Bacterial strain for producing pectinase and application of bacterial strain in hemp degumming

Abstract

The invention discloses a strain for producing pectinase and application of the strain in hemp degumming, and belongs to the technical field of microorganisms. The classification name of the strain for producing pectinase is talaromyces wortmannii, the preservation unit is China General Microbiological Culture Collection Center, the preservation name of the strain is HM01, the preservation number is CGMCC No, 41378, and the preservation date is June 20, 2024; the strain generates pectinase and does not generate cellulase, and the strain fermentation liquor is sprayed to the hemp stems, so that the strain can be colonized and grow on the epidermis of the hemp, and the degumming effect of the hemp can be improved; the device effectively improves the rain and dew degumming effect of the China-hemp, accelerates the rain and dew degumming speed, improves the separation effect of hemp skins and hemp stems of the China-hemp, and promotes the high-quality development of the China-hemp fiber industry.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and relates to a strain producing pectinase and application of the strain in hemp degumming. Background Art

[0002] Hemp is an annual herb of the genus Hemp of the family Hempseed. It is one of the earliest plants used in the textile industry. my country has a long history of hemp cultivation and ranks first in the world in terms of production. Hemp fiber is the bast tissue separated from hemp stalks, also commonly known as raw hemp. The chemical components of hemp fiber mainly include cellulose and colloid. The latter is mainly composed of non-cellulose components such as pectin, water-soluble substances, hemicellulose and lignin. Gum will affect the spinnability of hemp fiber, so raw hemp needs to be degummed before it can be used as a textile raw material.

[0003] At present, the commonly used degumming methods in industry include physical degumming, chemical degumming and biological degumming. Physical degumming is carried out by rolling, ultrasonic and gas explosion, but the degumming is not thorough and the energy consumption is generally high; chemical degumming is carried out by alkali treatment or oxidation treatment. The degumming effect is good and the process is relatively mature, but there are still problems such as high energy consumption and pollution; biological degumming is a method of using microorganisms or enzymes to specifically decompose the colloid in the raw hemp and retain the cellulose component. This method is simple in process, low in energy consumption, and green and environmentally friendly. However, there are currently few strains that can be used for hemp degumming, and the enzyme activity and enzyme yield are low. These factors limit the promotion and application of biological degumming technology.

[0004] Microorganisms commonly used in hemp degumming include fungi and bacteria. Compared with bacteria, fungi have obvious advantages in hemp degumming: first, fungi have large enzyme production and high enzyme activity; second, fungi have strong decomposition ability for anti-degradation components such as lignin, while bacteria generally cannot degrade lignin; third, fungi are more likely to colonize on the surface of hemp after spraying, and produce spores to spread, which can play a degumming role for a long time. Therefore, on the whole, fungi have stronger degumming ability and better effect. However, fungi also have some obstacles in application: on the one hand, fungi have richer enzyme systems, and they often produce cellulase when decomposing colloids, which will cause hemp fiber hydrolysis and damage its spinnability; on the other hand, fungi will form mycelial balls when cultured in liquid, and this granular structure will block pipes and nozzles, which is not conducive to the use of equipment for field spraying. Therefore, the development of new fungal resources suitable for degumming applications has become a hot research direction. Summary of the invention

[0005] In view of the shortcomings of the existing technology, a strain producing pectinase and its application in hemp degumming are provided. The strain only produces pectinase but not cellulase. Spraying the fermentation liquid of the strain on hemp stalks can make the strain colonize and grow on the hemp epidermis, thereby improving the degumming effect of the hemp. The strain effectively improves the rain and dew degumming effect of hemp, accelerates the rain and dew degumming speed, improves the separation effect of hemp bark and hemp stalks, and promotes the high-quality development of the hemp fiber industry.

[0006] The strain producing pectinase is classified and named Talaromyces wortmannii, the preservation unit is the General Microbiology Center of China Microbiological Culture Collection Administration, the preservation address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the strain preservation name is: HM01, the preservation number is: CGMCC No. 41378, and the preservation date is: June 20, 2024.

[0007] The invention discloses an application of a strain producing pectinase, and utilizes Talaromyces wortmannii HM01 to remove pectin from hemp.

[0008] Furthermore, pectinase produced by fermentation of Talaromyces wortmannii HM01 was used to depectinize hemp.

[0009] Further, the process of producing pectinase by Talaromyces wortmannii HM01 is as follows:

[0010] a1. Inoculate the strain Talaromyces wortmannii HM01 into the seed liquid culture medium and place it in a shaker. Cultivate the shaker at a constant temperature of 28°C and a rotation speed of 180 rpm for 48 hours to obtain the seed liquid;

[0011] a2. The seed solution was transferred to the fermentation medium at a ratio of 0.5% (v / v) and then placed in a shaker. The shaker was kept at a constant temperature of 28°C and a speed of 180 rpm for 96 hours to obtain an enzyme-active fermentation liquid;

[0012] a3. Centrifuge the enzyme-active fermentation broth at a relative centrifugal force of 13,000 g for 5 minutes, collect the supernatant, and measure the pectinase activity in the supernatant to reach 324 U / mL.

[0013] Furthermore, the seed liquid culture medium contains the following components: pectin 10 g / L, (NH4)2SO4 1.5 g / L, K2HPO4 1.0 g / L, KCl 0.5 g / L, MgSO4 0.5 g / L, and FeSO4 0.1 g / L.

[0014] Furthermore, the fermentation medium components are: 10 g / L tangerine peel powder, 1.5 g / L (NH4)2SO4, 1.0 g / L K2HPO4, 0.5 g / L KCl, 0.5 g / L MgSO4, and 0.1 g / L FeSO4.

[0015] Further, the screening process of Talaromyces wortmannii HM01 for pectinase production is as follows:

[0016] b1. Enrichment culture of Bacillaceae Watermanii: Select hemp stalks with mildew spots and mold on the surface, peel off the epidermis, cut into small segments of about 5 mm, place in sterile saline and shake thoroughly, then spread gradient dilutions on potato dextrose agar (PDA) medium, culture at 28°C for 3 days, and pick out strains that meet the morphological characteristics of fungi from PDA for initial screening.

[0017] b2. Primary screening: The strains obtained from the primary screening were purified, and the strains were inoculated in the center of the solid cellulose culture medium. After culturing at 28°C for 5 days, Congo red solution was added to the surface of the solid cellulose culture medium for staining. The strains that could not grow on the cellulose culture medium or had no obvious transparent zone were selected for rescreening;

[0018] b3. Rescreening: Inoculate the strain obtained from the initial screening in the center of the solid pectin culture medium. After culturing at 28°C for 5 days, add iodine solution to the surface of the solid pectin culture medium for staining. Measure and calculate the ratio of the transparent zone diameter to the colony diameter. Select the strain with the largest ratio as the screened strain.

[0019] The invention discloses an application of pectinase in hemp degumming, wherein the strain Talaromyces wortmannii HM01 is inoculated into a seed liquid culture medium, and the culture medium is cultured at a shaking speed of 180 rpm and a temperature of 28°C for 48 hours; then the obtained seed liquid is transferred into a PDA culture medium at a ratio of 0.5% (v / v), and the culture medium is cultured at a shaking speed of 180 rpm and a temperature of 28°C for 96 hours to obtain a degumming fermentation liquid; the degumming fermentation liquid is sprayed on hemp stalks, and the degumming fermentation liquid causes obvious strain colonization on the epidermis of the hemp stalks, and improves the degumming effect.

[0020] Further, the specific steps of using the degumming fermentation liquid to treat hemp stalks are as follows:

[0021] c1. Microbial colonization analysis: Cut hemp stalks into 20 cm lengths and lay them flat in a container; put the degumming fermentation liquid into a spray bottle and spray it on the surface of the hemp stalks at a ratio of 0.1 mL of fermentation liquid / g of hemp; then leave the hemp stalks at room temperature for 3 days to observe the microbial colonization on the surface of the hemp;

[0022] c2. Degumming effect determination: centrifuge the degumming fermentation liquid at 13000g and 4℃ for 5min, and take the supernatant; wash the hemp epidermis with clean water and dry it, then immerse the epidermis in the fermentation supernatant at a ratio of 1:1 (w / v), and incubate it on a shaker at 28℃ and 100rpm for 5h to achieve degumming treatment.

[0023] Furthermore, the incubated hemp epidermis was washed with clean water and dried, and the dry weight of the hemp epidermis before and after treatment was weighed to calculate the degumming rate, degumming rate = [(dry weight before treatment - dry weight after treatment) / dry weight before treatment] × 100%.

[0024] Beneficial effects: a. The strain Talaromyces wortmannii HM01 used in the present invention has a fast growth rate and a short fermentation cycle. It has high pectinase activity in a fermentation medium with tangerine peel powder as a carbon source and can be used as a strain resource for producing pectinase; b. The strain Talaromyces wortmannii HM01 used in the present invention can produce highly active pectinase in a PDA medium, but almost no cellulase, which can effectively avoid the damage to hemp fiber caused by spraying the crude fermentation liquid on hemp stalks; c. The strain Talaromyces wortmannii HM01 used in the present invention does not form mycelium balls in the PDA medium, and can be directly loaded on a spraying pot, agricultural machinery, unmanned aerial vehicle and other equipment for spraying without additional treatment, and will not block pipelines and nozzles; d. The strain Talaromyces wortmannii HM01 used in the present invention belongs to a fungus, can effectively colonize on the surface of hemp stalks, and can produce spores to spread between hemp stalks, and can also achieve good degumming effect when applied in an open air environment; e. The strain Talaromyces used in the present invention wortmannii HM01 was isolated in situ from hemp straw in the hemp growing area of ​​Heilongjiang Province and has strong adaptability to the hemp growing environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a colony morphology diagram of the pectinase-producing strain Talaromyces wortmannii HM01 of the present invention;

[0026] Figure 2 This is a diagram showing the results of ITS fragment amplification of the pectinase-producing strain of the present invention;

[0027] Figure 3 A phylogenetic tree diagram of the strain producing pectinase of the present invention;

[0028] Figure 4 This is a schematic diagram of the colonization effect of the pectinase-producing strain Talaromyces wortmannii HM01 in the embodiment of the present invention on the epidermis of hemp. DETAILED DESCRIPTION

[0029] The embodiments of the present invention are further described below in conjunction with the contents of the accompanying drawings:

[0030] The present invention discloses a strain that produces pectinase. The strain that produces pectinase is classified and named as Talaromyces wortmannii. The preservation unit is the General Microbiology Center of China Microbiological Culture Collection Administration Committee. The preservation address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The strain preservation name is: HM01, the preservation number is: CGMCC No. 41378, and the preservation date is: June 20, 2024. The strain was isolated in situ from hemp straw in Heilongjiang Province and has good adaptability to the hemp planting environment. It is beneficial to improve the rain and dew degumming effect of hemp, accelerate the rain and dew degumming speed, improve the separation effect of hemp skin and hemp stalks, and promote the high-quality development of the hemp fiber industry. Specifically, Figure 1 shown.

[0031] 1. Screening of pectinase producing bacteria

[0032] a. Fungal enrichment culture: Select hemp stalks with mildew spots and mold on the surface, peel off the epidermis, cut into small pieces of about 5 mm, place in sterile physiological saline and shake thoroughly, then spread gradient dilutions on potato dextrose agar (PDA) medium, culture at 28°C for 3 days, and select strains that meet the morphological characteristics of fungi for initial screening.

[0033] b. Primary screening: Purify the fungi obtained in the previous step, then inoculate them in the center of a solid cellulose culture medium. After culturing at 28°C for 5 days, add Congo red solution to the surface of the culture medium for staining. Select strains that cannot grow on the cellulose culture medium or do not have an obvious transparent zone for rescreening.

[0034] c. Rescreening: Inoculate the strain obtained from the initial screening in the center of a solid pectin culture medium. After culturing at 28°C for 5 days, add iodine solution to the surface of the culture medium for staining. Measure and calculate the ratio of the transparent zone diameter to the colony diameter, and select the strain with the largest ratio for subsequent experiments.

[0035] 2. Identification of pectinase producing bacteria

[0036] a. Morphological identification: On PDA medium, typical colonies were formed after culturing at 28°C for 5 days. The colonies were nearly round, with regular outlines, dense and compact appearance, dark green on the front, white on the edges, light green on the back, and produced orange-yellow substances.

[0037] b. Molecular biological identification: Take 50 mg of fresh mycelium, add liquid nitrogen to grind, and use a kit to extract its genomic DNA. Use the genomic DNA as a template, use universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') to perform PCR, and amplify a fragment of about 700 bp in length. The amplification results of the ITS fragment of the strain are as follows: Figure 2 As shown; after sequencing the fragment, the sequence was subjected to BLAST comparison analysis in NCBI. The results showed that the ITS sequence of the strain had the highest consistency with the Talaromyces wortmannii ITS sequence, reaching 98%; the phylogenetic tree of the ITS sequence was constructed using MEGA7.0 software, as shown Figure 3 As shown, it was found that the strain was most closely related to Talaromyces wortmannii. Combined with morphological characteristics, the isolated strain was identified as Talaromyces wortmannii.

[0038] The ITS fragment sequencing results of the strain were:

[0039] .

[0040] 3. Enzyme activity determination

[0041] a. Extraction of crude enzyme solution: The strain Talaromyces wortmannii HM01 was inoculated into a seed solution culture medium, and cultured on a shaker at 180 rpm and 28° C. for 48 h. The resulting seed solution was then transferred to a fermentation medium at a ratio of 0.5% (v / v), and cultured on a shaker at 180 rpm and 28° C. for 96 h to obtain an enzyme-active fermentation solution. Finally, the fermentation solution was centrifuged at 13000 g and 4° C. for 5 min, and the supernatant was collected as the enzyme solution. The supernatant was incubated in a boiling water bath for 30 min, and after cooling, it was used as an inactivation control of the enzyme solution.

[0042] b. Pectinase activity assay: Use pH 5.0 phosphate buffer to prepare 0.2% pectin solution as substrate, and dilute the enzyme solution appropriately with the same buffer; add 100 μL substrate solution and 50 μL enzyme dilution solution to a 1.5 mL centrifuge tube, mix well and react at 50°C for 10 minutes, then immediately add 200 μL DNS reagent to terminate the reaction; mix the system well and place it in a boiling water bath for 5 minutes, cool it down and dilute it with 1 mL of deionized water; measure the absorbance of the reaction solution at 520 nm, and then calculate the product release and enzyme activity based on the standard curve. In this system, the amount of enzyme required to produce 1 μmol of product per minute is defined as 1 activity unit, and the results show that the pectinase activity of the enzyme-active fermentation broth reaches 324 U / mL.

[0043] c. Cellulase activity determination: Use pH 5.0 phosphate buffer to prepare 0.2% sodium carboxymethyl cellulose solution as substrate, and dilute the enzyme solution appropriately with the same buffer; take 100 μL substrate solution and 50 μL enzyme dilution solution and add them to a 1.5 mL centrifuge tube, mix well and react at 50°C for 10 minutes, then immediately add 200 μL DNS reagent to terminate the reaction; mix the system and place it in a boiling water bath for 5 minutes, cool it down and dilute it with 1 mL of deionized water; measure the absorbance of the reaction solution at 520 nm, and then calculate the product release and enzyme activity based on the standard curve. In this system, the amount of enzyme required to produce 1 μmol of product per minute is defined as 1 activity unit, and the results show that the enzyme-active fermentation broth contains almost no cellulase activity.

[0044] 4. Treatment of hemp stalks with Talaromyces wortmannii degumming fermentation liquid

[0045] a. Microbial colonization analysis: Cut hemp stalks into about 20 cm lengths and lay them flat in a container; put the degumming fermentation liquid into a spray bottle and spray it on the surface of the hemp stalks at a ratio of 0.1 mL of fermentation liquid / g of hemp; then leave the hemp stalks at room temperature for 3 days to observe the microbial colonization on the surface of the hemp.

[0046] b. Degumming effect determination: centrifuge the degumming fermentation liquid at 13000g and 4℃ for 5min, take the supernatant; wash the hemp epidermis with clean water and dry it, then immerse the epidermis in the fermentation supernatant at a ratio of 1:1 (w / v), incubate it at 28℃ and 100rpm for 5h; wash the treated epidermis with clean water and dry it. Weigh the dry weight of the epidermis before and after treatment to calculate the degumming rate:

[0047] Degumming rate = [(dry weight before treatment - dry weight after treatment) / dry weight before treatment] × 100%

[0048] Example 1: Screening and identification of pectinase-producing bacteria and enzyme fermentation

[0049] 5. Prepare culture medium:

[0050] a. Seed liquid culture medium: pectin 10g / L, (NH4)2SO4 1.5g / L, K2HPO4 1.0g / L, KCl 0.5g / L, MgSO40.5g / L, FeSO4 0.1g / L, pH natural, sterilized at 115℃ for 30min;

[0051] b. Fermentation medium: tangerine peel powder 10g / L, (NH4)2SO4 1.5g / L, K2HPO4 1.0g / L, KCl 0.5g / L, MgSO40.5g / L, FeSO4 0.1g / L, pH natural, sterilized at 115℃ for 30min;

[0052] c. PDA culture medium: weigh 200g of potato cubes, add water and boil for 30 minutes, filter with eight layers of gauze, add 20g of glucose to the filtrate and stir to dissolve, cool and adjust the volume to 1L, set the pH to natural, and sterilize at 115℃ for 30min.

[0053] Example 2: Pectinase-producing bacteria improve hemp degumming effect

[0054] 6. Prepare culture medium

[0055] a. Seed liquid culture medium: pectin 10g / L, (NH4)2SO4 1.5g / L, K2HPO4 1.0g / L, KCl 0.5g / L, MgSO40.5g / L, FeSO4 0.1g / L, pH natural, sterilized at 115℃ for 30min;

[0056] b. PDA culture medium: weigh 200g of potato cubes, add water and boil for 30 minutes, filter with eight layers of gauze, add 20g of glucose to the filtrate and stir to dissolve, cool and adjust the volume to 1L, set the pH to natural, and sterilize at 115℃ for 30min.

[0057] 7. Preparation of degumming fermentation liquid

[0058] The strain Talaromyces wortmannii HM01 was inoculated into a seed liquid culture medium and cultured in a shaking incubator at 180 rpm and 28° C. for 48 h. The resulting seed liquid was then transferred to a PDA culture medium at a ratio of 0.5% (v / v) and cultured in a shaking incubator at 180 rpm and 28° C. for 96 h to obtain a degumming fermentation liquid.

[0059] 8. If Figure 4 As shown, hemp stalks are treated with degumming fermentation liquid

[0060] a. Microbial colonization analysis: Cut hemp stalks into about 20 cm lengths and lay them flat in a container; put the degumming fermentation liquid into a spray bottle and spray it on the surface of the hemp stalks at a ratio of 0.1 mL of fermentation liquid / g of hemp; then leave the hemp stalks at room temperature for 3 days and observe the microbial colonization on the surface of the hemp. The results showed that spraying the fermentation liquid can cause obvious strain colonization on the epidermis and improve the degumming effect.

[0061] b. Degumming effect determination: The degumming fermentation liquid was centrifuged at 13000g and 4℃ for 5 minutes, and the supernatant was taken; the hemp epidermis was washed with clean water and dried, and then the epidermis was immersed in the fermentation supernatant at a ratio of 1:1 (w / v), and incubated at 28℃ and 100rpm for 5 hours; the treated epidermis was washed with clean water and dried. The dry weight of the epidermis before and after treatment was weighed to calculate the degumming rate, and the results showed that the degumming rate was significantly improved.

Claims

1. A strain producing pectinase, characterized in that: The strain producing pectinase is classified and named Talaromyces wortmannii, the preservation unit is the General Microbiology Center of China Microbiological Culture Collection Administration, the preservation address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the strain preservation name is: HM01, the preservation number is: CGMCC No. 41378, and the preservation date is: June 20, 2024.

2. An application of the pectinase-producing strain according to claim 1 to remove pectin from hemp using Talaromyces wortmannii HM01.

3. The use according to claim 2, characterized in that: Pectinase produced by fermentation of Talaromyces wortmanniiHM01 is used to depectinize hemp.

4. The use according to claim 3, characterized in that: The process of producing pectinase by Talaromyces wortmanniiHM01 is as follows: a1. Inoculate the strain Talaromyces wortmannii HM01 into the seed liquid culture medium and place it in a shaker. Cultivate the shaker at a constant temperature of 28°C and a rotation speed of 180 rpm for 48 hours to obtain the seed liquid; a2. The seed solution was transferred to the fermentation medium at a ratio of 0.5% (v / v) and then placed in a shaker. The shaker was kept at a constant temperature of 28°C and a speed of 180 rpm for 96 hours to obtain an enzyme-active fermentation liquid; a3. Centrifuge the enzyme-active fermentation broth at a relative centrifugal force of 13,000 g for 5 minutes, collect the supernatant, and measure the pectinase activity in the supernatant to reach 324 U / mL.

5. The use according to claim 4, characterized in that The components of the seed liquid culture medium are: pectin 10 g / L, (NH4)2SO4 1.5 g / L, K2HPO4 1.0 g / L, KCl 0.5 g / L, MgSO4 0.5 g / L, and FeSO4 0.1 g / L.

6. The use according to claim 4, characterized in that: The fermentation medium comprises: 10 g / L tangerine peel powder, 1.5 g / L (NH4)2SO4, 1.0 g / L K2HPO4, 0.5 g / L KCl, 0.5 g / L MgSO4 and 0.1 g / L FeSO4.

7. The use according to claim 4, characterized in that The screening process of Talaromyces wortmannii HM01 for pectinase production is as follows b1. Enrichment culture of Bacillaceae Watermanii: Select hemp stalks with mildew spots and mold on the surface, peel off the epidermis, cut into small segments of about 5 mm, place in sterile saline and shake thoroughly, then spread gradient dilutions on potato dextrose agar (PDA) medium, culture at 28°C for 3 days, and pick out strains that meet the morphological characteristics of fungi from PDA for initial screening. b2. Primary screening: The strains obtained from the primary screening were purified, and the strains were inoculated in the center of the solid cellulose culture medium. After culturing at 28°C for 5 days, Congo red solution was added to the surface of the solid cellulose culture medium for staining. The strains that could not grow on the cellulose culture medium or had no obvious transparent zone were selected for rescreening; b3. Rescreening: Inoculate the strain obtained from the initial screening in the center of the solid pectin culture medium. After culturing at 28°C for 5 days, add iodine solution to the surface of the solid pectin culture medium for staining. Measure and calculate the ratio of the transparent zone diameter to the colony diameter. Select the strain with the largest ratio as the screened strain.

8. An application of the pectinase according to claim 4 in hemp degumming, characterized in that: The strain Talaromyces wortmannii HM01 was inoculated into a seed liquid culture medium, and cultured at a shaking speed of 180 rpm and a temperature of 28°C for 48 hours. Then, the obtained seed liquid was transferred to a PDA culture medium at a ratio of 0.5% (v / v), and cultured at a shaking speed of 180 rpm and a temperature of 28°C for 96 hours to obtain a degumming fermentation liquid. The degumming fermentation liquid was sprayed on hemp stalks, and the degumming fermentation liquid caused obvious strain colonization phenomenon on the epidermis of the hemp stalks, and the degumming effect was improved.

9. The use according to claim 8, characterized in that The specific steps of using degumming fermentation liquid to treat hemp stalks are as follows: c1. Microbial colonization analysis: Cut hemp stalks into 20 cm lengths and lay them flat in a container; put the degumming fermentation liquid into a spray bottle and spray it on the surface of the hemp stalks at a ratio of 0.1 mL of fermentation liquid / g of hemp; then leave the hemp stalks at room temperature for 3 days to observe the microbial colonization on the surface of the hemp; c2. Degumming effect determination: centrifuge the degumming fermentation liquid at 13000g and 4℃ for 5min, and take the supernatant; wash the hemp epidermis with clean water and dry it, then immerse the epidermis in the fermentation supernatant at a ratio of 1:1 (w / v), and incubate it on a shaker at 28℃ and 100rpm for 5h to achieve degumming treatment.

10. The use according to claim 9, characterized in that The incubated hemp epidermis was washed with clean water and dried, and the dry weight of the hemp epidermis before and after treatment was weighed to calculate the degumming rate, degumming rate = [(dry weight before treatment - dry weight after treatment) / dry weight before treatment] × 100%.

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