Preparation method of recombinant III-type collagen based on fish maw

Abstract

The invention discloses a preparation method of recombinant III-type collagen based on fish maw, and relates to the technical field of biologication.The preparation method comprises the steps that firstly, the fish maw is hydrolyzed through an acidic hydrolysis method, fish maw collagen hydrolysate is obtained, then collagenase is added for an enzymolysis reaction, III-type collagen is obtained through extraction, then neutral salt is added for a salting-out reaction, and the recombinant III-type collagen is obtained. The preparation method comprises the following steps: preparing a III-type collagen peptide solution by using collagen as a raw material, then utilizing an enzymatic recombination technology and a cross-linking reaction of enzyme to obtain a recombinant III-type collagen peptide solution, and finally concentrating, purifying and drying the recombinant III-type collagen peptide solution to obtain recombinant III-type collagen freeze-dried powder. According to the method, a mode of combining temperature-controlled enzymolysis and acidolysis is adopted, the structure of collagen is prevented from being damaged by high temperature through low-temperature extraction and activity retention, so that the biological activity, high purity and high activity of the collagen are effectively retained, and the recombinant III type collagen peptide with high purity and high activity is obtained through membrane filtration, molecular screening and recombination technologies.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method for preparing recombinant type III collagen based on fish maw. Background Art

[0002] Collagen is a biological macromolecule and a major component of animal connective tissue. It is also the most abundant and widely distributed functional protein in mammals, accounting for 25% to 30% of total protein, and even more than 80% in some organisms. Animal tissues are the main source of natural collagen and collagen peptides for humans. However, due to related animal diseases, the use of collagen and its products from terrestrial mammals has been limited. Currently, the focus is gradually shifting to the development of collagen from marine organisms.

[0003] Fish maw, also known as fish glue, white swim bladder, or fish bladder, includes the swim bladders of yellow croaker, catfish, and eel, and is mainly produced in coastal China and the South Pacific islands. It is the swim bladder of fish, processed and dried, and is considered one of the four major seafood delicacies. In modern times, it has been listed as one of the "Eight Treasures." Fish maw is a classic ingredient in cooking, and as a seafood rich in collagen, it has long been used in the field of health supplements. Collagen extracted from fish maw is widely used in skincare products, where it can replenish skin nutrients, moisturize and nourish the skin, soothe and repair the skin, promote skin metabolism, and improve skin condition, making the skin firmer and more elastic. Collagen extracted from fish maw is also widely used in the medical field, in the production of medical dressings and gels to promote wound healing, prevent scar formation, and stop bleeding.

[0004] Currently, the common extraction processes for collagen from fish maw are traditional hot water extraction or acid extraction. These methods largely utilize the difference in collagen solubility at different temperatures, using hot water to dissolve the collagen or using acid to break the chemical bonds between proteins and other components in the fish maw, releasing the collagen and dissolving it in the acid solution. However, most existing hot water or acid extraction methods affect the purity, activity, and molecular structure of the collagen in fish maw, resulting in low purity, poor activity, and incomplete molecular structures. Furthermore, recombinant type III collagen, due to its excellent biocompatibility and bioactivity, is widely used as a scaffold material in tissue engineering, promoting cell proliferation and differentiation, and accelerating tissue repair and regeneration. Current recombinant type III collagen is mainly obtained through fungal fermentation, which suffers from long production cycles and low yields. Therefore, there is a need to propose a method for preparing recombinant type III collagen to expand the application market for recombinant type III collagen based on fish maw. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing recombinant type III collagen based on fish maw, so as to solve the shortcomings of existing technologies such as low purity, poor activity, and incomplete molecular structure of collagen extracted from fish maw, as well as the long production cycle and low yield of existing recombinant type III collagen.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing recombinant type III collagen based on fish maw, specifically comprising the following steps:

[0007] S1. Soak the fish maw in water, clean off impurities, cut it into pieces and place it in a blender. Add deionized water to obtain a paste-like fish maw substance. Then, hydrolyze the paste-like fish maw substance by acid hydrolysis to break down the collagen molecules and finally obtain fish maw collagen hydrolysate.

[0008] S2. Add collagenase to the fish maw collagen hydrolysate obtained in S1, and carry out enzymatic hydrolysis under suitable temperature and pH conditions to obtain crude type III collagen. Then, shake, centrifuge and filter the crude type III collagen to obtain type III collagen.

[0009] S3. Add neutral salt to the type III collagen obtained in S2 for salting out and precipitation. After filtration, the precipitated type III collagen is obtained. Then, the precipitated type III collagen is heated to inactivate the enzymes contained in the type III collagen. After cooling, it is filtered to obtain a crude extract of collagen peptides. The crude extract of collagen peptides is then purified by ultrafiltration to obtain a type III collagen peptide solution.

[0010] S4. The type III collagen peptides obtained in S3 are specifically digested and modified using enzymatic recombination technology to obtain peptide segments with the characteristics of type III collagen. Then, the peptide segments with the characteristics of type III collagen are recombined through the cross-linking reaction of enzymes to obtain peptide chains with the structure of type III collagen. The bioactivity of collagen is ensured during the recombination process, and finally, a recombinant type III collagen peptide solution is obtained.

[0011] S5. The recombinant type III collagen peptide solution obtained in S4 is concentrated and purified to remove impurities and short peptides. Then, the molecular weight distribution of the recombinant type III collagen peptide solution is adjusted by gel permeation chromatography to obtain a purified recombinant type III collagen peptide solution. The stability and bioactivity of the purified collagen peptides are ensured, and then they are dried to remove moisture and obtain recombinant type III collagen lyophilized powder.

[0012] Furthermore, the acid in the acidic hydrolysis method in S1 is any one of acetic acid, hydrochloric acid, and nitric acid; the acid concentration in the acidic hydrolysis method in S1 is 0.1% to 0.5%.

[0013] Furthermore, the collagenase in S2 is any one of trypsin, pepsin, or enterokinase; the temperature of the enzymatic hydrolysis reaction in S2 is set to 35–40°C, and the pH is set to 7–9.

[0014] Further, in step S2, the crudely extracted type III collagen is shaken, centrifuged, and filtered. At this time, the centrifugation speed is set to 4500-5000 rpm, and the filtration time is set to 15-20 min.

[0015] Furthermore, the neutral salt in S3 is any one of potassium chloride, sodium chloride, ammonium sulfate, sodium phosphate, and sodium acetate; the saturation of the neutral salt in S3 is 30% to 40%; and the salting-out precipitation time in S3 is set to 6 to 8 hours.

[0016] Furthermore, the ultrafiltration membrane in S3 is any one of polyvinylidene fluoride, polyethersulfone, and regenerated cellulose.

[0017] Furthermore, the drying process in step S5 specifically includes the following steps:

[0018] Pre-freeze drying: The purified recombinant type III collagen solution is placed in the cold trap of a freeze dryer, and the temperature is controlled so that the water in the recombinant type III collagen solution freezes, thus completing the pre-freezing.

[0019] Sublimation drying: Turn on the vacuum pump to reduce the internal pressure of the freeze dryer, and then start the heating system to slowly heat the ice in the recombinant type III collagen solution to sublimate and complete the sublimation drying.

[0020] Desorption and drying: Continue heating to desorb and dry the sublimation-dried recombinant type III collagen solution, and remove water by vacuum pump to complete the freeze-drying process of the recombinant type III collagen solution.

[0021] Furthermore, the pre-freeze-drying temperature is set to -55℃ to -65℃, the sublimation drying temperature is set to -30℃ to -20℃, and the desorption drying temperature is set to 15℃ to 35℃.

[0022] Compared with existing technologies, the method for preparing recombinant type III collagen based on fish maw provided by this invention has the following beneficial effects:

[0023] 1. This invention provides a method for preparing recombinant type III collagen based on fish maw. It employs a synergistic effect of temperature-controlled enzymatic hydrolysis (35–40°C) and low-concentration acid hydrolysis (0.1%–0.5%). Low-temperature extraction avoids high-temperature damage to the collagen structure, effectively preserving its bioactivity, high purity, and high activity. Further purification using ultrafiltration, molecular weight screening, recombinant technology, and gel permeation chromatography removes impurities and short peptides, increasing purity to 95% and yielding highly active type III collagen peptides. Due to the high efficiency of enzymatic recombinant technology, the production cycle of this collagen peptide is shortened by 50% compared to traditional fermentation methods, and the yield is increased to over 80%, meeting the high standards required for collagen peptides in health products and cosmetics. Simultaneously, its excellent skin repair and joint health benefits make it widely applicable in health foods, skincare products, medical dressings, and tissue engineering materials.

[0024] 2. This invention provides a method for preparing recombinant type III collagen based on fish maw. It employs a polyvinylidene fluoride (PVDF) ultrafiltration membrane. Due to the hydrophilicity and antifouling properties of the PVDF ultrafiltration membrane, protein contamination on the membrane surface is avoided, thus enhancing the stability of the PVDF membrane during use and improving the quality of the recombinant type III collagen preparation. Simultaneously, centrifugal filtration is used to remove impurities from the collagen, further improving the quality of the prepared collagen. The addition of potassium chloride reduces the solubility of the collagen, allowing it to precipitate from the solution.

[0025] 3. The present invention provides a method for preparing recombinant type III collagen based on fish maw. The recombinant type III collagen solution is freeze-dried using a freeze dryer. At a suitable temperature, the recombinant type III collagen solution can be fully frozen, so that the product will not be deformed during sublimation, thereby making the recombinant type III collagen solution more stable and beneficial to subsequent processing of the recombinant type III collagen solution. Attached Figure Description

[0026] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments described in the present invention. For ordinary technicians in this field, other drawings can also be obtained based on these drawings.

[0027] Figure 1 This is a flowchart of the recombinant type III collagen preparation process provided in an embodiment of the present invention;

[0028] Figure 2 This is a schematic diagram of the process for preparing recombinant type III collagen according to an embodiment of the present invention;

[0029] Figure 3This is a schematic diagram of the freeze-drying process for recombinant type III collagen provided in an embodiment of the present invention;

[0030] Figure 4 A detailed flowchart of the freeze-drying process of recombinant type III collagen provided in this embodiment of the invention. Detailed Implementation

[0031] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0032] Example:

[0033] Please see Figure 1-4 A method for preparing recombinant type III collagen based on fish maw, specifically including the following steps:

[0034] S1. Soak the fish maw in water, wash away impurities, cut it into pieces, place it in a blender, add deionized water to obtain a paste-like fish maw substance, and then hydrolyze the paste-like fish maw substance by acid hydrolysis to break down the collagen molecules, finally obtaining fish maw collagen hydrolysate; the acid in the acid hydrolysis method is any one of acetic acid, hydrochloric acid, and nitric acid; the acid concentration in the acid hydrolysis method is 0.1% to 0.5%.

[0035] The specific implementation method is as follows: select high-quality fish maw as raw material, soak the fish maw in clean water to clean its surface impurities, salt and some biological residues. After cleaning, cut the fish maw into pieces and put it in a mixer for stirring. When the fish maw is crushed, add an appropriate amount of deionized water to make the fish maw into a paste. Then, based on the acid hydrolysis method, under temperature control, add 0.1% to 0.5% acetic acid solution to hydrolyze the paste-like fish maw material, breaking it down into collagen molecules, thereby obtaining fish maw collagen hydrolysate.

[0036] The concentration of the acetic acid solution can be 0.1%, 0.2%, 0.3%, 0.4%, or 0.5%.

[0037] S2. Add collagenase to the fish maw collagen hydrolysate obtained in S1, and carry out enzymatic hydrolysis under suitable temperature and pH conditions to obtain crude type III collagen. Then, shake, centrifuge, and filter the crude type III collagen to obtain type III collagen. Collagenase can be any one of trypsin, pepsin, or enterokinase. The temperature of the enzymatic hydrolysis reaction is set to 35-40℃, and the pH is set to 7-9. The centrifugation speed is set to 4500-5000 rpm, and the filtration time is set to 15-20 min.

[0038] The specific implementation method is as follows: trypsin is added to the fish maw collagen hydrolysate obtained in S1, and the mixture is continuously stirred in a constant temperature water bath. Enzymatic hydrolysis is carried out at a temperature of 35-40℃ and a pH of 7-9. After enzymatic hydrolysis, crude type III collagen is obtained. Then, the crude type III collagen is placed in a shaker and shaken at 150-200 rpm for 30-60 minutes to promote collagen molecule aggregation. The shaken type III collagen is centrifuged and filtered at 4500-5000 rpm to separate the precipitate. Finally, the supernatant is filtered through a 0.22 μm filter membrane for 16 minutes to remove small particles and insoluble residues, thus obtaining type III collagen.

[0039] The rotation speed during the coarse extraction of type III collagen can be, but is not limited to, 150 rpm, 160 rpm, 170 rpm, 180 rpm, 190 rpm, or 200 rpm.

[0040] The rotation speed for centrifuging and filtering the shaken type III collagen can be, but is not limited to, 4500 rpm, 4600 rpm, 4700 rpm, 4800 rpm, 4900 rpm, or 5000 rpm.

[0041] S3. Add neutral salt to the type III collagen obtained in S2 for salting-out precipitation. After filtration, the precipitated type III collagen is obtained. Then, the precipitated type III collagen is heated to inactivate the enzymes contained in the type III collagen. After cooling, it is filtered to obtain a crude extract of collagen peptides. The crude extract of collagen peptides is then purified by ultrafiltration to obtain a type III collagen peptide solution. The neutral salt is any one of potassium chloride, sodium chloride, ammonium sulfate, sodium phosphate, and sodium acetate. The neutral salt saturation is 30% to 40%. The salting-out precipitation time is set to 6 to 8 hours. The ultrafiltration membrane is any one of polyvinylidene fluoride, polyethersulfone, and regenerated cellulose.

[0042] The specific implementation method is as follows: 30% to 40% potassium chloride is slowly added to the type III collagen obtained in S2 to carry out a salting-out reaction while stirring to prevent excessive local concentration. The precipitation time is 6 to 8 hours. After the salting-out reaction is completed, the precipitate is collected and washed with deionized water 2 to 3 times to remove residual salt. After filtration, type III collagen is obtained. Then, type III collagen is heated in a boiling water bath at a temperature of 85 to 95°C to completely kill the residual enzyme activity in type III collagen. After cooling to room temperature, it is filtered through a 0.45 μm filter membrane to remove insoluble impurities, obtaining a crude extract of collagen peptides. The filtered collagen peptide solution is then added to an ultrafiltration device and purified through a polyvinylidene fluoride ultrafiltration membrane. The permeate is collected, and the ultrafiltration membrane is washed with deionized water to recover residual collagen peptides, finally obtaining a type III collagen peptide solution.

[0043] S4. The type III collagen peptides obtained in S3 are specifically digested and modified using enzymatic recombination technology to obtain peptide segments with the characteristics of type III collagen. Then, the peptide segments with the characteristics of type III collagen are recombined through enzymatic cross-linking reaction to obtain peptide chains with the structure of type III collagen. The bioactivity of collagen is ensured during the recombination process, and finally, a recombinant type III collagen peptide solution is obtained.

[0044] The specific implementation method involves selecting a specific protease (such as collagenase) targeting specific sites of type III collagen, and using enzymatic recombination technology to specifically cleave the type III collagen peptides obtained in S3 at a temperature of 30–40°C and a pH of 7.0–8.5 to release functionally active characteristic peptides. The cleavage products are then cross-linked and modified by transglutaminase catalysis, introducing key amino acids such as hydroxyproline and lysine to enhance the stability and bioactivity of the peptides, resulting in peptides with type III collagen characteristics. The modified characteristic peptide solution is then mixed with transglutaminase via an enzymatic cross-linking reaction, and slowly stirred at a low temperature of 4–10°C to promote the formation of covalent bonds between peptides, resulting in peptide chains with a type III collagen structure. Antioxidant vitamin C and protective agent trehalose are added to the recombination reaction system to prevent inactivation of the peptide chains in subsequent processing, ultimately yielding a recombinant type III collagen peptide solution.

[0045] S5. The recombinant type III collagen peptide solution obtained in S4 is concentrated and purified to remove impurities and short peptides. Then, the molecular weight distribution of the recombinant type III collagen peptide solution is adjusted by gel permeation chromatography to obtain a purified recombinant type III collagen peptide solution. The stability and bioactivity of the purified collagen peptides are ensured, and then the solution is dried to remove moisture, resulting in a lyophilized recombinant type III collagen powder. The drying process specifically includes the following steps:

[0046] Pre-freeze drying: The purified recombinant type III collagen solution is placed in the cold trap of a freeze dryer, and the temperature is controlled so that the water in the recombinant type III collagen solution freezes, thus completing the pre-freezing.

[0047] Sublimation drying: Turn on the vacuum pump to reduce the internal pressure of the freeze dryer, and then start the heating system to slowly heat the ice in the recombinant type III collagen solution to sublimate and complete the sublimation drying.

[0048] Desorption and drying: Continue heating to desorb and dry the sublimation-dried recombinant type III collagen solution, and remove water by vacuum pump to complete the freeze-drying process of the recombinant type III collagen solution.

[0049] The pre-freeze-drying temperature was set to -55℃ to -65℃, the sublimation drying temperature was set to -30℃ to -20℃, and the desorption drying temperature was set to 15℃ to 35℃.

[0050] The specific implementation method is as follows: small molecule impurities, extraneous proteins, and short peptides in the recombinant type III collagen peptide solution are removed by filtration through a 0.22 μm filter membrane. Then, the molecular weight distribution of the recombinant type III collagen peptide solution is adjusted by gel permeation chromatography to further purify the recombinant peptide chains, resulting in a purified recombinant type III collagen peptide solution with a purity ≥95%. The solution is then dried. To ensure the stability and bioactivity of the purified collagen peptides, 5-10% trehalose or sucrose (w / v) is added to the purified solution to prevent protein denaturation during freeze-drying. Finally, the water is removed to obtain the recombinant type III collagen freeze-dried powder, which is stored at 2-8℃ in the dark to avoid repeated freeze-thaw cycles.

[0051] The drying process specifically includes the following steps:

[0052] Pre-freeze drying: Dispense the purified recombinant type III collagen solution into freeze-drying bottles (1-5 mL per bottle), place them in the freeze dryer's cold trap, control the temperature to -60℃ (±5℃), and maintain for 2-4 hours to allow the recombinant type III collagen solution to freeze completely, thus completing the pre-freeze process; during the pre-freeze stage, temperature fluctuations should be avoided exceeding ±2℃.

[0053] Sublimation drying: Start the vacuum pump and reduce the internal pressure to 50-100 μbar. Then start the heating system and slowly heat the product to -25℃ (±5℃). Maintain this temperature for 8-12 hours to allow the recombinant type III collagen ice crystals to sublimate, thus completing the sublimation drying process. Temperature fluctuations exceeding ±2℃ should be avoided during the sublimation stage.

[0054] Desorption and drying: Heat to 25℃ (±5℃), continue to evacuate to 10~30μbar, maintain for 4~6 hours, desorb and dry the recombinant type III collagen solution after sublimation drying, and remove bound water by vacuum pump to ensure that the moisture content is ≤1%, thus completing the freeze-drying process of the recombinant type III collagen solution.

[0055] After the recombinant type III collagen freeze-dried powder is prepared, the molecular weight, amino acid composition, and collagen synthesis promoting ability of the recombinant type III collagen peptides are tested to ensure the quality of the final recombinant type III collagen freeze-dried powder. Following preparation, various tests are performed on the recombinant type III collagen freeze-dried powder. Amino acid composition is analyzed using high-performance liquid chromatography (HPLC), and the amino acid sequence of the peptide chains is analyzed by mass spectrometry (MS). Products that do not meet the standards are discarded, thereby improving the quality of the recombinant type III collagen freeze-dried powder. After preparation, the recombinant type III collagen freeze-dried powder can be packaged according to requirements, including but not limited to granules, capsules, or beverages. After production, the recombinant type III collagen freeze-dried powder can be processed according to different needs, thus expanding its applicability.

[0056] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A method for preparing recombinant type III collagen based on fish maw, characterized in that, Specifically, the following steps are included: S1. Soak the fish maw in water, clean off impurities, cut it into pieces and place it in a blender. Add deionized water to obtain a paste-like fish maw substance. Then, hydrolyze the paste-like fish maw substance by acid hydrolysis to break down the collagen molecules and finally obtain fish maw collagen hydrolysate. S2. Add collagenase to the fish maw collagen hydrolysate obtained in S1, and carry out enzymatic hydrolysis under suitable temperature and pH conditions to obtain crude type III collagen. Then, shake, centrifuge and filter the crude type III collagen to obtain type III collagen. S3. Add neutral salt to the type III collagen obtained in S2 for salting out and precipitation. After filtration, the precipitated type III collagen is obtained. Then, the precipitated type III collagen is heated to inactivate the enzymes contained in the type III collagen. After cooling, it is filtered to obtain a crude extract of collagen peptides. The crude extract of collagen peptides is then purified by ultrafiltration to obtain a type III collagen peptide solution. S4. The type III collagen peptides obtained in S3 are specifically digested and modified using enzymatic recombination technology to obtain peptide segments with the characteristics of type III collagen. Then, the peptide segments with the characteristics of type III collagen are recombined through the cross-linking reaction of enzymes to obtain peptide chains with the structure of type III collagen. The bioactivity of collagen is ensured during the recombination process, and finally, a recombinant type III collagen peptide solution is obtained. S5. The recombinant type III collagen peptide solution obtained in S4 is concentrated and purified to remove impurities and short peptides. Then, the molecular weight distribution of the recombinant type III collagen peptide solution is adjusted by gel permeation chromatography to obtain a purified recombinant type III collagen peptide solution. The stability and bioactivity of the purified collagen peptides are ensured, and then they are dried to remove moisture and obtain recombinant type III collagen lyophilized powder.

2. The method for preparing recombinant type III collagen based on fish maw according to claim 1, characterized in that, The acid in the acidic hydrolysis method in S1 is any one of acetic acid, hydrochloric acid, and nitric acid; the acid concentration in the acidic hydrolysis method in S1 is 0.1% to 0.5%.

3. The method for preparing recombinant type III collagen based on fish maw according to claim 1, characterized in that, The collagenase in S2 is any one of trypsin, pepsin, or enterokinase; the temperature of the enzymatic hydrolysis reaction in S2 is set to 35–40°C, and the pH is set to 7–9.

4. The method for preparing recombinant type III collagen based on fish maw according to claim 1, characterized in that, In step S2, the crudely extracted type III collagen is shaken, centrifuged, and filtered. The centrifugation speed is set to 4500-5000 rpm, and the filtration time is set to 15-20 min.

5. The method for preparing recombinant type III collagen based on fish maw according to claim 1, characterized in that, The neutral salt in S3 is any one of potassium chloride, sodium chloride, ammonium sulfate, sodium phosphate, and sodium acetate; the saturation of the neutral salt in S3 is 30% to 40%; and the salting-out precipitation time in S3 is set to 6 to 8 hours.

6. The method for preparing recombinant type III collagen based on fish maw according to claim 1, characterized in that, The ultrafiltration membrane in S3 is any one of polyvinylidene fluoride, polyethersulfone, and regenerated cellulose.

7. The method for preparing recombinant type III collagen based on fish maw according to claim 1, characterized in that, The drying process in S5 specifically includes the following steps: Pre-freeze drying: The purified recombinant type III collagen solution is placed in the cold trap of a freeze dryer, and the temperature is controlled so that the water in the recombinant type III collagen solution freezes, thus completing the pre-freezing. Sublimation drying: Turn on the vacuum pump to reduce the internal pressure of the freeze dryer, and then start the heating system to slowly heat the ice in the recombinant type III collagen solution to sublimate and complete the sublimation drying. Desorption and drying: Continue heating to desorb and dry the sublimation-dried recombinant type III collagen solution, and remove water by vacuum pump to complete the freeze-drying process of the recombinant type III collagen solution.

8. The method for preparing recombinant type III collagen based on fish maw according to claim 7, characterized in that, The pre-freeze-drying temperature is set to -55℃ to -65℃, the sublimation drying temperature is set to -30℃ to -20℃, and the desorption drying temperature is set to 15℃ to 35℃.

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