Penicillium citrinum and its application in cotton stalk degumming

Abstract

This invention belongs to the field of microbial technology and relates to a strain of *Penicillium citrinum* and its application in cotton stalk degumming. The strain provided by this invention is *Penicillium citrinum* isolated from the Tiangong space station in China. Penicillium citrinum KJZ101. Penicillium lemon KJZ101 can secrete highly active pectinase in enzyme-producing fermentation media; Penicillium citrinum Spraying the fermentation broth of KJZ101 onto cotton stalks allows the strain to colonize and grow on the epidermis, promoting degumming of the cotton stalks through the action of pectinase. Studies on the growth and enzyme production characteristics of the strain revealed that it prefers to utilize pectin rather than cellulose, which helps avoid damage to the cotton stalk fibers during degumming. This strain exhibits dispersed suspension growth in PDA liquid medium without aggregating into large mycelial balls, which is beneficial for drone spraying. In summary, this invention enriches the resources of pectinase-producing strains, improves the separation efficiency of cotton stalk fibers from the stalk bark, reduces the difficulty and cost of cotton stalk degumming, and promotes the high-quality development of the cotton stalk fiber industry.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and relates to a strain of Penicillium citrinum and its application in cotton stalk degumming. Background Technology

[0002] Cotton stalks are a major byproduct of cotton crops and have wide applications in papermaking and agricultural fertilizers. The bast layer of cotton stalks is rich in high-quality long fibers, suitable for high-value-added fields such as papermaking; while the core material can be used in fuels, fertilizers, and other applications with lower requirements. Therefore, dehulling (i.e., separating the bast and the core material) is a key pretreatment step for achieving raw material grading, purification, and improving the economic value of products. Currently, industrially, dehulling machines are mainly used for physical dehulling of cotton stalks. However, due to the tight bond between the core material and the bast layer, there is still considerable room for improvement in the efficiency, effectiveness, and energy consumption of machine dehulling. The core material and the bast layer are mainly bonded by pectin; removing the pectin would significantly reduce the difficulty and cost of the dehulling process.

[0003] Biological degumming is a method that utilizes microorganisms or enzymes to specifically degrade pectin, thereby achieving degumming. Compared to chemical methods, biological degumming is simpler, less energy-intensive, and more environmentally friendly. Compared to bacteria, fungi have advantages such as higher enzyme yield, higher enzyme activity, and easier colonization, thus their degumming ability is generally stronger and more effective. However, there are also some obstacles to the application of fungi: on the one hand, fungi have a richer enzyme system, and when they decompose pectin, they often produce cellulase simultaneously, which may lead to hydrolysis of the fibers in the phloem and core, impairing the utilization value of the fibers; on the other hand, some fungi tend to aggregate into clumps during liquid culture, and this granular structure can clog pipes and nozzles, hindering field spraying. Therefore, developing novel fungal resources suitable for degumming processes has become a current research hotspot. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this paper presents a strain of Penicillium citrinum and its application in cotton stalk degumming. This strain was isolated to the Chinese space station, exhibits good pectinase activity, and does not form balls during fermentation, making it suitable for large-scale field spraying.

[0005] To achieve the above technical objectives, this invention discloses a strain of *Penicillium citrinum*, wherein *Penicillium citrinum*... Penicillium lemon The strain was isolated from the Tiangong space station in China. The depository is the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The strain name is KJZ101, the accession number is CGMCC No. 42422, and the deposit date is January 9, 2026.

[0006] An application of Penicillium citrinum, Penicillium citrinum Penicillium citrinumKJZ101 removes pectin from cotton stalks.

[0007] Furthermore, using Penicillium citrinum Penicillium citrinum The pectinase produced by fermentation of KJZ101 can depectinize hemp.

[0008] Application of a pectinase in hemp degumming, Penicillium citrinum Penicillium citrinum KJZ101 was inoculated into PDA medium and cultured at 200 rpm and 28℃ for 24 h to obtain degummed fermentation broth. The degummed fermentation broth was sprayed onto cotton stalks, and obvious bacterial colonization occurred on the surface of the cotton stalks, which improved the degumming effect.

[0009] Furthermore, the specific steps for treating cotton stalks using degumming fermentation broth are as follows: a1. Microbial colonization analysis: Cotton stalks were cut into 8cm lengths and laid flat in a petri dish. The degummed fermentation broth was sprayed onto the surface of the cotton stalks at a ratio of 0.5mL fermentation broth / g cotton stalks. The cotton stalks were then left to stand at room temperature for 3 days to observe the microbial colonization on the surface of the cotton stalks. a2. Degumming effect determination: The degumming fermentation broth was diluted at 10000... g Centrifuge at 4℃ for 10 min and collect the supernatant; wash the cotton stalk skin with clean water and dry it, then immerse the cotton stalk skin in the fermentation supernatant at a ratio of 1:1 (w / v) and incubate at 28℃ and 100 rpm for 6 h to achieve degumming.

[0010] Furthermore, the treated cotton stalks were washed with clean water and dried. The dry weight of the cotton stalks 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%.

[0011] A type of Penicillium citrinum Penicillium citrinum The analytical method for KJZ101 is as follows: b1. Qualitative analysis of pectinase activity: [The text abruptly ends here, likely due to an incomplete Penicillium citrinum KJZ101 was inoculated onto solid pectin medium and cultured. After incubation, the cells were stained with Lugol's iodine solution to qualitatively analyze the extracellular pectinase activity. b2. Quantitative analysis of pectinase activity: [The text abruptly ends here, likely due to an incomplete sentence or missing information.] Penicillium citrinumKJZ101 was inoculated into enzyme-producing fermentation medium and cultured on a shaker at a constant temperature to obtain enzyme-producing fermentation broth. The supernatant of the fermentation broth was collected by centrifugation as enzyme solution. A 0.2% pectin solution was prepared using pH 5.0 phosphate buffer as substrate, and the enzyme solution was diluted. 100 μL of substrate solution and 50 μL of enzyme dilution were added to a centrifuge tube and mixed well. The mixture was then reacted at 50℃ for 10 min, and 200 μL of DNS reagent was added immediately to terminate the reaction. The system was mixed and placed in a boiling water bath for 5 min. After cooling, 1 mL of deionized water was added to dilute the reaction solution. The absorbance of the reaction solution at 520 nm was measured, and the product release and enzyme activity were calculated according to the standard curve. b3. Pectin-cellulose metabolism analysis: Penicillium... Penicillium citrinum KJZ101 was inoculated into metabolic medium and cultured on a shaker at a constant temperature; the culture solution was then incubated at 4℃ and 10,000 mL / min. g Centrifuge for 10 min under the specified conditions, place the supernatant in a dialysis bag with a molecular weight cutoff of 1000 Da for dialysis, and then freeze-dry. Weigh 0.1 g of the freeze-dried powder, mix it with 1 mL of 72% H2SO4, incubate at 30℃ for 1 h, then dilute to 30 mL, and incubate the system at 121℃ for 1 h to obtain the hydrolysate. Determine the content of galacturonic acid and glucose in the hydrolysate by high performance liquid chromatography (HPLC), and compare it with the uninoculated control medium to analyze the metabolic preferences of the strain.

[0012] Furthermore, the solid pectin culture medium consists of: 10 g / L pectin, 1.5 g / L (NH4)2SO4, 1.0 g / L K2HPO4, 0.5 g / L KCl, 0.5 g / L MgSO4, 0.1 g / L FeSO4, and 18 g / L agar.

[0013] Furthermore, the enzyme-producing fermentation medium consists of: 10 g / L dried 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.

[0014] Furthermore, the metabolic culture medium consisted of: 5 g / L pectin, 5 g / L carboxymethyl cellulose, 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] Beneficial effects: The Penicillium citrinum described in this invention Penicillium citrinum KJZ101, isolated from China's Tiangong space station, underwent long-term mutations induced by the low-radiation and weightless conditions of the space environment, resulting in unique metabolic characteristics compared to terrestrial strains. This was confirmed by testing of the *Penicillium citrinum* strain described in this invention. Penicillium citrinum KJZ101 produces pectinase with high activity; a fermentation broth containing 792 U / mL of pectinase can be harvested after 48 hours of culture. This invention describes the *Penicillium citrinum* species... Penicillium lemon KJZ101 grows rapidly; it reaches the requirements for surface spraying on cotton stalks within 24 hours in PDA liquid medium, and can colonize the cotton stalk surface after spraying. This invention describes the *Penicillium citrinum* species. Penicillium citrinum KJZ101 does not aggregate and grow in PDA medium, and the culture medium can be directly sprayed by drones, avoiding clogging of drone nozzles. This invention describes the *Penicillium citrinum* species. Penicillium citrinum KJZ101 prioritizes the use of pectin rather than cellulose, which helps improve the degumming effect of cotton stalks and avoids damage to the cotton stalk fibers. Attached Figure Description

[0016] Figure 1 Penicillium citrinum in the embodiments of the present invention Penicillium citrinum Colony morphology diagram of KJZ101.

[0017] Figure 2 Penicillium citrinum in the embodiments of the present invention Penicillium citrinum Phylogenetic analysis diagram of KJZ101.

[0018] Figure 3 Penicillium citrinum in the embodiments of the present invention Penicillium citrinum Qualitative diagram of the pectin decomposition activity of KJZ101.

[0019] Figure 4 Penicillium citrinum in the embodiments of the present invention Penicillium citrinum Image showing the planting effect of KJZ101 on the cotton stalk epidermis. Detailed Implementation

[0020] The embodiments of the present invention will be further described below with reference to the accompanying drawings: This invention discloses a strain of Penicillium citrinum, wherein Penicillium citrinum... Penicillium citrinum The strain was isolated from the Tiangong space station in China. The depository is the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The strain name is KJZ101, the accession number is CGMCC No. 42422, and the deposit date is January 9, 2026.

[0021] Using Penicillium citrinum Penicillium citrinum The fermentation broth of KJZ101 was used to remove pectin from cotton stalks, and the results showed good application effects.

[0022] 1. Penicillium citrinum Penicillium citrinum Identification of KJZ101: Morphological identification: As shown in the figure, typical colonies were formed after incubation at 28°C for 3 days on PDA medium. Figure 1 On the left side, the colony is nearly circular with a regular outline. It is dark green on the front, with grayish-white hyphae visible in the center and white edges. Figure 1 As can be seen on the right, the back of the culture medium is yellowish-green and has a wrinkled shape.

[0023] b. Molecular biological identification: 50 mg of fresh mycelium was ground in liquid nitrogen, and genomic DNA was extracted using a kit. Using this genomic DNA as a template, PCR was performed using universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') to amplify the ITS fragment. After sequencing the fragment, BLAST alignment analysis was performed using NCBI, and the results showed that the strain's ITS sequence was consistent with... Penicillium citrinum NRRL1841 The ITS sequence showed the highest identity, reaching 99.41%; a phylogenetic tree of the ITS sequence was constructed using MEGA 7.0 software, revealing that this strain was related to... Penicillium citrinum The strain was most closely related. Based on morphological characteristics, the isolated strain was identified as... Penicillium citrinum ,like Figure 2 As shown.

[0024] 2. Penicillium citrinum Penicillium citrinum Analysis of the growth and enzyme activity of KJZ101: a. Qualitative analysis of pectinase activity: The bacterial strain was inoculated onto solid pectin medium and incubated at 28°C for 3 days. The plates were then stained with Lugol's iodine solution, and the extracellular pectinase activity was qualitatively analyzed by observing the formation of the clear zone. Figure 3 As shown. The pectin culture medium consists of: 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, FeSO4 0.1 g / L, and agar 18 g / L.

[0025] b. Quantitative analysis of pectinase activity: The strain was inoculated into the enzyme-producing fermentation medium and cultured in a shaker at 200 rpm and 28℃ for 48 h to obtain the enzyme-producing fermentation broth; the fermentation broth was then incubated at 4℃ and 10000... gCentrifuge for 10 min under the specified conditions, and use the supernatant as the enzyme solution. Prepare a 0.2% pectin solution using 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 react at 50℃ for 10 min. Immediately add 200 μL of DNS reagent to terminate the reaction. Mix the system well and place it in a boiling water bath for 5 min. After cooling, add 1 mL of deionized water to dilute. Measure the absorbance of the reaction solution at 520 nm, and calculate the product release and enzyme activity according to 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. The enzyme-producing fermentation medium consists of: 10 g / L dried 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.

[0026] c. Pectin-cellulose metabolism analysis: The strain was inoculated into metabolic medium and cultured in a shaker at 200 rpm and 28℃ for 48 h; the culture solution was then incubated at 4℃ and 10000... g Centrifuge for 10 min under the specified conditions, and dialyze the supernatant in a dialysis bag with a molecular weight cutoff of 1000 Da, followed by lyophilization. Weigh 0.1 g of the lyophilized powder, mix it with 1 mL of 72% H2SO4, incubate at 30℃ for 1 h, then dilute to 30 mL, and incubate the system at 121℃ for 1 h to obtain the hydrolysate. HPLC was used to determine the galacturonic acid and glucose content in the hydrolysate, and the results were compared with the uninoculated control medium to analyze the metabolic preferences of the strain. The metabolic medium consisted of: pectin 5 g / L, carboxymethyl cellulose 5 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.

[0027] 3. Utilizing Penicillium citrinum Penicillium citrinum KJZ101 degumming fermentation broth treatment of cotton stalks: a. Microbial colonization analysis: Cotton stalks were cut into approximately 8 cm lengths and laid flat in a petri dish; the degummed fermentation broth was poured into a spray bottle and sprayed onto the surface of the cotton stalks at a ratio of 0.5 mL fermentation broth / g cotton stalk; subsequently, the cotton stalks were left to stand at room temperature for 3 days, and the microbial colonization on their surface was observed. Figure 4 As shown.

[0028] b. Degumming effect test: The degumming fermentation broth was diluted at 10000... gCentrifuge at 4℃ for 10 min and collect the supernatant. Wash and dry the cotton stalk outer skin with water, then immerse the outer skin in the fermentation supernatant at a 1:1 (w / v) ratio and incubate at 28℃ and 100 rpm for 6 h. Wash and dry the treated outer skin with water. Weigh the dry weight of the outer skin before and after treatment to calculate the degumming rate: Degumming rate = [(dry weight before treatment - dry weight after treatment) / dry weight before treatment] × 100% 5. Culture medium preparation: PDA medium: Weigh 200 g of potato chunks, 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. After cooling, bring the volume to 1 L. Set the pH to natural and sterilize at 115℃ for 30 min. When preparing PDA solid medium, add 20 g of agar powder to the above PDA medium. After sterilization, pour into plates.

[0029] 6. Preparation of degummed fermentation broth: [The text abruptly ends here, likely due to an incomplete sentence or missing information.] Penicillium citrinum KJZ101 cells were inoculated onto PDA plates and incubated statically at 28°C for 72 h. Subsequently, cells were picked from the plates and transferred to PDA liquid medium, and cultured on a shaker at 200 rpm and 28°C for 24 h to obtain the degummed fermentation broth.

[0030] 7. Treating cotton stalks with degumming fermentation liquid: a. Microbial colonization analysis: Cotton stalks were cut into lengths of approximately 8 cm and laid flat in a container. The degumming fermentation broth was poured into a spray bottle and sprayed onto the surface of the cotton stalks at a ratio of 0.5 mL fermentation broth / g cotton stalk. The cotton stalks were then left to stand at room temperature for 3 days, and the microbial colonization on their surface was observed. The results showed that spraying the fermentation broth could induce significant bacterial colonization on the surface and improve the degumming effect.

[0031] b. Degumming effect test: The degumming fermentation broth was diluted at 10000... g Centrifuge at 4℃ for 10 min and collect the supernatant; wash and dry the cotton stalk epidermis with water, then immerse the epidermis in the fermentation supernatant at a ratio of 1:1 (w / v) and incubate at 28℃ and 100 rpm for 6 h; wash and dry the treated epidermis with water. Weigh the epidermis before and after treatment to calculate the degumming rate, and the results show that the degumming rate is significantly improved.

Claims

1. A strain of Penicillium citrinum, characterized by: The Penicillium citrinum Penicillium citrinum The strain was isolated from the Tiangong space station in China. The depository is the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The strain name is KJZ101, the accession number is CGMCC No. 42422, and the deposit date is January 9, 2026.

2. An application of Penicillium citrinum as described in claim 1, characterized in that: Penicillium citrinum Penicillium citrinum KJZ101 removes pectin from cotton stalks.

3. The application according to claim 2, characterized in that: Using Penicillium citrinum Penicillium citrinum The pectinase produced by fermentation of KJZ101 can depectinize hemp.

4. An application of the pectinase described in claim 3 in hemp degumming, characterized in that, Penicillium citrinum Penicillium citrinum KJZ101 was inoculated into PDA medium and cultured at 200 rpm and 28℃ for 24 h to obtain degummed fermentation broth. The degummed fermentation broth was sprayed onto cotton stalks, and obvious bacterial colonization occurred on the surface of the cotton stalks, which improved the degumming effect.

5. The application according to claim 4, characterized in that, The specific steps for treating cotton stalks using degumming fermentation broth are as follows: a1. Microbial colonization analysis: Cotton stalks were cut into 8cm lengths and laid flat in a petri dish. The degummed fermentation broth was sprayed onto the surface of the cotton stalks at a ratio of 0.5 mL fermentation broth / g cotton stalk. The cotton stalks were then left to stand at room temperature for 3 days to observe the microbial colonization on the surface of the cotton stalks. a2. Degumming effect determination: The degumming fermentation broth was diluted at 10000... g Centrifuge at 4℃ for 10 min and collect the supernatant; wash the cotton stalk skin with clean water and dry it, then immerse the cotton stalk skin in the fermentation supernatant at a ratio of 1:1 (w / v) and incubate at 28℃ and 100 rpm for 6 h to achieve degumming.

6. The application according to claim 5, characterized in that, Wash the treated cotton stalks with clean water and let them dry. Weigh the dry weight of the cotton stalks before and after treatment to calculate the degumming rate: Degumming rate = [(dry weight before treatment - dry weight after treatment) / dry weight before treatment] × 100%.

7. The Penicillium citrinum of claim 1 Penicillium citrinum The analytical method for KJZ101 is characterized by, The steps are as follows: b1. Qualitative analysis of pectinase activity: [The text abruptly ends here, likely due to an incomplete sentence or a missing section.] Penicillium citrinum KJZ101 was inoculated onto solid pectin medium and cultured. After incubation, the cells were stained with Lugol's iodine solution to qualitatively analyze the extracellular pectinase activity. b2. Quantitative analysis of pectinase activity: [The text abruptly ends here, likely due to an incomplete sentence or missing information.] Penicillium citrinum KJZ101 was inoculated into enzyme-producing fermentation medium and cultured on a shaker at a constant temperature to obtain enzyme-producing fermentation broth. The supernatant of the fermentation broth was collected by centrifugation as enzyme solution. A 0.2% pectin solution was prepared using pH 5.0 phosphate buffer as substrate, and the enzyme solution was diluted. 100 μL of substrate solution and 50 μL of enzyme dilution were added to a centrifuge tube and mixed well. The mixture was then reacted at 50℃ for 10 min, and 200 μL of DNS reagent was added immediately to terminate the reaction. The system was mixed and placed in a boiling water bath for 5 min. After cooling, 1 mL of deionized water was added to dilute the reaction solution. The absorbance of the reaction solution at 520 nm was measured, and the product release and enzyme activity were calculated according to the standard curve. b3. Pectin-cellulose metabolism analysis: Penicillium... Penicillium citrinum KJZ101 was inoculated into metabolic culture medium and cultured in a shaker at a constant temperature; The culture medium was incubated at 4℃ for 10,000... g Centrifuge for 10 min under the specified conditions, place the supernatant in a dialysis bag with a molecular weight cutoff of 1000 Da for dialysis, and then freeze-dry. Weigh 0.1 g of the freeze-dried powder, mix it with 1 mL of 72% H2SO4, incubate at 30℃ for 1 h, then dilute to 30 mL, and incubate the system at 121℃ for 1 h to obtain the hydrolysate. Determine the content of galacturonic acid and glucose in the hydrolysate by high performance liquid chromatography (HPLC), and compare it with the uninoculated control medium to analyze the metabolic preferences of the strain.

8. The analytical method according to claim 7, characterized in that, The solid pectin culture medium consists of: 10 g / L pectin, 1.5 g / L (NH4)2SO4, 1.0 g / L K2HPO4, 0.5 g / L KCl, 0.5 g / L MgSO4, 0.1 g / L FeSO4, and 18 g / L agar.

9. The analytical method according to claim 7, characterized in that, The enzyme-producing fermentation medium consisted of: 10 g / L dried 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.

10. The analytical method according to claim 7, characterized in that, The metabolic culture medium consisted of: 5 g / L pectin, 5 g / L carboxymethyl cellulose, 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.

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