阅读说明：本技术 一种铁皮石斛多糖的提取纯化方法 (Extraction and purification method of dendrobium officinale polysaccharide ) 是由 邵平 千佩玉 潘杰峰 于 2020-04-25 设计创作，主要内容包括：一种铁皮石斛多糖的提取纯化方法,包括：(1)取铁皮石斛粉末充分分散于纯水中得到粗原料液；(2)粗原料液通过微滤膜去除不溶性杂质得到渗透液1和截留液1；(3)渗透液1通过大孔超滤膜进行超滤处理并收集渗透液2和截留液2；(4)截留液2中加入可食用碱金属无机盐的水溶液进行稀释后通过大孔超滤膜进行超滤处理得到渗透液3和截留液3；(5)渗透液2和渗透液3合并加入电渗析装置中脱盐,并收集淡室溶液和浓室溶液；(6)淡室溶液通过小孔超滤膜进行超滤处理,收集截留液4与渗透液4；(7)截留液4经冷冻干燥制得铁皮石斛多糖。本发明方法在提高铁皮石斛多糖的提取率的同时解决了铁皮石斛中有毒重金属富集的问题。(A method for extracting and purifying dendrobium officinale polysaccharide comprises the following steps: (1) fully dispersing dendrobium officinale powder in pure water to obtain a crude raw material liquid; (2) removing insoluble impurities from the crude raw material solution through a microfiltration membrane to obtain a penetrating fluid 1 and a trapped fluid 1; (3) the penetrating fluid 1 is subjected to ultrafiltration treatment by a macroporous ultrafiltration membrane, and a penetrating fluid 2 and a trapped fluid 2 are collected; (4) adding an aqueous solution of edible alkali metal inorganic salt into the trapped fluid 2, diluting, and performing ultrafiltration treatment by a macroporous ultrafiltration membrane to obtain a penetrating fluid 3 and a trapped fluid 3; (5) combining the penetrating fluid 2 and the penetrating fluid 3, adding the mixture into an electrodialysis device for desalting, and collecting a light chamber solution and a thick chamber solution; (6) performing ultrafiltration treatment on the solution in the dilute chamber through a small-pore ultrafiltration membrane, and collecting trapped fluid 4 and penetrating fluid 4; (7) and (4) freeze-drying the trapped fluid 4 to obtain the dendrobium officinale polysaccharide. The method provided by the invention improves the extraction rate of the dendrobium officinale polysaccharide and solves the problem of enrichment of toxic heavy metals in the dendrobium officinale.)

1. a method for extracting and purifying dendrobium officinale polysaccharide comprises the following steps:

(1) fully dispersing dendrobium officinale powder in pure water to obtain a crude raw material liquid;

(2) removing insoluble impurities from the crude raw material liquid obtained in the step (1) through a microfiltration membrane to obtain penetrating fluid 1 and trapped fluid 1;

(3) performing ultrafiltration treatment on the penetrating fluid 1 collected in the step (2) through a macroporous ultrafiltration membrane with the molecular weight cut-off of 100-800kDa, and collecting a penetrating fluid 2 and a cut-off fluid 2;

(4) adding 0.5-1.5mol/L of edible alkali metal inorganic salt aqueous solution into the trapped fluid 2 collected in the step (3) for dilution, fully stirring and dissolving to obtain polysaccharide crude raw material liquid, performing ultrafiltration treatment by using a macroporous ultrafiltration membrane with the trapping molecular weight of 100-800kDa, and collecting a penetrating fluid 3 and a trapped fluid 3;

(5) combining the penetrating fluid 2 and the penetrating fluid 3 obtained after the treatment in the steps (3) and (4) and adding the combined penetrating fluid and the penetrating fluid into an electrodialysis device for desalting, and collecting a light chamber solution and a thick chamber solution;

(6) performing ultrafiltration treatment on the dilute solution obtained in the step (5) through an ultrafiltration membrane with the molecular weight cutoff of 3-8 kDa, and collecting a trapped fluid 4 and a penetrating fluid 4;

(7) and (4) freezing and drying the trapped fluid 4 obtained in the step (6) to obtain the dendrobium officinale polysaccharide.

2. The extraction and purification method according to claim 1, wherein: in the step (1), the concentration of the crude feed solution is 10 to 30 wt%.

3. The extraction and purification method according to claim 1, wherein: in the step (2), diluting the obtained trapped fluid 1 with pure water, then treating the trapped fluid with a microfiltration membrane, wherein the number of times of repeated treatment with the microfiltration membrane is more than 2 (preferably 2-6), and then combining the permeated fluid 1; the microfiltration membrane treatment conditions are as follows: the operation temperature is 25-50 ℃, the transmembrane pressure is 0.3-0.6MPa, and the flow rate of the feed liquid is 0.2-0.5L/min.

4. The extraction and purification method according to claim 1, wherein: in steps (3) and (4), the ultrafiltration membrane has a molecular weight cut-off of 300-800kDa, preferably 500 kDa.

5. The extraction and purification method according to claim 1, wherein: diluting the trapped fluid 2 obtained in the step (3) with pure water, and then repeatedly performing ultrafiltration membrane treatment, wherein the repeated ultrafiltration times are 2-5 times; the ultrafiltration treatment conditions in the step (3) are as follows: the operation temperature is 25-50 ℃, the transmembrane pressure is 0.3-0.6MPa, and the flow rate of the feed liquid is 0.2-0.5L/min.

6. The extraction and purification method according to claim 1, wherein: in the step (4), the edible alkali metal inorganic salt is NaCl and Na₂SO₄、KCl、K₂SO₄One or a mixture of several of them.

7. The extraction and purification method according to claim 1, wherein: in the step (4), the aqueous solution of the edible alkali metal inorganic salt is 0.5-1.5M NaCl aqueous solution or 0.5-1M Na aqueous solution₂SO₄An aqueous solution, preferably 0.5M NaCl aqueous solution or Na₂SO₄An aqueous solution.

8. The extraction and purification method according to claim 1, 6 or 7, wherein: diluting the trapped fluid 3 obtained in the step (4) with the aqueous solution of the edible alkali metal inorganic salt, and then repeatedly performing ultrafiltration membrane treatment, wherein the repeated ultrafiltration times are 2-5 times; when the trapped fluid 2 or 3 is diluted by the aqueous solution of edible alkali metal inorganic salt, the volume ratio of the trapped fluid 2 or 3 to the aqueous solution of inorganic salt is 3: 8-12; the ultrafiltration treatment conditions in the step (4) are as follows: the pH of the feed liquid is 4-10 (preferably pH 6-7), the operation temperature is 25-50 deg.C (preferably 30-35 deg.C), transmembrane pressure is 0.3-0.6MPa, and the flow rate of the feed liquid is 0.2-0.5L/min.

9. The extraction and purification method according to claim 1, wherein: in the step (6), the ultrafiltration membrane has a molecular weight cut-off of 3-5kDa, preferably 5 kDa.

10. The extraction and purification method according to claim 1, wherein: in the step (6), diluting the obtained trapped fluid 3 with pure water, and then performing ultrafiltration membrane treatment again, wherein the total treatment times of the ultrafiltration membrane is 2-6 times; the ultrafiltration treatment conditions were: the temperature is 25-50 ℃, the transmembrane pressure is 0.3-0.6MPa, and the flow rate of the feed liquid is 0.2-0.5L/min.

(I) technical field

The invention belongs to the technical field of natural polysaccharide extraction and purification, and relates to a method for extracting and purifying dendrobium officinale polysaccharide.

(II) background of the invention

Dendrobe has a long history in the field of medicinal health care in China, and the further processing of dendrobium officinale mostly extracts the main active ingredient, namely water-soluble dendrobe polysaccharide, but researches show that the dendrobium officinale contains polysaccharide partially dissolved in salt solution and also has the effects of antioxidation, antitumor and the like, and the neglect of the salt-soluble polysaccharide in the dendrobium officinale causes resource waste and causes higher processing cost. The membrane separation technology is used as a physical purification method with lower cost, no impurities are introduced, the operation condition is mild, the influence of the processing process on active ingredients can be effectively reduced, but the ultrafiltration extraction only allows molecules with the aperture smaller than that of an ultrafiltration membrane to pass, and the molecular weight of part of polysaccharide is increased because intermolecular complexation is generated between the polysaccharide and metal ions, so the part of dendrobium polysaccharide is wasted in the processing process by the traditional ultrafiltration extraction, and meanwhile, the problem of how to extract the dendrobium officinale polysaccharide dissolved in a salt solution by the traditional ultrafiltration technology is not solved.

On the other hand, soil, water source and the like are polluted, heavy metals are usually enriched in plants, the heavy metals are easy to accumulate in human bodies and can be retained in the human bodies for a long time, and the good adsorbability of polysaccharide to metal ions can cause the problem that the heavy metal ions in the polysaccharide exceed the standard, so that the quality of the polysaccharide and the health of the human bodies are seriously influenced. However, the existing polysaccharide extraction technology does not pay attention to the problem that heavy metal exceeds the standard, and the existing common heavy metal ion removal technology is mostly used for wastewater treatment (such as a precipitation method or an adsorption method), and the methods can reduce the content of partial heavy metal ions to a certain extent, but the method for removing the metal ions complexed with the polysaccharide has certain difficulty, and easily generates waste residues, thereby not only causing environmental pollution, but also causing resource waste.

Patent CN102532332A discloses a preparation method of dunaliella salina polysaccharide, which comprises preparing crude notoginseng polysaccharide from brown algae by filtering and ethanol precipitation, purifying fucoidin by ultrafiltration technology, and desalting polysaccharide solution by electrodialysis. However, the fucoidin content prepared by the method is low and is only about 35%, and the purity of the final product polysaccharide is not explicitly described in the patent. Published patent 106317243A discloses a method for preparing Lycium barbarum polysaccharides, which comprises extracting Lycium barbarum polysaccharides by ultrafiltration and reverse osmosis concentration, desalting by electrodialysis, and performing ultrafiltration and electrodialysis with four stages of different membrane apertures to obtain polysaccharides with desalting rate of 92% and purity of 65%. The method has the advantages of high yield of polysaccharide, low purity of polysaccharide and complex operation, and simultaneously, the patent provides microelement indexes (heavy metal ions Pb should be less than 0.5mg/Kg and As should be less than 0.3mg/Kg) in the lycium barbarum polysaccharide, but the patent does not show the content of the heavy metal ions in the lycium barbarum polysaccharide prepared by the method.

The US8097086 patent discloses a method for producing syrup with high fructose content, which partially removes sorbitol and salt ions in the syrup by means of main ultrafiltration and electrodialysis, with certain advantages, but the method of the invention only adopts a combined mode of two separation methods for purification, and the extraction steps are complicated, and the patent does not solve the problem of recovery of alcohol and phenol removed by ultrafiltration. US6372049 discloses a process for preparing refined syrup by electrochemical treatment, ultrafiltration, electrodialysis, ion exchange techniques, which carries out low-efficiency ultrafiltration by diffusion capacity rich in high molecular components and salts, causing serious pollution of ultrafiltration membranes and serious waste of resources.

In order to improve the problems faced in extracting and purifying the dendrobium officinale polysaccharide, the purification process of the natural polysaccharide needs to be further optimized and improved.

Disclosure of the invention

The invention provides the extraction and purification method of the dendrobium officinale polysaccharide, which is simple to operate, environment-friendly, safe, efficient, low in cost and suitable for industrial production, aiming at the existing problems, the extraction rate of the dendrobium officinale polysaccharide is improved, and the problem of toxic heavy metal enrichment in dendrobium officinale is solved.

The technical scheme adopted by the invention is as follows:

a method for extracting and purifying dendrobium officinale polysaccharide comprises the following steps:

(1) fully dispersing dendrobium officinale powder in pure water to obtain a crude raw material liquid;

(2) removing insoluble impurities from the crude raw material liquid obtained in the step (1) through a microfiltration membrane to obtain penetrating fluid 1 and trapped fluid 1;

(3) performing ultrafiltration treatment on the penetrating fluid 1 collected in the step (2) through a macroporous ultrafiltration membrane with the molecular weight cut-off of 100-800kDa, and collecting a penetrating fluid 2 and a cut-off fluid 2;

(4) adding 0.5-1.5mol/L of aqueous solution of edible alkali metal inorganic salt into the trapped fluid 2 collected in the step (3) for dilution, fully stirring and dissolving to obtain crude polysaccharide raw material liquid (the crude polysaccharide raw material liquid also contains amino acid, polyphenol and partial protein), performing ultrafiltration treatment by using a macroporous ultrafiltration membrane with the trapped molecular weight of 100-800kDa, and collecting penetrating fluid 3 and trapped fluid 3;

(5) combining the penetrating fluid 2 and the penetrating fluid 3 obtained after the treatment in the steps (3) and (4) and adding the combined penetrating fluid and the penetrating fluid into an electrodialysis device for desalting, and collecting a light chamber solution and a thick chamber solution;

(6) performing ultrafiltration treatment on the dilute solution obtained in the step (5) through an ultrafiltration membrane with the molecular weight cutoff of 3-8 kDa, and collecting a trapped fluid 4 and a penetrating fluid 4; the step can remove substances such as oligosaccharide, monosaccharide, polyphenol, flavone and the like in the dilute solution;

(7) and (4) freezing and drying the trapped fluid 4 obtained in the step (6) to obtain the dendrobium officinale polysaccharide.

In step (1) of the present invention, the concentration of the crude liquid is preferably 10 to 30% by weight.

In step (2) of the present invention, the preferable conditions for the microfiltration membrane treatment are: the operation temperature is 25-50 ℃, the transmembrane pressure is 0.3-0.6MPa, and the flow rate of the feed liquid is 0.2-0.5L/min. Due to the design of the microfiltration membrane device, a certain volume of trapped fluid which does not enter the subsequent treatment steps is inevitably generated by each microfiltration, and dendrobium officinale polysaccharide contained in the trapped fluid is wasted, so that the trapped fluid 1 is preferably diluted by pure water and then treated by the microfiltration membrane for more than 2 times, and then the penetrating fluid 1 is combined. The operation can reduce the residual quantity of dendrobium officinale polysaccharide in the trapped fluid to the maximum extent, which is beneficial to improving the extraction rate of polysaccharide, but in consideration of economy, the number of times of repeated treatment of the microfiltration membrane is preferably 2-6 times, and most preferably 5 times. When the trapped fluid 1 is diluted by pure water, the volume ratio of the trapped fluid 1 to the pure water for dilution is preferably 3: 8-12.

In step (3) of the present invention, the cut-off molecular weight of the ultrafiltration membrane affects the polysaccharide extraction rate and the polysaccharide quality, and preferably the cut-off molecular weight of the macroporous ultrafiltration membrane is 300-800kDa, and most preferably 500kDa, such that a higher polysaccharide extraction rate, a higher polysaccharide purity and a lower heavy metal content can be obtained. Preferably, in step (3), the ultrafiltration conditions are: the operation temperature is 25-50 ℃, the transmembrane pressure is 0.3-0.6MPa, and the flow rate of the feed liquid is 0.2-0.5L/min. Due to the design of the ultrafiltration membrane device, a certain volume of trapped fluid which does not enter the subsequent treatment step is inevitably generated by each ultrafiltration, and the dendrobium officinale polysaccharide contained in the trapped fluid is wasted, so that the trapped fluid 2 obtained in the step (3) is preferably diluted by pure water and then subjected to ultrafiltration membrane treatment repeatedly for 2-5 times, and the residual amount of the dendrobium officinale polysaccharide in the trapped fluid 2 can be reduced to the maximum extent by the operation, and the polysaccharide extraction rate is favorably improved. When the trapped fluid 2 is diluted by pure water, the volume ratio of the trapped fluid 2 to the pure water for dilution is preferably 3: 8-12. However, after the ultrafiltration treatment in step (3), some polysaccharides could not be extracted, including: the polysaccharide which is partially insoluble in water but dissolved in salt solution also has the effects of oxidation resistance, tumor resistance and the like; and polysaccharides that have been unable to pass through ultrafiltration membranes due in part to an increase in molecular weight due to intermolecular complexation with metal ions. In order to improve the extraction rate of polysaccharide, the invention carries out the treatment of the step (4) on the trapped fluid 2 after the ultrafiltration treatment.

In step (4) of the present invention, the edible alkali metal inorganic salt added is preferably NaCl, Na₂SO₄、KCl、K₂SO₄One or more of them, and particularly preferably the aqueous solution of the edible alkali metal inorganic salt is 0.5-1.5M NaCl solution or 0.5-1M Na solution₂SO₄Aqueous solution, most preferably 0.5M aqueous NaCl or Na₂SO₄An aqueous solution. Trapped fluid 2 is diluted with edible alkali metal inorganic salt aqueous solution and then is ultrafiltered, which is helpful for improving the extraction rate of polysaccharide on the one hand and reducing polysaccharide retardation caused by a pollution layer formed on the membrane surface in the subsequent membrane treatment on the other hand, and because the concentration of feed liquid is too high, the concentration polarization phenomenon is more easily formed on the membrane surface. Edible alkali metal non-valent salt with certain concentration is added into the trapped fluid, which is helpful for continuously extracting polysaccharide which can be dissolved in the salt solution and increasing the extraction rate of the polysaccharide; on the other hand, edible alkali metal inorganic salts make divalent or higher valentThe binding force of harmful metal ions (such as copper, arsenic, lead, mercury and cadmium ions) and polysaccharide is reduced, so that part of bivalent or high-valence metal ions complexed with the polysaccharide are converted into a free state, and the molecular weight of the polysaccharide originally complexed with the bivalent or high-valence harmful metal ions is reduced, so that the polysaccharide can easily pass through the ultrafiltration membrane. Therefore, in the step (4), more divalent or high-valent harmful metal ions are converted into a free state by adding edible inorganic salt with a certain concentration so as to be convenient for subsequent electrodialysis removal, and more polysaccharides can enter the penetrating fluid 3 through subsequent ultrafiltration treatment, so that the polysaccharide extraction rate is improved. Similar to step (3), in step (4), the molecular weight cut-off of the macroporous ultrafiltration membrane is preferably 300-800kDa, most preferably 500 kDa. Preferably, in the step (4), the ultrafiltration treatment conditions are as follows: the pH of the feed liquid is 4-10 (preferably 6-7), the operation temperature is 25-50 deg.C (preferably 30-35 deg.C), transmembrane pressure is 0.3-0.6MPa, and the flow rate of the feed liquid is 0.2-0.5L/min. The pH of the feed solution can be adjusted by adding hydrochloric acid or sodium hydroxide. And (3) preferably diluting the trapped fluid 3 obtained in the step (4) by using an inorganic salt aqueous solution and then repeatedly performing ultrafiltration membrane treatment for 2-5 times due to the design of an ultrafiltration membrane device, so that the residual quantity of dendrobium officinale polysaccharide in the trapped fluid 3 can be reduced to the maximum extent and the polysaccharide extraction rate is improved. When the trapped fluid 2 or 3 is diluted by the inorganic salt aqueous solution, the volume ratio of the trapped fluid 2 or 3 to the inorganic salt aqueous solution is preferably 3:8-12, and most preferably 3: 10.

In the step (5), the penetrating fluid 2 and the penetrating fluid 3 are combined for electrodialysis treatment, because the electrodialysis is that charged particles in the solution generate transmembrane migration under the action of electric field force, monovalent ions and divalent or high-valent ions have competitive transmission in the electrodialysis process, and the addition of edible inorganic ions is beneficial to the separation of the divalent or high-valent heavy metal ions, so that the divalent or high-valent heavy metal ions in the feed liquid and inorganic salts (such as NaCl and Na) added in the later period can be effectively removed through the electrodialysis treatment₂SO₄Etc.).

In step (5) of the invention, the electrodialysis device comprises a direct current power supply, an anode plate connected with the positive pole of the power supply, a cathode plate connected with the negative pole of the direct current power supply and a membrane stack arranged between the anode plate and the cathode plate, wherein the membrane stack and the anode plate and the cathode plate respectively form an anode chamber and a cathode chamber, the membrane stack is formed by sequentially arranging and assembling cation exchange membranes and anion exchange membranes at intervals, the outermost membrane is a cation exchange membrane, two adjacent membranes are separated by a partition plate, one group of the adjacent cation exchange membranes, the anion exchange membranes and the cation exchange membranes form an electrodialysis unit with two compartments (dense chamber/dilute chamber), and at least one electrodialysis unit is arranged in the membrane stack; the anode chamber and the cathode chamber are respectively connected with the electrode solution tank through pipelines to form a loop (namely, the electrode solution in the electrode solution tank can flow into the anode chamber and the cathode chamber by using a circulating pump, and then the material liquid in the anode chamber and the cathode chamber flows back to the electrode solution tank), and the dilute chamber and the concentrated chamber are respectively connected with the dilute solution tank and the concentrated solution tank through pipelines to form respective loops. In the present invention, before starting the electrodialysis, an electrode solution (preferably a 3 wt.% sodium sulfate solution), a mixed solution of a permeate 2 and a permeate 3, and pure water are injected into an electrode solution tank, a thin solution tank, and a thick solution tank, respectively. The skilled person can screen the membranes and set the appropriate electrodialysis operating conditions as the case may be. Generally, the screening of the membranes of the electrodialysis unit is based on the resistance and current efficiency of the stack, preferably lower resistance and higher current efficiency, which means lower process costs; and the electrodialysis condition is determined according to the material liquid property and the membrane resistance, and corresponding voltage and current are selected within the limit current density. For the invention, no special requirements are required for the selection of the cation exchange membrane and the anion exchange membrane, and the conventional commercially available cation exchange membrane and anion exchange membrane can be selected according to the actual situation. The invention has no special requirements on electrodialysis conditions, for example, the electrodialysis operation conditions can be set as follows: the electrodialysis operation is generally stopped when the conductivity value tends to be stable, the time length is usually not more than 1h, and the conductivity is not more than 2mS/cm, so as to ensure that the added salt is removed.

In the step (6) of the present invention, the ultrafiltration membrane preferably has a molecular weight cut-off of 3-5kDa, more preferably 5kDa, which allows for better removal of oligosaccharides, monosaccharides, polyphenols, flavonoids, etc. Preferably, in step (6), the ultrafiltration conditions are: the temperature is 25-50 ℃, the transmembrane pressure is 0.3-0.6MPa, and the flow rate of the feed liquid is 0.2-0.5L/min. In order to remove substances such as oligosaccharides, monosaccharides, polyphenols, and flavones as much as possible, it is preferable to dilute the obtained retentate 3 with pure water and then perform the ultrafiltration membrane treatment again, and the total number of ultrafiltration membrane treatments is 2 or more, but in view of economy, it is preferable that the total number of ultrafiltration membrane treatments is 2 to 6, and most preferably 3. When the trapped fluid 3 is diluted with pure water, the volume ratio of the trapped fluid 3 to the pure water for dilution is preferably 3: 8-12.

In step (7) of the present invention, the retentate 4 may be concentrated and then freeze-dried. There is no special requirement for the concentration mode, such as nanofiltration, rotary evaporation and the like, which can achieve the concentration purpose of the invention, but the nanofiltration concentration is preferably adopted in consideration of the requirement of industrial production. The invention has no special requirements on the nanofiltration membrane and the operating parameters adopted by nanofiltration concentration, and can realize the concentration purpose. For example, in the embodiment of the invention, a nanofiltration membrane with the molecular weight cut-off (mwco) of 3kDa-83kDa is adopted, and the operation conditions are as follows: room temperature, transmembrane pressure of 0.3-0.6MPa, and feed liquid flow rate of 0.2-0.5L/min.

The advantages and the beneficial effects of the invention are as follows:

(1) according to the extraction and purification method of the dendrobium officinale polysaccharide, provided by the invention, after the water-soluble polysaccharide in the dendrobium officinale is extracted by adopting an ultrafiltration technology, inorganic salt with a certain mass concentration is added into the trapped fluid for continuous extraction and the electrodialysis operation is matched, so that on one hand, the polysaccharide yield is improved, and on the other hand, the heavy metal ions in the polysaccharide are effectively removed.

(2) The method for preparing the dendrobium officinale polysaccharide based on the ultrafiltration and electrodialysis membrane technologies has the characteristics of environmental protection, safety, high efficiency, continuous operation, low cost and the like, and is suitable for industrial production.

(IV) description of the drawings

Fig. 1 is a conventional process route for preparing dendrobium polysaccharides.

Fig. 2 is a process route for preparing dendrobium polysaccharides adopted by the invention.

Fig. 3 is a schematic view of the principle of an electrodialysis apparatus employed in an embodiment of the present invention, in which a thin-liquid tank, a thick-liquid tank, an electrode-liquid tank, a circulation pump, and the like are not shown.

Fig. 4 is a plot of the content of metal ions in the extract over time during electrodialysis as in example 1.

Fig. 5 is a plot of the conductivity of the concentrate and dilute cells over time during electrodialysis in example 1.

(V) detailed description of the preferred embodiments

The technical solution of the present invention is further described below by using specific examples, but the scope of the present invention is not limited thereto.