Method for resolving and pulverizing residual plant tissues on eucommia ulmoides gum by using biological enzyme

Abstract

The present application belongs to the technical field of processing eucommia ulmoides, and particularly relates to a method for resolving and pulverizing residual hard-to-degrade plant tissues on eucommia ulmoides glue by using biological enzymes. The present application uses eucommia ulmoides shell crude glue as raw material, and resolves the raw material by using three biological enzymes in the following order: acid protease, alkaline xylanase and acid xylanase. Then, trace colored tissue debris and plant pigments attached to the G glue filament are removed by ultrasonic cleaning with a surfactant to obtain original flocculent eucommia ulmoides pure white fine glue. The acid protease enzymolysis conditions are as follows: pH 3.0, 55 DEG C oscillation for 48 h. The alkaline xylanase enzymolysis conditions are as follows: pH 7.2, 50 DEG C oscillation for 48 h. The acid xylanase enzymolysis conditions are as follows: pH 4.8, 50 DEG C oscillation for 72 h. The present application successfully separates and pulverizes the brown hard shell in the inner layer of eucommia ulmoides from the eucommia ulmoides glue filament, removes all non-glue plant tissues such as the outer epidermis and the hard shell in the inner layer of eucommia ulmoides shell, and obtains high-purity eucommia ulmoides glue after ultrasonic cleaning with a surfactant, with a purity as high as 98.94%.

Description

Technical Field

[0001] This invention belongs to the field of Eucommia ulmoides processing technology, and specifically relates to a method for disintegrating and pulverizing residual plant tissue on Eucommia ulmoides gum using bio-enzyme engineering technology. Background Technology

[0002] Eucommia gum is a rare biomaterial in the world possessing multiple properties and functions similar to rubber and plastics. In my country, it is an important basic material and a key strategic material. Its unique natural physicochemical properties, mechanical strength, and original morphology allow it to retain its original characteristics and functions in applications, unaffected by impurities, solvents, or other substances. The highest gum content is found in the Eucommia fruit shell, from which long-filamentous, flocculent Eucommia gum with a purity of over 99% can be extracted for its applications in national defense and artificial organs.

[0003] Unlike existing enzymatic hydrolysis for extracting natural plant products, this study on pulverized plant tissues found that the substrates for enzymatic hydrolysis are structurally specific, meaning they are ubiquitous within structural tissues. Only when biological enzymes erode these structural substrates, causing numerous defects and breaks in the originally continuous and ordered plant tissue, transforming it into a disordered and discontinuous tissue, does the structure disintegrate and the tissue pulverize.

[0004] The structural substrates of Eucommia ulmoides plants mainly consist of two polymers: proteins and xylan from hemicellulose. From seed germination, plants continuously undergo metabolic activities of combination and decomposition under the catalysis of various functional enzymes, resulting in growth, development, and the formation of new tissues while older tissues degenerate. Enzyme proteins are retained in situ, becoming structural proteins. Xylan, commonly known as hemicellulose, has widely distributed xylan residues in plant tissues and is ubiquitous in woven structures; it is a structural substrate that can be degraded by enzymes.

[0005] The following literature has disclosed relevant methods for extracting Eucommia gum:

[0006] CN 105670001 A discloses a method for extracting Eucommia gum from Eucommia ulmoides fruit shells, which involves (1) mechanical crushing; (2) sealed ozone oxidation; repeating steps (1) to (2) until there are basically no wood fibers; (3) adding 1 to 10 drops of anhydrous ethanol for rinsing; (4) drying the flocculent residue, oxidizing it again with ozone, drying it, and sieving it. The flocculent residue is washed with water until there is no obvious floating powder; (5) drying the flocculent residue to obtain Eucommia ulmoides crude gum; (6) extracting Eucommia ulmoides crude gum with petroleum ether at a weight ratio of 1:1 to 3 in a 75°C water bath, filtering while hot, combining the filtrates, freezing and placing them until a white filamentous precipitate appears, filtering, and then washing with methanol to obtain Eucommia ulmoides refined gum.

[0007] The disadvantages of the above patent are: 1. Steps (1) and (2) severely reduce the degree of polymerization of Eucommia ulmoides and can only obtain small molecule Eucommia ulmoides gum, while removing the advantages of high degree of polymerization and tear resistance of Eucommia ulmoides gum; 2. Step (6) changes the naturally pure white flocculent polymer state of Eucommia ulmoides gum, and the gum fibers lose the chemical modification properties and cannot be synthesized with reinforcing groups.

[0008] CN 109602783 A discloses an enzyme-assisted extraction method for active ingredients from Eucommia ulmoides leaves. The method includes: (1) removing impurities and pulverizing Eucommia ulmoides leaves; (2) enzymatic extraction, mixing the pulverized Eucommia ulmoides leaves from (1) with an enzyme solution and placing them in a reactor, ensuring the enzyme solution covers the Eucommia ulmoides leaves, and performing an enzymatic hydrolysis reaction; the enzyme solution includes a main enzyme and a coenzyme; the main enzyme is cellulase; the coenzyme is one or more of xylanase, pectinase, and keratinase; (3) separation, after the enzymatic hydrolysis reaction in (2) is completed, centrifuging is performed to separate the hydrolysate into solid and liquid components, obtaining a hydrolysate supernatant containing active ingredients.

[0009] The shortcomings of patent CN 109602783 A are as follows: Eucommia gum is the natural product with the highest content of biological materials in tree leaves, but it cannot withstand mechanical crushing. Crushed Eucommia gum loses its industrial value, and the raw material cost is entirely concentrated on the enzymatic hydrolysis extract, which is too expensive for the market to bear. Moreover, these enzymatic processes cannot achieve the disintegration and pulverization of plant tissues, resulting in low extraction efficiency of drug components.

[0010] CN 110467734 A discloses a method for purifying Eucommia ulmoides seed bark crude gum into a water solution to prepare Eucommia ulmoides refined gum. The steps are as follows: (1) using Eucommia ulmoides winged fruit seed bark as raw material, the plant tissue is hydrolyzed with biological enzymes to extract solid residues to obtain Eucommia ulmoides crude gum; (2) using biological enzymes to degrade the Eucommia ulmoides crude gum to remove the residual cell walls on the Eucommia ulmoides gum; (3) after removing the cell walls, a wetting surfactant is added; (4) sonication is performed to peel off the residual Eucommia ulmoides plant tissue fragments from the Eucommia ulmoides gum; (5) the sonicated Eucommia ulmoides crude gum with the cell walls removed is heated and stirred at 65°C to disperse the Eucommia ulmoides gum loosely and spread out in the water solution to release the residual Eucommia ulmoides plant tissue fragments that were wrapped in the gum and peeled off.

[0011] The shortcomings of patent CN 110467734 A are: 1. The purity of the crude eucommia gum obtained in step (1) is too low, less than 70%; 2. The cell wall is composed of cellulose, hemicellulose and lignin, which are intertwined and obstruct each other, and it is not easy to completely decompose it by enzymes in step (2); 3. The wetting surfactants in steps (3) and (4) can only remove oil-soluble organic pigments and resinous substances, and are powerless against hydrophilic plant tissue fragments; 4. In step (5), the eucommia begins to soften at 65°C and becomes more intertwined, and heating cannot loosen the gum. Summary of the Invention

[0012] To address the aforementioned technical problems, this invention provides a method for breaking down and pulverizing residual plant tissue on Eucommia ulmoides gum using biological enzymes, without mechanically crushing the gum or leaving organic solvent residues. This invention successfully separates and pulverizes the inner brownish-red hard pericarp of Eucommia ulmoides from the gum filaments using acidic protease; it successfully pulverizes the outermost pericarp tissue of the Eucommia ulmoides capsule using alkaline xylanase, thus removing the outer layer of the capsule; and it successfully pulverizes the brownish-red hard inner pericarp—the obstacle to enzymatic degradation and purification within the Eucommia ulmoides capsule—using acidic xylanase. This yields soft, grayish-green Eucommia ulmoides capsule tablets, which are then ultrasonically cleaned with surfactants to obtain pure, flocculent Eucommia ulmoides gum.

[0013] The method provided by this invention for disintegrating and pulverizing residual plant tissue on Eucommia ulmoides gum using biological enzymes involves using crude Eucommia ulmoides fruit shell gum as raw material and disintegrating it with three kinds of biological enzymes in the following order: acidic protease, alkaline xylanase, and acidic xylanase. The substrates for enzymatic hydrolysis are structural protein substrates and xylan substrates in structural hemicellulose.

[0014] (1) Mix acidic protease with crude gum from Eucommia ulmoides fruit shell, add a pH buffer solution containing acidic protease, and then hydrolyze by shaking in a water bath. Filter to obtain the hydrolysate A and the gum-containing solid residue A.

[0015] (2) Solid residue A is subjected to two repeated enzymatic hydrolysis under the same enzymatic hydrolysis conditions as in step (1), and solid residue B is obtained by filtration. Solid residue B is then subjected to three repeated enzymatic hydrolysis and filtered to obtain solid residue C.

[0016] (3) The obtained solid residue C was mixed with alkaline xylanase, and after adding a pH buffer solution containing alkaline xylanase, it was enzymatically hydrolyzed by shaking in a water bath and filtered to obtain solid residue D.

[0017] (4) Under the same enzymatic hydrolysis conditions as in step (3), solid residue D is subjected to a second enzymatic hydrolysis, and solid residue E is obtained by filtration.

[0018] (5) The obtained solid residue E is mixed with acidic xylanase, and after adding a pH buffer solution containing acidic xylanase, it is enzymatically hydrolyzed by shaking in a water bath and then filtered to obtain solid residue F.

[0019] (6) The solid residue F is subjected to the same enzymatic hydrolysis conditions as in step (5) for a second repeated enzymatic hydrolysis, and the solid residue G is obtained by filtration.

[0020] (7) Filter, wash and collect the solid residue G;

[0021] (8) Use surfactant-assisted ultrasonic cleaning to remove trace amounts of discolored tissue debris and plant pigments from the solid residue G glue fibers collected in (7) to obtain native flocculent Eucommia ulmoides pure white glue.

[0022] Preferably, in step (1) above, the weight ratio of acidic protease to crude gum from Eucommia ulmoides fruit shell is 3:8-15; 7-10 times the weight of a pH buffer solution containing active acidic protease is added; the acidic protease content in the pH buffer solution is 2%-8% (W / V); the pH is 2.5-3.5; and the solution is shaken in a constant temperature water bath at 53-58°C for 40-55 hours.

[0023] Preferably, in the above (1), the weight ratio of acidic protease to crude gum from Eucommia ulmoides fruit shell is 3:10; the pH of acidic protease is 3.0, and it is shaken in a constant temperature water bath at 55°C for 48 hours.

[0024] Preferably, in step (3) above, the weight ratio of alkaline xylanase to solid residue C is 0.1:4-7; 7-10 times the weight of pH buffer solution containing active alkaline xylanase is added; the alkaline xylanase content in the pH buffer solution is 3%-10% (W / V); the pH of alkaline xylanase is 7.0-7.5, and the solution is shaken in a constant temperature water bath at 45-52℃ for 40-55 hours.

[0025] Preferably, in step (3) above, the weight ratio of alkaline xylanase to solid residue C is 0.1:5; the pH of alkaline xylanase is 7.2, and it is shaken in a constant temperature water bath at 50°C for 48 hours.

[0026] Preferably, in step (5) above, the weight ratio of acidic xylanase to solid residue E is 0.1:2-6; 7-10 times the weight of pH buffer solution containing active acidic xylanase is added; the acidic xylanase content in the pH buffer solution is 4%-10% (W / V); the pH of acidic xylanase is 4.5-5.3; and the mixture is shaken in a constant temperature water bath at 45-52℃ for 72 hours.

[0027] Preferably, in the above (5), the weight ratio of acidic xylanase to solid residue E is 0.1:4; the pH of acidic xylanase is 4.8, and it is shaken in a constant temperature water bath at 50°C for 72 hours.

[0028] Preferably, in (7) above, filtration is performed using a 25-40 mesh sieve.

[0029] Preferably, in (7) above, a 30-mesh sieve is used for filtration.

[0030] Preferably, the surfactant mentioned in (8) above is a surfactant with high wetting properties, such as sodium dodecyl sulfate; the ultrasonic power is 3 to 4.5 × 10⁻⁶. 4 Hz.

[0031] Surfactants can effectively spread and disperse hydrophobic eucommia gum, and have a strong elution ability for various plant debris and pigments adsorbed on the surface of eucommia gum filaments, restoring the original color of eucommia gum.

[0032] Preferably, the ultrasound used in (8) above is intermittent ultrasound.

[0033] The present invention utilizes the immense pressure and vibration generated by ultrasonic cavitation to cause vibrational fatigue damage to cell debris and tissue fragments, which are then forcefully detached from the Eucommia ulmoides fibers and rupture, differentiating into the aqueous solution. With each activation of the ultrasound, dissolved air converges to form air bubbles, which rapidly and repeatedly rub against the surface of the fibers. These gaseous air bubbles continuously scrub and clean the surface of the Eucommia ulmoides fibers, removing fine debris and purifying the surface. This invention employs an intermittent ultrasonic method, repeatedly activating the ultrasound to generate a large number of air bubbles that vibrate and rub against the surface of the Eucommia ulmoides fibers, cleaning debris and pigment patches while polishing the fiber surface.

[0034] The beneficial effects of this invention are as follows:

[0035] 1. The original flocculent Eucommia ulmoides pure white gum prepared by this invention has a purity of up to 98.94%, which is about 2-3% higher than the purity of Eucommia ulmoides gum prepared by existing technology; it has fewer impurities and is less likely to form stress concentration points; the high purity makes Eucommia ulmoides gum easier to combine and mix with ordinary rubber, and it has more advantages in national defense and artificial organs.

[0036] 2. This invention discovers three bioenzymes with the function of pulverizing plant tissues: alkaline xylanase, acidic xylanase, and acidic protease. Based on numerous repeated experiments demonstrating the degradation and erosion of major organs of Eucommia ulmoides by acidic protease, this invention finds that the pulverizing function of acidic protease in plant tissues significantly promotes and enhances the extraction rate of natural products from enzyme degradation; acidic protease can break down and erode tissue components containing structural proteins.

[0037] 3. Alkaline xylanase successfully pulverized the outermost pericarp tissue of Eucommia ulmoides fruit shell, thus removing the outer pericarp tissue of the capsule; acidic xylanase successfully pulverized the hard, brownish-red inner pericarp, the obstacle to enzymatic degradation and purification inside the Eucommia ulmoides capsule, to obtain soft, grayish-green Eucommia ulmoides capsule tablets; and pure white flocculent Eucommia ulmoides gum was obtained by ultrasonic cleaning with surfactant.

[0038] 4. The experiment on the biochemical pulverization of the outer pericarp and the brownish-brown inner hard pericarp by alkaline xylanase and acidic xylanase verified the new theory of bioenzymatic extraction of natural products: "The bioenzymatic degradation of structural substrates is the basic enzymatic biochemical process that leads to the disintegration of substrate structure and pulverization of tissue, and is the basis for the efficient extraction of natural products by enzyme biochemistry." This confirms the feasibility of the new theory and has guiding significance for the production of Eucommia ulmoides pericarp gum. Attached Figure Description

[0039] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below.

[0040] Figure 1These are photographs showing the disintegration and pulverization of plant tissues caused by the degradation and erosion of structural substrates by acidic proteases.

[0041] Figure 2 A is the fruit shell raw material; B is the Eucommia ulmoides capsule with the outermost brown fruit peel remaining after the drug components have been extracted by the biodegradation of acidic protease, pectinase and cellulase.

[0042] Figure 3 A is Figure 2 B is a completely white Eucommia ulmoides capsule whose pericarp has been degraded by alkaline xylanase; B is the brownish-red hard inner pericarp attached to the Eucommia ulmoides capsule.

[0043] Figure 4 This is a biological enzyme control experiment in which acidic xylanase erodes hemicellulose and pulverizes the brownish-brown inner hard pericarp tissue.

[0044] Figure 5 The image shows the brownish-red inner hard shell of the fruit after it has been eroded, broken down, and pulverized by acidic xylanase in an aqueous solution (A), and the powdered tissue after it has been washed and separated from Eucommia ulmoides capsules through a 30-mesh stainless steel sieve and dried (B).

[0045] Figure 6 A is the inner layer of the capsule's pericarp tissue structure; the innermost bottom layer is a white, spatially reticulated layer of Eucommia ulmoides gum, the surface layer is a hard, smooth inner pericarp layer that wraps the kernel, and the middle layer between the inner and outer layers is the tissue layer formed by Eucommia ulmoides gum embedded with gum threads (B).

[0046] Figure 7 Photo A shows the integrated raw material of the eucommia gum shell, which is a brownish-red hard fruit shell. Photo B shows the eucommia gum cambium layer structure embedded with gum filaments, which has been eroded and powdered by acidic protease.

[0047] Figure 8 A shows the inner pericarp tissue of the Eucommia ulmoides capsule after it has been peeled off; B shows the separation of the inner brownish-red hard pericarp from the Eucommia ulmoides gum filaments after being eroded and degraded by acidic protease; C shows the exposed, free inner brownish-red hard pericarp.

[0048] Figure 9 It is a thin, flocculent, soft, capsule-shaped gelatinous sheet with gray and light green pigments, formed by the ablation and pulverization of acidic xylanase.

[0049] Figure 10 After the surfactant disperses the Eucommia ulmoides gum fibers and washes away the attached plant debris and pigments, pure white Eucommia ulmoides gum floats on the upper layer of the aqueous solution (Figure 1).

[0050] Figure 11 Eucommia capsules, made by bio-enzymatic degradation, erosion, and pulverization of plant tissues, are purified into pure white flocculent Eucommia ulmoides extract by surface-active assisted ultrasonic washing to remove plant debris and pigments. Detailed Implementation

[0051] To enable those skilled in the art to better understand the present invention, it will now be further described in conjunction with specific embodiments. The following examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0052] The raw material is crude gum from Eucommia ulmoides fruit shell, with a gum content of ≤80%.

[0053] (1) Biological enzymes and biochemical preparations

[0054] 537-acidic protease (50,000 U / g) is a commercially available feed-grade edible enzyme preparation;

[0055] Alkaline xylanase (SUKAXY ALK, 42,000 U / g);

[0056] Acidic xylanase (SUKAXY ACID, 200,000 U / g) is a commercially available pulp improver: Shandong Sukehan Bioengineering Co., Ltd.

[0057] Glycine (A111465), ≥99.0%: Shanghai Aladdin Biochemical Technology Co., Ltd.

[0058] (2) Experimental apparatus

[0059] SHA-CA Digital Display Water Bath Thermostatic Oscillator: Changzhou Putian Instrument Manufacturing Co., Ltd.;

[0060] AL04 electronic balance (0.0001 g): Mettler Toledo Instruments GmbH, Germany; UV754N UV-Vis spectrophotometer: Shanghai Instrument & Electronics Analytical Instrument Co., Ltd.

[0061] pHS-3E pH meter: Shanghai Instrument & Electronics Scientific Instruments Co., Ltd.;

[0062] SB-400DTY Ultrasonic Sweep Cleaner: Ningbo Xinzhi Biotechnology Co., Ltd.; B-260 Water Bath: Shanghai Yarong Biochemical Instrument Factory;

[0063] Model 500 1% Electronic Platform Scale: Bangyi Precision Measuring Instruments (Shanghai) Co., Ltd.;

[0064] Thermo Heraeus Multifuge X3R General Purpose Benchtop Centrifuge: Thermo Fisher Scientific. XW-80A Vortex Mixer;

[0065] Thermo Scientific TM Variable volume pipettes and pipette tips: Thermo Fisher Scientific (China) Co., Ltd.;

[0066] 10-40 mesh stainless steel standard sieve: Shaoxing Shangyu Shengchao Instrument Equipment Co., Ltd.

[0067] (3) Index Measurement

[0068] Determination of hydrolyzed amino acids: The amino acid determination method using ninhydrin colorimetric method was adopted, referring to "Comparison of Four Methods for Determining the Degree of Hydrolysis of Rice Protein" Vol.42, No.5, 35-37;

[0069] Determination of hydrolyzed amino acids: For the determination of hydrolyzed xylan and other monosaccharides, refer to "Study on the determination of xylose content using different DNS reagents".

[0070] (4) Experiment Content

[0071] In the enzymatic extraction of natural medicines and eucommia gum from the bark, leaves, and fruit shells of Eucommia ulmoides, the first stage is drug extraction, simultaneously obtaining active drug components and solid crude eucommia gum. The drug components dissolve in the enzymatic hydrolysate, while the eucommia gum strands remain in solid form. The second stage is eucommia gum purification, where the residue fragments remaining in the crude gum strands are subjected to deep residue pulverization and purification to obtain the eucommia gum.

[0072] In this invention, the effects of enzymatic hydrolysis on the extraction rate of four natural compounds—aucubin, genipin, genipinic acid, and chlorogenic acid—after enzymatic hydrolysis of crude Eucommia ulmoides pericarp samples using a comprehensive enzymatic extraction method were specifically investigated. The results are as follows:

[0073] Table 1. Results of determination of the content of four natural compounds in the sample.

[0074]

[0075] As shown in the table above, the extraction rate of acidic protease is 2 to 13 times that of pectinase and cellulase, and its enzymatic hydrolysis solution contains the highest content of drug components. (This is in conjunction with Table 1.) Figure 1 Experimental photos show that the acidic protease's ability to break down and pulverize Eucommia ulmoides tissue during extraction is unmatched by mechanical pulverization. Further degradation by pectinase and cellulase extracts the intracellular drug components, yielding a solid residue known as Eucommia ulmoides crude gum.

[0076] The increased contact area between the powdered tissue and the solution leads to a significant increase in the dissolution rate, amount, and types of drug components. Tissue disintegration and powdering are the fundamental reasons for the high extraction rate, revealing the limitations of traditional enzymatic extraction theories. The structural disintegration and tissue powdering phenomenon of acidic proteases is unique to acidic enzymes and is a key condition for high extraction rates.

[0077] However, acidic proteases cannot break down the brownish-brown inner hard pericarp containing no structural proteins, leaving behind this brownish-brown hard pericarp, which becomes a bottleneck in the purification of crude Eucommia ulmoides capsules. Alkaline xylanase is capable of degrading and eroding... Figure 2B shows the bioenzyme of xylan in the outermost layer of the capsule; the inventors further utilized alkaline xylanase to... Figure 2 B. After the outer pericarp is eroded and powdered, it is obtained Figure 3 The presence of a white capsule containing a hard, brownish-red inner shell (A) confirms that alkaline xylanase possesses a strong ability to degrade and pulverize the outermost pericarp, but is ineffective against the hard, brownish-red inner shell within the capsule. Acidic xylanase, however, exhibits significantly better activity against the hard, brownish-red inner shell within the capsule. Figure 4 The erosion and pulverization function; further, by Figure 4 It can be seen that, compared with the control standard solution, acidic xylanase hydrolyzes and releases xylose monosaccharides with the highest molecular weight, and has the function of efficiently degrading structural xylans. Figure 5 The pulverized tissue clearly demonstrates the ablative and pulverizing effect of acidic xylanase.

[0078] The enzymatic hydrolysis experiments shown in Table 1 above clearly demonstrate that: biological enzymes must be able to freely approach and locate on their substrates; this is a necessary and sufficient condition for the enzymatic hydrolysis of plant tissues. The overall structure of dense, blocky plant tissues must be broken down and pulverized to expose the substrate so that the enzyme can approach and locate it. In acidic proteases, in the two layers of pericarp tissue within the capsule, such as... Figure 6 B and Figure 7 B shows the experimental phenomena of erosion and pulverization of the eucommia gum-forming tissue layer embedded with eucommia gum fibers. The inventors realized the mechanism of enzymatic hydrolysis in eliminating pulverization of plant tissues: the hydrolysis of structural substrates is the key to the disintegration of plant structure and pulverization of tissues; the key to this invention is that the enzymatic hydrolysis target we selected is a structural substrate.

[0079] Example 1

[0080] (1) Mix 3 parts of acidic protease with pH 3.0 with 10 parts of crude gum from Eucommia ulmoides fruit shell, add 10 times the weight of pH buffer solution containing 6% (W / V) active acidic protease, and shake in a constant temperature water bath at 55℃ for 48h. After filtration, separate the enzymatic hydrolysate A and the gum-containing solid residue A.

[0081] (2) Solid residue A is subjected to two repeated enzymatic hydrolysis under the same enzymatic hydrolysis conditions as in step (1), and solid residue B is obtained by filtration. Solid residue B is then subjected to three repeated enzymatic hydrolysis and filtered to obtain solid residue C.

[0082] (3) Add 5 times the weight of alkaline xylanase with pH 7.2 to the obtained solid residue C and mix. Add 10 times the weight of pH buffer solution containing 8% (W / V) active alkaline xylanase and shake in a constant temperature water bath at 50°C for 48 hours. After filtration, obtain solid residue D.

[0083] (4) The solid residue D is subjected to the same enzymatic hydrolysis conditions as step (3) and then filtered to obtain solid residue E.

[0084] (5) Add 4 times the weight of acidic xylanase with pH 4.8 to the obtained solid residue E and mix. Add 8 times the weight of pH buffer solution containing 8% (W / V) active alkaline xylanase and shake in a constant temperature water bath at 50°C for 72 hours. After filtration, obtain solid residue F.

[0085] (6) The solid residue F is subjected to a second enzymatic hydrolysis under the same enzymatic hydrolysis conditions as step (5), and the solid residue G is obtained by filtration.

[0086] (7) Filter, wash and collect the solid residue G;

[0087] (8) Use surfactant-assisted ultrasonic cleaning to clean the trace amounts of discolored tissue debris and plant pigments on the solid residue G glue fiber collected in (7) to obtain the original flocculent Eucommia ulmoides pure white glue.

[0088] Examples 2-5

[0089] The pH of the biological enzyme and the temperature during enzymatic hydrolysis are shown in the table below. Other steps are the same as in Example 1.

[0090] Table 2 Enzymatic hydrolysis conditions for each embodiment

[0091]

[0092] Comparative Example 1

[0093] The difference from Example 1 is that the enzymatic hydrolysis order is alkaline xylan hydrolysis, acidic protease hydrolysis, and acidic xylan hydrolysis; the enzymatic hydrolysis conditions and other steps are the same as in Example 1.

[0094] Comparative Example 2

[0095] The difference from Example 1 is that the enzymatic hydrolysis order is acidic xylan hydrolysis, acidic protease hydrolysis, and alkaline xylan hydrolysis; the enzymatic hydrolysis conditions and other steps are the same as in Example 1.

[0096] Comparative Example 3

[0097] The difference from Example 1 is that the enzymatic hydrolysis order is acidic xylan hydrolysis, basic xylan hydrolysis, and acidic protease hydrolysis; the enzymatic hydrolysis conditions and other steps are the same as in Example 1.

[0098] Comparative Example 4

[0099] The difference from Example 1 is that the enzymatic hydrolysis order is alkaline xylan hydrolysis, acidic xylan hydrolysis, and acidic protease hydrolysis; the enzymatic hydrolysis conditions and other steps are the same as in Example 1.

[0100] Table 3. Purity of Eucommia ulmoides gum prepared in each experimental group

[0101] project purity% Example 1 98.12 Example 2 98.94 Example 3 98.04 Example 4 98.08 Example 5 98.10 Comparative Example 1 95.18 Comparative Example 2 94.76 Comparative Example 3 95.05 Comparative Example 4 95.25

[0102] The eucommia ulmoides refined gum prepared in the embodiments of the present invention has a very high purity, reaching up to 98.94%, which is about 2-3% higher than the purity of eucommia ulmoides fruit shell refined gum prepared by the existing technology.

[0103] After changing the enzymatic hydrolysis order of the three enzymes in Comparative Examples 1-4, the purity of the prepared Eucommia ulmoides fruit shell gum was consistently around 95%, which was lower than the purity of the Eucommia ulmoides gum prepared in Examples 1-5 (approximately 98%). This is because in the first stage of the comparative experiment, acidic protease was not used to separate the inner brownish-red hard fruit shell of Eucommia ulmoides from the gum filaments, and the channel for xylanase to degrade and erode the brownish-red hard fruit shell was not opened, resulting in insufficient enzymatic hydrolysis of the crude Eucommia ulmoides fruit shell gum, thus leading to a relatively low purity of the extracted gum.

[0104] The high-purity Eucommia ulmoides fruit shell gum prepared by this invention is easier to combine with ordinary Brazilian rubber; it can be widely used in various sectors of the national economy, including the rubber industry, aerospace, national defense, shipbuilding, chemical industry, medical, and sports. Furthermore, due to its high purity and low impurity content, there are no stress concentration release points.

Claims

1. A method for using biological enzymes to break down and pulverize residual plant tissue on Eucommia ulmoides gum, characterized in that, Includes the following steps: (1) Mix acidic protease with pH 2.6 and crude gum of Eucommia ulmoides fruit shell at a weight ratio of 3:10, add 10 times the weight of pH buffer solution containing 6% (W / V) active acidic protease, and hydrolyze in a constant temperature water bath at 53℃ for 48h. Filter to obtain the separated hydrolysate A and solid residue A. (2) Solid residue A is subjected to a second enzymatic hydrolysis under the same conditions as in step (1), and after filtration, solid residue B is obtained. Then, solid residue C is obtained by a third enzymatic hydrolysis. (3) Mix solid residue C with alkaline xylanase at pH 7.0 at a weight ratio of 1:5, add 10 times the weight of pH buffer solution containing 8% (W / V) active alkaline xylanase, shake in a constant temperature water bath at 45℃ for 48h, and filter to obtain solid residue D; (4) Under the same conditions as in step (3), solid residue D is enzymatically hydrolyzed a second time and filtered to obtain solid residue E; (5) Mix solid residue E with acidic xylanase at pH 4.5 at a weight ratio of 1:4, add 8 times the weight of pH buffer solution containing 8% (W / V) active acidic xylanase, shake in a constant temperature water bath at 52℃ for 72h, and filter to obtain solid residue F; (6) The solid residue F is enzymatically hydrolyzed a second time under the same conditions as in step (5), and filtered to obtain solid residue G; (7) Filter and wash the solid residue G through a 30-mesh sieve; (8) Using surfactant-assisted ultrasonic cleaning, trace amounts of discolored tissue debris and plant pigments on the surface of the adhesive fibers are removed to obtain virgin flocculent Eucommia ulmoides pure white adhesive. The ultrasonic power is 3~4.5×10⁻⁶. 4 Hz.

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