraction, and cooperates with modified activated carbon for impurity removal treatment, to achieve efficient acquisition of plant polysaccharide extract and improve the extraction rate and purity of the extract.

[0005] Chinese patent CN119371567A discloses an extraction method of plant polysaccharides and plant polysaccharides. The method uses enzymatic hydrolysis under the stimulation of a micro-DC electric field, which can significantly improve the extraction rate of plant polysaccharides and ensure the activity of plant polysaccharides.

[0006] The Chinese patent CN119060214A uses ultrasonic treatment to extract polysaccharides from eucommia leaf dry powder, which has high extraction efficiency and good effect, obtains plant crude polysaccharides, then dissolves the plant crude polysaccharides, and uses a purification adsorbent to adsorb the plant crude polysaccharide solution. The adsorption of the purification adsorbent has excellent impurity removal effect and is easy to use, improves the efficiency of extracting plant polysaccharides, and fully removes impurities in the plant crude polysaccharides, with almost no loss of polysaccharides, and improves the quality of the extracted plant polysaccharides.

[0007] Therefore, there is currently no method that can achieve efficient cell wall breaking and active ingredient preservation through a physical crushing-biological enzymatic hydrolysis-acoustic cavitation release three-level synergistic mechanism. Therefore, it is of great practical significance to develop an efficient, environmentally friendly, low-cost extraction and purification method that can simultaneously ensure high extraction rate and high purity of marine polysaccharides. SUMMARY

[0008] To address the deficiencies of the prior art, the present invention aims to provide a marine polysaccharide extraction and purification method with high extraction efficiency, good product purity, and green process. Specifically, a three-level physical-biological synergistic breaking technology is used to achieve efficient cell wall breaking and active ingredient preservation through a physical crushing-biological enzymatic hydrolysis-acoustic cavitation release three-level synergistic mechanism.

[0009] For the first time, the present invention provides an application of a three-level physical-biological synergistic breaking technology in the extraction of plant polysaccharides.

[0010] Specifically, the three-level physical-biological synergistic breaking technology described in the present invention is a combination of enzymatic hydrolysis, low-frequency ultrasonic-assisted extraction, and membrane separation and purification technology.

[0011] Preferably, the enzymatic hydrolysis uses a cellulase / xylanase complex system for enzymatic hydrolysis.

[0012] Preferably, the low-frequency ultrasonic-assisted extraction conditions are 40 kHz and 300-500 W.

[0013] Preferably, the membrane separation and purification has a molecular weight cutoff range of 5-100 kDa.

[0014] Furthermore, using the above-mentioned synergistic technology, the present invention also provides a high-efficiency green extraction method for plant marine polysaccharides based on ultramicro-pulverization-enzymatic hydrolysis coupling ultrasonic, which specifically includes the following detailed steps: (a) Raw material pretreatment: after cleaning the seaweed, it is cryogenically pulverized to a particle size of 10-50 μm in a liquid nitrogen environment; In this step, the ultramicro-pulverization technology is used, specifically the liquid nitrogen cryogenic (-40℃) cell wall embrittlement, and the mechanical shearing force reduces the particle size to 10-50 μm, increasing the specific surface area by 50 times and breaking the macroscopic diffusion barrier; (b) Complex enzymatic hydrolysis: the crushed raw material is mixed with a complex enzyme solution, the complex enzyme comprises cellulase 2.0-3.5 U / mg of raw material, xylanase 1.5-2.5 U / mg of raw material, and the reaction is carried out at pH 4.5-5.5 and temperature 35-40℃ for 30 min; This step is to use low-temperature enzymatic hydrolysis technology, and the complex enzyme system specifically hydrolyzes the cellulose / hemicellulose network at 35-40℃ to dissolve the cell wall skeleton; (c) Ultrasonic extraction: after enzymatic hydrolysis, the material is added with water at a solid-liquid ratio of 1:15-1:25, and ultrasonic treatment is carried out at a frequency of 40 kHz±5 kHz and a power density of 80-120 W / L for 20-30 min, during which the pulse mode is working for 5 s and intermittent for 2 s, and the temperature is controlled to be ≤40℃. This step is pulse ultrasonic, which is 40 kHz low-frequency ultrasonic, and can promote the dissolution of polysaccharide. The pulse mode avoids local overheating.

[0015] (d) Membrane separation and purification: sequentially pass through 0.22 μm microfiltration membrane to remove impurities, ultrafiltration membrane to cut off polysaccharides with a target molecular weight, nanofiltration membrane to desalt and concentrate, and finally dry to obtain the finished product.

[0016] Further, the above step (a) cryogenic crushing is carried out at-40℃ to-50℃, and the amount of liquid nitrogen is 1.5-2 times the mass of the raw material.

[0017] Further, the above step (b) complex enzymatic hydrolysis additionally adds lysozyme 0.8-1.2 U / mg of raw material for hydrolysis of β-1, 3-glucan.

[0018] Further, the above step (d) the ultrafiltration membrane cuts off the molecular weight of 5-100 kDa; further, the components of 10-50 kDa are preferentially selected for the development of blood glucose-lowering products.

[0019] The key steps involved in the extraction method of the present application are: 1. Raw material pretreatment Select brown algae (kelp, kelp) or red algae (long hair, seaweed), and after acceptance, cleaning and drying, store for use or crush. Ultrafine crushing uses a liquid nitrogen cryogenic crusher, and the control temperature is ≤-40℃. After crushing, the particle size reaches 10-50 μm, which is beneficial to break through the physical barrier of the cell wall and improve the polysaccharide extraction rate.

[0020] 2. Complex enzymatic hydrolysis Prepare a complex enzyme system (cellulase: 2.0-3.5 U / mg of raw material, xylanase: 1.5-2.5 U / mg of raw material, lysozyme: 0.8-1.2 U / mg of raw material), and control the enzymatic hydrolysis conditions: pH 4.5-5.5, temperature 35-40℃ (low-temperature protection of activity), time 30 min.

[0021] 3. Low frequency ultrasound extraction Low frequency ultrasound enhances the extraction rate of polysaccharides. The operating conditions are: frequency 40 kHz ± 5 kHz, power 80-120 W / L, prevent local overheating by pulse mode (work 5 s / intermittent 2 s), cooperate with circulating water cooling system to control the extraction temperature to 35℃ ± 2℃, ultrasonic extraction time: 20-30 min, polysaccharide dissolution rate > 95%.

[0022] 4. Membrane separation and purification Polysaccharide separation and purification adopts ceramic membrane-roll type membrane combination: first stage: 0.22 μm microfiltration membrane removes impurities; second stage: 50 kDa ultrafiltration membrane retains target polysaccharide; third stage: 5 kDa nanofiltration membrane desalination and concentration.

[0023] 5. Drying The drying method adopts spray drying or true freeze drying.

[0024] Spray drying: inlet air temperature 170℃, outlet air temperature 80℃, feed rate 10 mL / min, atomization pressure 0.3 MPa. Freeze drying: temperature -40 to -50℃, vacuum degree 10-20P, freeze drying for 12-24 hours.

[0025] Advantages of the present application: The present application innovatively adopts three-stage physical-biological synergistic wall breaking technology, breaks through the dependence on organic solvents in traditional process, realizes the whole process without organic solvents and zero ethanol addition, the polysaccharide yield is increased to 38.7% ± 1.2% (kelp) and 32.5% ± 0.8% (gracilaria), the extraction time is shortened to 45 min, and the energy consumption is reduced by 40%. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 is the process step diagram of the present application.

[0027] Figure 2 is the raw material state diagram after ultrafine grinding.

[0028] Figure 3 is one kind of marine crude polysaccharide (3a) after extraction and marine polysaccharide (3b) after purification.

[0029] Figure 4 is the polysaccharide molecular weight distribution diagram (HPSEC method) after purification.

[0030] Figure 5 is the α-glucosidase inhibition rate curve. DETAILED DESCRIPTION

[0031] The specific embodiments of the present application are further described below, and it is to be understood that the description is used to help understand the present application and does not constitute a limitation of the present application. In addition, the technical features involved in the various example embodiments of the present application described below can be combined with each other as long as they do not conflict with each other.

[0032] Example 1: Efficient and green extraction method for kelp polysaccharide extraction (targeted extraction of hypoglycemic active components) 1. Raw material processing: Take dry kelp, rinse with ultrapure water after removing sand and stones.

[0033] 2. Liquid nitrogen cryogenic grinding: In a -40℃ liquid nitrogen environment, use an air flow grinder to process to D50=32.5μm (measured by Malvern particle size analyzer Mastersizer 3000).

[0034] 3. Enzymatic hydrolysis: Prepare a composite enzyme solution: cellulase 3.0 U / mg raw material, xylanase 2.0 U / mg raw material in pH 5.0 citric acid buffer, solid-liquid ratio 1:15, 38℃ oscillation reaction for 30min.

[0035] 4. Ultrasonic enhanced extraction: Ultrasonic frequency 40kHz ± 2kHz, power density: 100 W / L (based on solution volume), pulse mode: work 5s / intermittent 2s, internal cooling coil temperature control 35℃ ± 0.5℃, continuous ultrasonic 25min, centrifugation (8000rpm, 10min) to take the supernatant.

[0036] 5. Membrane separation and purification: First, 0.22μm ceramic membrane filtration to remove impurities, operating pressure 0.2MPa; then 50kDa polyether sulfone roll membrane ultrafiltration enrichment, intercept target polysaccharide (10-50kDa hypoglycemic active components); finally, nanofiltration desalination, 5kDa nanofiltration membrane to remove ions, concentrated to solid content 15%.

[0037] 6. Freeze-drying. Vacuum freeze-drying at -50℃ to obtain porous polysaccharide powder.

[0038] 7. Result verification:

[0039] Example 2: Efficient and green extraction method for Gracilaria duraucal extract 1. Raw material processing: manually remove shell impurities, rinse 3 times with ultrapure water.

[0040] 2. Liquid nitrogen cryogenic grinding: liquid nitrogen temperature -45℃, feed rate 200g / min, control the particle size after grinding to below 30μm (measured by Malvern particle size analyzer Mastersizer 3000).

[0041] 3. Enzymatic lysis of cell wall: the complex enzyme system is: xylanase 2.5 U / mg raw material, cellulase: 1.8 U / mg raw material, beta-glucanase: 0.5 U / mg raw material, in 0.1 mol / L acetic acid-sodium acetate buffer (pH 5.2) at pH 5.2, with solid-liquid ratio 1:18 (w / v) in 38℃ constant temperature shaking water bath for 30 min.

[0042] 4. Ultrasonic extraction: Ultrasonic enhanced extraction: after enzymolysis, the material is directly ultrasonically treated without solid-liquid separation. Ultrasonic frequency 40kHz±1kHz, pulse mode 38℃±0.5℃, continuous ultrasonic treatment for 20min, centrifugation (8000rpm, 10min) to take the supernatant.

[0043] 5. Membrane separation and purification: first, 0.22μm ceramic membrane filtration to remove impurities, transmembrane pressure 0.15MPa; then 300kDa polyether sulfone membrane ultrafiltration enrichment, cut off target carrageenan (200-500kDa), finally use 2kDa nanofiltration membrane to remove salt, concentrated to solid content 18%.

[0044] 6. Drying: spray drying is used, inlet air temperature 160℃, outlet air temperature 75℃, white carrageenan polysaccharide powder is obtained.

[0045] 7. Result verification:

[0046] The embodiments of the present application are not limited to the above, and are only the preferred embodiments of the present application, and are not other forms of limitations on the present application. Any skilled person in the art can use the above disclosed technical content to make changes or modifications into equivalent embodiments with equivalent changes. However, any simple modification, equivalent change and modification made on the above embodiments according to the technical essence of the present application, without departing from the technical scheme content of the present application, still belongs to the protection scope of the present application technical scheme.

Claims

1. Application of three-stage physical-biological synergistic cell wall breaking technology in extracting plant polysaccharides.

2. Use according to claim 1, characterized in that, The three-stage physical-biological synergistic cell wall breaking technology is enzyme hydrolysis, low-frequency ultrasonic assisted extraction, and membrane separation and purification technology.

3. Use according to claim 2, characterized in that, The enzyme hydrolysis uses a cellulase / xylanase complex system.

4. Use according to claim 2, characterized in that, The low-frequency ultrasonic assisted extraction conditions are 40 kHz and 300-500 W.

5. Use according to claim 2, characterized in that, The membrane separation and purification has a molecular weight cut-off range of 5-100 kDa.

6. A highly efficient green extraction method of plant marine polysaccharides based on ultramicro-pulverization-enzymolysis coupling ultrasonic, characterized in that, The method comprises the following steps: (a) raw material pretreatment: after cleaning, the seaweed is cryogenically pulverized to a particle size of 10-50 μm in a liquid nitrogen environment; (b) complex enzymolysis: the pulverized raw material is mixed with a complex enzyme solution, the complex enzyme contains cellulase 2.0-3.5 U / mg of raw material and xylanase 1.5-2.5 U / mg of raw material, and the reaction is carried out at pH 4.5-5.5 and a temperature of 35-40°C for 30 min; (c) ultrasonic extraction: after enzyme hydrolysis, the material is added with water at a solid-liquid ratio of 1:15-1:25, and ultrasonic treatment is carried out at a frequency of 40 kHz±5 kHz and a power density of 80-120 W / L for 20-30 min, during which the pulse mode is working for 5 s and intermittent for 2 s, and the temperature is controlled at ≤40°C; (d) membrane separation and purification: sequentially passing through a 0.22 μm microfiltration membrane to remove impurities, an ultrafiltration membrane to cut off polysaccharides of a target molecular weight, and a nanofiltration membrane to desalt and concentrate, and finally drying to obtain the finished product.

7. The method of claim 6, wherein: The cryogenic pulverization in step (a) is carried out at -40°C to -50°C, and the amount of liquid nitrogen is 1.5-2 times the mass of the raw material.

8. The method of claim 6, wherein: In step (b) of complex enzymolysis, additional lysozyme 0.8-1.2 U / mg of raw material is added to hydrolyze β-1,3-glucan.

9. The method of claim 6, wherein: In step (d), the ultrafiltration membrane has a molecular weight cut-off of 5-100 kDa.

10. The method of claim 6, wherein: The nanofiltration concentrated water produced in step (d) of membrane separation and purification is recovered and used in the extraction process of step (c).

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