A strain producing pectinase and its application in hemp degumming

Abstract

The present invention discloses a strain that produces pectinase and its application in hemp degumming, belonging to the field of microbial technology. The strain that produces pectinase is classified as Talaromyces wortmannii, and the deposit unit is the General Microbiology Center of the China Microorganism Culture Collection Administration Committee. The strain deposit name is: HM01, the deposit number is: CGMCC No. 41378, and the deposit date is: June 20, 2024; the strain produces pectinase but does not produce cellulase. Spraying the fermentation liquid of the strain on hemp stalks can make the strain colonize and grow on the epidermis of hemp, thereby improving the degumming effect of hemp; it effectively improves the rain and degumming effect of hemp, accelerates the rain and degumming speed, improves the separation effect of hemp skin and hemp stalk, and promotes the high-quality development of the hemp fiber industry.

Description

Technical Field

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

[0002] Hemp, an annual herbaceous plant of the genus Hemp in the family Hempaceae, was 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 production. Hemp fiber, also commonly referred to as raw hemp, is the bast tissue separated from hemp stalks. Hemp fiber's chemical components primarily include cellulose and pectin, the latter consisting primarily of non-cellulosic components such as pectin, water-soluble substances, hemicellulose, and lignin. Pectin affects the spinnability of hemp fiber, so raw hemp requires degumming 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 methods such as 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. Its degumming effect is good and the process is relatively mature, but there are still problems such as high energy consumption and large pollution; biological degumming is a method that uses the action of microorganisms or enzymes to specifically decompose the gum 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 for hemp degumming include fungi and bacteria. Compared to bacteria, fungi offer distinct advantages in hemp degumming: First, they produce large amounts of enzymes with high enzyme activity; second, they are highly capable of degrading resistant components like lignin, which bacteria generally cannot degrade; third, after spraying, fungi are more likely to colonize the hemp surface and produce spores for spread, allowing for prolonged degumming. Therefore, overall, fungi offer greater degumming capabilities and effectiveness. However, fungi also present several obstacles to their application. First, because they possess a richer enzyme repertoire, they often produce cellulase when decomposing colloids, which can hydrolyze hemp fibers and impair their spinnability. Second, fungi form mycelial pellets when cultured in liquid form. These granular structures can clog pipes and nozzles, hindering field spraying equipment. Therefore, developing novel fungal resources suitable for degumming applications has become a hot research topic. Summary of the Invention

[0005] In response to the shortcomings of the existing technology, a strain that produces pectinase and its application in hemp degumming are provided. The strain only produces pectinase but not cellulase. Spraying the fermentation liquid of the strain onto hemp stalks can allow the strain to 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 pectinase-producing strain is classified and named Talaromyces wortmannii. The preservation unit is the General Microbiology Center of the China Culture Collection Administration Committee, and the preservation address is the 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 pectinase-producing strain to remove pectin from hemp by utilizing Talaromyces wortmannii HM01.

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

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

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

[0011] a2. Transfer the seed solution to the fermentation medium at a ratio of 0.5% (v / v) and place it in a shaker. Incubate the shaker at a constant temperature of 28°C and a speed of 180 rpm for 96 hours to obtain an enzyme-active fermentation broth.

[0012] a3. Centrifuge the 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 comprises 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: tangerine peel powder 10g / L, (NH4)2SO4 1.5g / L, K2HPO4 1.0g / L, KCl0.5g / L, MgSO4 0.5g / L, FeSO4 0.1g / L.

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

[0016] b1. Enrichment culture of Talaromyces 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 physiological 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 morphologically meet the fungal characteristics from the PDA for initial screening.

[0017] b2. Primary screening: Purify the strains obtained from the primary screening and inoculate the strains 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 solid cellulose culture medium for staining. Select strains that cannot grow on the cellulose culture medium or do not have a clear clearing zone for rescreening.

[0018] b3. 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 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, comprising inoculating the bacterial strain Talaromyces wortmannii HM01 into a seed liquid culture medium, culturing the culture medium at a shaking speed of 180 rpm and a temperature of 28° C. for 48 hours, then transferring the obtained seed liquid into a PDA culture medium at a ratio of 0.5% (v / v), culturing the culture medium at a shaking speed of 180 rpm and a temperature of 28° C. for 96 hours to obtain a degumming fermentation liquid; spraying the degumming fermentation liquid onto hemp stalks, thereby causing obvious bacterial colonization on the epidermis of the hemp stalks and improving the degumming effect.

[0020] Furthermore, the specific steps of treating hemp stalks with degumming fermentation liquid are as follows:

[0021] c1. Microbial colonization analysis: Hemp stalks were cut into 20 cm lengths and laid flat in a container. The degummed fermentation liquid was placed in a spray bottle and sprayed onto the surface of the hemp stalks at a ratio of 0.1 mL of fermentation liquid per gram of hemp. The hemp stalks were then left at room temperature for 3 days to observe the microbial colonization on the surface of the hemp.

[0022] c2. Degumming effect determination: The degumming fermentation liquid was centrifuged at 13,000 g and 4°C for 5 min, and the supernatant was collected. The hemp epidermis was washed with clean water and dried, and then immersed in the fermentation supernatant at a ratio of 1:1 (w / v). The degumming treatment was achieved by incubating the epidermis in a shaker at 28°C and 100 rpm for 5 h.

[0023] Furthermore, the incubated hemp skin was washed with clean water and dried, and the dry weight of the hemp skin 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 PDA culture medium, but almost no cellulase, which can effectively avoid damage to hemp fiber after the crude fermentation liquid is sprayed onto hemp stalks; c. The strain Talaromyces wortmannii HM01 used in the present invention does not form mycelial balls in PDA culture medium and can be directly loaded into watering cans, agricultural machinery, drones and other equipment for spraying without additional treatment, without clogging pipelines and nozzles; d. The strain Talaromyces wortmannii HM01 used in the present invention is a fungus, can effectively colonize on the surface of hemp stalks, and can produce spores that spread among the stalks. It 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 This is 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 hemp epidermis in an embodiment of the present invention. DETAILED DESCRIPTION

[0029] The embodiments of the present invention are further described below with reference to the accompanying drawings:

[0030] The present invention discloses a strain that produces pectinase. The strain that produces pectinase is classified as Talaromyces wortmannii. The preservation unit is the General Microbiology Center of China Microorganism Culture Collection Administration Committee, and 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. 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 degumming speed, improve the separation effect of hemp skin and hemp stalks, and promote the high-quality development of the hemp fiber industry. 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 segments 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 fungus obtained in the previous step and then inoculate it 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 a clear clearing zone for rescreening.

[0034] c. Rescreening: Inoculate the strains 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 diameter of the transparent zone to the diameter of the colony. Select the strain with the largest ratio for subsequent experiments.

[0035] 2. Identification of pectinase-producing bacteria

[0036] a. Morphological Identification: Typical colonies formed on PDA medium at 28°C for 5 days. The colonies were nearly round, with regular outlines, dense and compact, dark green on the front with white edges, and light green on the back, producing an orange-yellow substance.

[0037] b. Molecular Biological Identification: 50 mg of fresh mycelium was collected and ground with liquid nitrogen. Genomic DNA was extracted using a kit. Using the genomic DNA as a template, PCR was performed using universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'). A fragment of approximately 700 bp in length was obtained. The amplification results of the ITS fragment of the strain are shown in the following table. 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, the strain was found to be 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 liquid culture medium and cultured on a shaker at 180 rpm and 28°C for 48 h. The resulting seed liquid was then transferred to a fermentation medium at a ratio of 0.5% (v / v). The culture was shaken at 180 rpm and 28°C for 96 h to obtain an enzyme-active fermentation liquid. Finally, the fermentation liquid was centrifuged at 13,000 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 cooled to serve as an enzyme inactivation control.

[0042] b. Pectinase activity assay: Prepare a 0.2% pectin solution as substrate in pH 5.0 phosphate buffer and dilute the enzyme solution appropriately with the same buffer. Add 100 μL of substrate solution and 50 μL of enzyme dilution to a 1.5 mL centrifuge tube, mix well, and incubate at 50°C for 10 minutes. Immediately terminate the reaction by adding 200 μL of DNS reagent. After mixing well, incubate in a boiling water bath for 5 minutes. After cooling, dilute with 1 mL of deionized water. Measure the absorbance of the reaction solution at 520 nm, and calculate the product release and enzyme activity using a standard curve. In this system, 1 activity unit is defined as the amount of enzyme required to produce 1 μmol of product per minute. The results showed that the pectinase activity of the fermentation broth reached 324 U / mL.

[0043] c. Cellulase activity assay: Prepare a 0.2% sodium carboxymethyl cellulose solution in pH 5.0 phosphate buffer as the substrate, and dilute the enzyme solution appropriately with the same buffer. Add 100 μL of substrate solution and 50 μL of enzyme dilution to a 1.5 mL centrifuge tube, mix well, and incubate at 50°C for 10 minutes. Immediately terminate the reaction by adding 200 μL of DNS reagent. Mix well, incubate in a boiling water bath for 5 minutes, cool, and dilute with 1 mL of deionized water. Measure the absorbance of the reaction solution at 520 nm, and calculate the amount of product released and enzyme activity using a standard curve. In this system, 1 activity unit is defined as the amount of enzyme required to produce 1 μmol of product per minute. The results show that the 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 approximately 20 cm lengths and lay them flat in a container. Pour the degummed fermentation liquid into a spray bottle and spray it onto the surface of the hemp stalks at a ratio of 0.1 mL of fermentation liquid per gram of hemp. Then, let the hemp stalks stand at room temperature for 3 days and 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 5 minutes, and take the supernatant; wash the hemp skin with clean water and dry it, then immerse the skin in the fermentation supernatant at a ratio of 1:1 (w / v) and incubate it at 28℃ and 100rpm for 5 hours; wash the treated skin with clean water and dry it. Weigh the dry weight of the skin 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, MgSO4 0.5g / 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, MgSO4 0.5g / L, FeSO4 0.1g / L, pH natural, sterilized at 115℃ for 30min;

[0052] c. PDA culture medium: Weigh 200 g of potato cubes, add water and boil for 30 minutes, filter through eight layers of gauze, add 20 g of glucose to the filtrate and stir to dissolve, cool and dilute to 1 L, adjust the pH to natural, and sterilize at 115°C for 30 minutes.

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

[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, MgSO4 0.5g / L, FeSO4 0.1g / L, pH natural, sterilized at 115℃ for 30min;

[0056] b. PDA medium: Weigh 200 g of potato cubes, add water and boil for 30 minutes, filter through eight layers of gauze, add 20 g of glucose to the filtrate and stir to dissolve, cool and dilute to 1 L, adjust the pH to natural, and sterilize at 115°C for 30 minutes.

[0057] 7. Preparation of degumming fermentation broth

[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 lengths of about 20 cm and lay them flat in a container. Pour the degumming fermentation liquid into a spray bottle and spray it onto the surface of the hemp stalks at a ratio of 0.1 mL of fermentation liquid per gram of hemp. Then, let the hemp stalks stand 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 bacterial colonization on the epidermis and improve the degumming effect.

[0061] b. Degumming Effect Measurement: The degummed fermentation broth was centrifuged at 13,000g and 4°C for 5 minutes, and the supernatant was collected. The hemp skins were rinsed with water and air-dried. Subsequently, the skins were immersed in the fermentation supernatant at a 1:1 (w / v) ratio and incubated at 28°C and 100 rpm for 5 hours. The treated skins were rinsed with water and air-dried. The dry weight of the skins before and after treatment was weighed to calculate the degumming rate, which showed a significant increase.

Claims

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

2. An application of the pectinase-producing strain according to claim 1, using Talaromyces watermanii ( Talaromyces wortmannii ) HM01 is used for pectin removal from hemp.

3. The use according to claim 2, characterized in that Waterman's Talaromyces ( Talaromyces wortmannii ) Pectinase produced by HM01 fermentation is used to remove pectin from hemp.

4. The use according to claim 3, characterized in that Waterman's Talaromyces ( Talaromyces wortmannii ) The process of HM01 producing pectinase is as follows: a1. Talaromyces wortmannii HM01 was inoculated into the seed liquid culture medium and placed in a shaker. The shaker was cultured at a constant temperature of 28°C and a rotation speed of 180 rpm for 48 hours to obtain the seed liquid. a2. Transfer the seed solution to the fermentation medium at a ratio of 0.5% (v / v) and place it in a shaker. Incubate the shaker at a constant temperature of 28°C and a speed of 180 rpm for 96 hours to obtain an enzyme-active fermentation broth. a3. The enzyme active fermentation liquid was heated at a relative centrifugal force of 13000. g The mixture was centrifuged for 5 min under the conditions of 40 °C, and the supernatant was collected. The pectinase activity in the supernatant was measured 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 the following components: 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. 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. The resulting seed liquid was then 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, which caused obvious strain colonization on the epidermis of the hemp stalks and improved the degumming effect.

8. The use according to claim 7, characterized in that The specific steps of using degumming fermentation liquid to treat hemp stalks are as follows: c1. Microbial colonization analysis: Hemp stalks were cut into 20 cm lengths and laid flat in a container. The degummed fermentation liquid was placed in a spray bottle and sprayed onto the surface of the hemp stalks at a ratio of 0.1 mL of fermentation liquid per gram of hemp. The hemp stalks were then left at room temperature for 3 days to observe the microbial colonization on the surface of the hemp. c2. Degumming effect determination: The degumming fermentation liquid was heated at 13000 g and 4°C for 5 minutes, and the supernatant was taken; the hemp skin was washed with clean water and dried, and then the skin was immersed in the fermentation supernatant at a ratio of 1:1 (w / v), and incubated on a shaker at 28°C and 100 rpm for 5 hours to achieve degumming.

9. The use according to claim 8, characterized in that The incubated hemp skin was washed with clean water and dried, and the dry weight of the hemp skin before and after treatment was weighed to calculate the degumming rate, which was degumming rate = [(dry weight before treatment - dry weight after treatment) / dry weight before treatment] × 100%.

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