Separation method of bark suppository cells

Abstract

The invention discloses a segregation method of bark suppository cells, and belongs to the technical field of plant cell segregation treatment. The isolation method of the bark suppository cells comprises the following steps: (1) cooking bark particles to remove impurities, so as to obtain impurity-removed suppository particles; (2) adding the impurity-removed suberect particles into a segregation reagent, and reacting for 40-60 minutes under the condition of 55-65 DEG C, so as to obtain segregated suberect cells; the segregation reagent is composed of a sodium hypochlorite solution with the mass concentration of 1%-10% and a hydrogen peroxide solution with the mass concentration of 0.5%-20%; (3) sequentially carrying out ultrasonic dispersion and freeze-drying treatment on the isolated suppository cells to obtain a suppository cell aggregate; and dispersing the suberect cell aggregate by using a weak polar reagent to obtain a complete single suberect cell. According to the method, the wooden suppository cells are separated through the synergistic oxidation effect of the sodium hypochlorite and the hydrogen peroxide solution, and efficient and complete separation of the wooden suppository cells in a short time is achieved.

Description

Technical Field

[0001] The invention relates to the technical field of plant cell separation and processing, and in particular to a method for separating bark cork cells. Background Art

[0002] Bark plays a role of barrier and protection in living trees because it has a large number of cork cells with closed-cell hollow structures. However, in daily life, bark is usually treated as a forestry waste resource and is burned or buried for composting, with low economic value. In order to give full play to the role of cork cells and realize the high-value utilization of bark, it is necessary to isolate and obtain independent hollow closed-cell cork cells from bark. However, this type of cell has significant differences from traditional plant wood cells due to its wall structure and chemical composition, and cannot be obtained by applying the common plant wood cell isolation method.

[0003] At present, common plant cell separation (i.e., separation) methods mainly include mechanical dissection, enzymatic hydrolysis, and chemical methods. These methods mainly achieve cell separation by removing pectin and lignin in the intercellular layer. However, the cell wall also contains a large amount of lignin. These methods usually remove lignin in the cell wall, causing damage to the cell structure, especially when the cell wall is thin, the structure is more likely to break. Therefore, these separation methods are mainly suitable for thick-walled woody cells (cell walls greater than 2μm) whose main components are rich in cellulose, lignin, and hemicellulose. It is worth emphasizing here that bark cork cells are essentially different from common plant woody cells in terms of wall structure and chemical composition. On the one hand, bark cork cells are thin-walled cells with a wall thickness of only 1 to 1.5μm, which are easily destroyed by the strong forces of mechanical separation methods, chemical reagents, etc., and it is difficult to maintain a complete cell structure. On the other hand, this type of bark cells is highly corky, with a three-layer cell wall structure, with the inner and outer layers accounting for less than 20%, and the main components are lignin and polysaccharides; the thickness of the middle layer accounts for more than 80%, and the main component is suberin, with a content of even more than 50%, and it forms a cross-linked network structure with wax, which is highly dense, hydrophobic, acid-resistant, and alkali-averse. Therefore, common acid, alkali, and organic plant cell separation reagents are difficult to play a good role in separating bark cork cells. In addition, there are few reports on the separation method of bark cork cells. Therefore, the invention of an efficient and rapid separation method for this type of cells is an important prerequisite for achieving high-value utilization of bark. Summary of the invention

[0004] In view of the above-mentioned prior art, the purpose of the present invention is to provide a method for isolating bark cork cells. The present invention accurately achieves rapid, efficient and complete separation of cork cells through the synergistic oxidation of sodium hypochlorite and hydrogen peroxide solution.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] A method for isolating bark cork cells comprises the following steps:

[0007] (1) steaming and removing impurities from the bark particles to obtain impurity-removed cork particles;

[0008] (2) adding the impurity-removed cork particles to a separation reagent, reacting at 55-65° C. for 40-60 minutes to obtain separated cork cells; the separation reagent is composed of a sodium hypochlorite solution with a mass concentration of 1-10% and a hydrogen peroxide solution with a mass concentration of 0.5%-20%;

[0009] (3) The isolated cork cells are subjected to ultrasonic dispersion and freeze-drying treatment in sequence to obtain cork cell aggregates; the cork cell aggregates are then dispersed with a weakly polar reagent to obtain complete single cork cells.

[0010] Preferably, in step (1), the temperature for steaming and removing impurities is 75-85° C., and the processing time is 2-4 hours; during this period, the mixture is filtered while hot every 1 hour.

[0011] Steaming can remove dust and impurities such as sand and gravel on the surface of cork particles, and can initially soften the cork cell walls.

[0012] Preferably, in step (2), in the separation reagent, the mass ratio of the sodium hypochlorite solution with a mass concentration of 1%-10% to the hydrogen peroxide solution with a mass concentration of 0.5%-20% is 5:1 to 15:1.

[0013] In the separation reagent of the present invention, the sodium hypochlorite solution reacts with the hydrogen peroxide solution to generate single linear oxygen with significant oxidative activity, which can quickly oxidize the pectin in the middle layer of the cork cells and a small amount of lignin in the outer wall in a short time, but is difficult to penetrate into the hydrophobic suberin / wax network structure, that is, it is difficult to destroy the suberin middle layer of the cell wall, thereby realizing rapid, efficient and complete separation of cells.

[0014] Preferably, in step (3), the operating frequency of ultrasonic dispersion is 20-60 KHz, the power is 600-800 W, and the ultrasonic time is 2-10 min.

[0015] The ultrasonic process significantly affects the structural integrity and uniformity of the isolated cells in the liquid. Generally, the higher the ultrasonic frequency and the greater the power, the more thorough the cell dispersion, but the easier the structure is to be destroyed. The ultrasonic process conditions of the present invention are used to help evenly disperse the cork cell aggregates that adhere to each other after the isolation treatment and retain the integrity of the cell structure.

[0016] Preferably, in step (3), the freeze-drying treatment is carried out at a temperature of -20°C to -60°C and for a time of 36 to 48 hours.

[0017] Freeze drying can remove the water between the isolated cork cells and maintain the closed-pore cavity structure of the cork cells.

[0018] Preferably, in step (3), the weak polar reagent is selected from one or any combination of solvents such as methanol, ethanol, acetone, benzene, dichloromethane, chloroform, ether, ethyl acetate, etc.; the amount of the weak polar reagent added is 5%-10% of the mass of the cork cell aggregates.

[0019] By treating the cork cell aggregates with a weak polarity reagent, the freeze-dried cork cell aggregates can be dispersed into independent cork cell individuals by taking advantage of the low polarity and volatility of the weak polarity reagent.

[0020] Beneficial effects of the present invention:

[0021] (1) The present invention proposes for the first time a method for isolating cork cells based on the synergistic oxidation of sodium hypochlorite and hydrogen peroxide solution, achieving a high yield and complete separation of cork cells in a short period of time. This method not only retains the natural hollow closed-cell structure of cork cells, but also transforms the bark material from hard macroscopic particles into flexible microscopic cells, thereby obtaining microscopic structural units with high specific surface area and soft elastic properties, providing a basic guarantee for the precise cutting and directional assembly of macroscopic high-performance material structures. In addition, the separation treatment removes part of the hydrophobic wax on the surface of the cork cells, improves the surface hydrophilicity, and helps to improve its interfacial compatibility when compounded with other polar materials.

[0022] (2) The isolated cork cells prepared by the present invention have broad application prospects in multiple fields, and are particularly suitable for occasions with high requirements for micro-nano size, specific surface area, interface compatibility, and surface modification, such as 3D printing, bark-based composite materials, and biomass activated carbon adsorption, catalysis, new energy, energy-saving intelligence, etc. In addition, the method has low cost, simple process, easy to realize industrial production, and has good economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 : Photograph of Quercus variabilis bark (cork) particles used in the present invention.

[0024] Figure 2 : Optical microscope photograph of the cork cells after ultrasonic dispersion in Example 1.

[0025] Figure 3 : Scanning electron micrograph of a complete single cork cell prepared in Example 1.

[0026] Figure 4 : Photograph of the cork oak bark (cork) granular material after treatment in Comparative Example 1.

[0027] Figure 5 : Optical microscope photograph of the cork cells after ultrasonic dispersion in Comparative Example 1.

[0028] Figure 6 : Photograph of the cork oak bark (cork) granular material after treatment in Comparative Example 2.

[0029] Figure 7 : Optical microscope photograph of the cork cells after ultrasonic dispersion in Comparative Example 2.

[0030] Figure 8 : Optical microscope photograph of the cork cells after ultrasonic dispersion in Comparative Example 3.

[0031] Fig. 9 : Optical microscope photograph of the cork cells after ultrasonic dispersion in Comparative Example 4. DETAILED DESCRIPTION

[0032] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0033] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0034] The test materials used in the examples and comparative examples of the present invention are all conventional test materials in the art and can be purchased through commercial channels. The experimental methods without detailed conditions are carried out in accordance with conventional test methods or the operating instructions recommended by the supplier. Among them:

[0035] The cork particles used in Example 1 of the present invention are bark of Quercus variabilis ( Figure 1 ), purchased from Shandong Ledesi Cork Development Co., Ltd., with a specific mesh size of 40-60 mesh.

[0036] Embodiment 1:

[0037] The sieved and dried 40-60 mesh Quercus variabilis bark (cork) particles were steamed in a water bath at 80°C for 3 hours, and filtered while hot every 1 hour to obtain impurity-free cork particles.

[0038] Add the impure cork particles to the separation reagent, and separate them at 60°C for 1 hour (replace the separation reagent every 30 minutes during this period) to obtain separated cork cells. The separation reagent is composed of a 3% sodium hypochlorite solution and a 3% hydrogen peroxide solution in a mass ratio of 10:1, and the ratio of the impure cork particles to the separation reagent is 1g:55ml.

[0039] After washing the isolated cork cells with water, ultrasonic dispersion was performed. The working frequency of ultrasonic dispersion was 40KHz, the power was 720W, and the ultrasonic time was 5min. The isolated cork cells after ultrasonic dispersion were observed under an optical microscope. The results are as follows: Figure 2 As shown, the results showed that most of the cork cells had been resolved and dispersed, and the cells were highly intact (hollow and closed), with a yield of more than 80%.

[0040] The isolated cork cells after ultrasonic dispersion were freeze-dried for 48 hours to obtain cork cell aggregates; the cork cell aggregates were dispersed with anhydrous ethanol, and the amount of anhydrous ethanol added was 5% of the mass of the cork cell aggregates. The mixture was stirred continuously, and a complete single hollow cork cell ( Figure 3 ).

[0041] Embodiment 2:

[0042] The bark of the French sycamore tree was crushed with a grinder and passed through a 40-60 mesh sieve to obtain the French sycamore tree bark particles; the particles were steamed in a water bath at 85° C. for 2 h, and filtered while hot every 1 h to obtain the impure cork particles.

[0043] The impure cork particles were added to the separation reagent, and the separation treatment was carried out at 55°C for 60 minutes (the separation reagent was replaced every 30 minutes during the period) to obtain the separated cork cells. The separation reagent was composed of a 3% sodium hypochlorite solution and a 3% hydrogen peroxide solution in a mass ratio of 10:1, and the ratio of the impure cork particles to the separation reagent was 1g:55ml.

[0044] After washing the isolated cork cells with water, ultrasonic dispersion was performed. The working frequency of ultrasonic dispersion was 40KHz, the power was 720W, and the ultrasonic time was 10min. The isolated cork cells after ultrasonic dispersion were observed under an optical microscope. The results are as follows: Figure 5 As shown, complete hollow closed-cell cells were isolated from the bark of sycamore trees with a yield of more than 75%.

[0045] The isolated cork cells after ultrasonic dispersion were freeze-dried for 48 hours to obtain cork cell aggregates; the cork cell aggregates were dispersed with anhydrous ethanol, the amount of anhydrous ethanol added was 10% of the mass of the cork cell aggregates, and stirring was continued to obtain complete single hollow cork cells after the reagent volatilized.

[0046] Comparative Example 1:

[0047] The difference from Example 1 is that the separation reagent in Example 1 is replaced by a hydrogen peroxide solution with a mass concentration of 3%.

[0048] Photos of processed materials Figure 4 As shown; the photo of the isolated cork cells after ultrasonic dispersion under an optical microscope is shown Figure 5 The results showed that: when using only 3% mass concentration hydrogen peroxide solution for separation treatment, the cork cells could not be separated and still maintained the natural honeycomb cell structure, indicating that the cork cells were difficult to be effectively separated.

[0049] Comparative Example 2:

[0050] The difference from Example 1 is that the separation reagent in Example 1 is replaced by a sodium hypochlorite solution with a mass concentration of 3%.

[0051] Photos of processed materials Figure 6 As shown; the photo of the isolated cork cells after ultrasonic dispersion under an optical microscope is shown Figure 7 The results showed that when using a 3% sodium hypochlorite solution alone for separation, only part of the cork cells were separated, and most of them were still in agglomerated state, indicating that the cork cells were difficult to be effectively separated.

[0052] Comparative Example 3:

[0053] The difference from Example 1 is that the separation reagent in Example 1 is replaced by a sodium hydroxide solution with a mass concentration of 10%.

[0054] The photo of the separated cork after ultrasonic dispersion under an optical microscope is as follows: Figure 8 The results showed that the cork cells were severely damaged and cell fragments could be directly observed, indicating that the alkaline separation system seriously affected the structure of the cork cells.

[0055] Comparative Example 4:

[0056] The difference from Example 1 is that the separation reagent in Example 1 is replaced by an acidic separation reagent composed of a sulfuric acid solution with a mass concentration of 8% and a nitric acid solution with a mass concentration of 3% in a mass ratio of 1:1.

[0057] The photo of the separated cork after ultrasonic dispersion under an optical microscope is as follows: Fig. 9 The results showed that the cork cells could not be separated, indicating that the common acid separation system could not separate the cork cells by swelling the cell wall and dissolving the pectin in the middle layer.

[0058] Test example:

[0059] The same batch of cork cell particles were taken and separated by the methods of Example 1, Comparative Example 1 and Comparative Example 2, respectively. The separated cork cells after ultrasonic dispersion were observed under an optical microscope. Five optical microscope fields of the same magnification ratio were randomly selected for each treatment. The number of separated intact cork cells was counted under the optical microscope field, and the yield of intact cork cells was calculated; yield = (number of cells with intact structure and completely separated / total number of cells in the field) * 100%. The results are shown in Table 1.

[0060] Table 1: Number of intact cork cells under different isolation treatment conditions

[0061] deal with Number of intact and isolated cork cells Yield Example 1 <![CDATA[ 62 ]]> (62 / 75)82.7％ Comparative Example 1 <![CDATA[ 2 ]]> <![CDATA[( 2 / 67 ) 2 . 9 %]]> Comparative Example 2 <![CDATA[ 24 ]]> <![CDATA[( 24 / 89 ) 26 . 9 %]]>

[0062] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for isolating bark cork cells, characterized in that: The following steps are involved: (1) steaming and removing impurities from the bark particles to obtain impurity-removed cork particles; (2) adding the impurity-removed cork particles to a separation reagent, reacting at 55-65° C. for 40-60 minutes to obtain separated cork cells; the separation reagent is composed of a sodium hypochlorite solution with a mass concentration of 1%-10% and a hydrogen peroxide solution with a mass concentration of 0.5%-20%; (3) subjecting the isolated cork cells to ultrasonic dispersion and freeze-drying treatments in sequence to obtain cork cell aggregates; The cork cell aggregates were dispersed with a weakly polar reagent to obtain intact single cork cells.

2. The separation method according to claim 1, characterized in that In step (1), the temperature for steaming and removing impurities is 75-85° C., and the processing time is 2-4 hours; during this period, the mixture is filtered while hot every 1 hour.

3. The separation method according to claim 1, characterized in that: In step (2), in the separation reagent, the mass ratio of the sodium hypochlorite solution with a mass concentration of 1%-10% to the hydrogen peroxide solution with a mass concentration of 0.5%-20% is 5:1-15:

1.

4. The separation method according to claim 1, characterized in that: In step (3), the operating frequency of ultrasonic dispersion is 20-60KHz, the power is 600-800W, and the ultrasonic time is 2-10min.

5. The separation method according to claim 1, characterized in that: In step (3), the freeze-drying treatment temperature is -20°C to -60°C, and the time is 36-48 hours.

6. The separation method according to claim 1, characterized in that: In step (3), the weak polar reagent is selected from one or any combination of solvents such as methanol, ethanol, acetone, benzene, dichloromethane, chloroform, ether, ethyl acetate, etc.; the addition amount of the weak polar reagent is 5%-10% of the mass of the cork cell aggregates.

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