White-spore aspergillus niger and application thereof

Abstract

This application belongs to the field of cellulose-degrading strains, specifically disclosing a white-spore Aspergillus niger and its applications. The white-spore Aspergillus niger is classified as... Aspergillus niger The name is Aspergillus niger B704 was deposited on January 6, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 65633. This application discloses a white-sporeed Aspergillus niger. Aspergillus niger B704 exhibits cellulose and hemicellulose degradation rates of 82% and 60%, respectively, significantly higher than existing white-spore Aspergillus niger. It also possesses the synergistic characteristics of "high enzyme production + white spores," achieving an effective combination of "morphological markers" and "functional specificity," making it suitable for applications in agriculture, food industry, and bioenergy.

Description

Technical Field

[0001] This application belongs to the field of cellulose-degrading strains, specifically relating to a white-spore Aspergillus niger and its applications. Background Technology

[0002] In existing technologies, *Aspergillus niger* is a commonly used cellulase-producing strain, and most research focuses on increasing cellulase production. Existing high-cellulase-producing *Aspergillus niger* strains (such as mutagenized or genetically engineered strains) mostly produce black spores (due to the presence of melanin), but black spores may consume metabolic resources due to melanin accumulation (Donatella Tesei, Encyclopedia, 2022). In *Aspergillus niger* breeding research, researchers have also obtained spore color mutants, such as white spores, through spontaneous or induced mutations. However, existing white-spore *Aspergillus niger* strains generally suffer from low cellulase production and slow growth rates. Moreover, most researchers focus on the morphological variation mechanisms of white-spore *Aspergillus niger* mutants (such as the deletion of melanin synthesis genes), with the core objective being to use spore color as a "visual screening marker" (facilitating strain purification). Research on how to improve the enzyme production capacity of white-spore *Aspergillus niger* is severely lacking.

[0003] Therefore, it would be very meaningful to screen for a white-sporeed Aspergillus niger with high cellulase production. Summary of the Invention

[0004] The purpose of this application is to address at least one deficiency of the prior art. Based on this, the first aspect of this application provides a white-spore Aspergillus niger, the classification of which is named... Aspergillus niger The name is Aspergillus niger B704 was deposited on January 6, 2025, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC) with accession number GDMCC No. 65633. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0005] The cellulose degradation rate and hemicellulose degradation rate of *Aspergillus niger* var. *white* in this application can reach 82% and 60%, respectively, which are significantly higher than those of existing *Aspergillus niger* var. *white*. Aspergillus nigerB704 possesses the synergistic characteristics of "high enzyme production + white spores," overcoming the complex steps required for enzyme activity testing and other verification processes in strain screening of black-spore Aspergillus niger. It also overcomes the problem that while white-spore Aspergillus niger is easily distinguishable by morphological observation, its lack of high-yield characteristics makes it difficult to directly use in industrial production. This achieves an effective combination of "morphological markers" and "functional specificity," making it applicable to agriculture (straw degradation), food industry (cellulose processing), and bioenergy (biomass conversion). Technically, it enables one-step, efficient degradation of biomass, simplifying breeding and identification; industrially, it reduces costs and promotes the large-scale utilization of biomass resources; in terms of safety, it reduces occupational exposure and pollution risks, making it suitable for the food and feed industries; and it also enriches genetic resources, contributing to safe industrial development. Furthermore, in the field of Aspergillus niger breeding, current methods mainly rely on traditional mutagenesis techniques such as ultraviolet mutagenesis and nitrosoguanidine (NTG) chemical mutagenesis, or spontaneous mutation or mutagenesis through genetic engineering techniques to modify strains. High-yielding strains obtained through these traditional mutagenesis breeding methods often suffer from poor genetic stability. Although genetically engineered strains have high yields, they require strict aseptic operation and gene editing tools, resulting in high production costs. Moreover, some engineered strains have weak adaptability to industrial fermentation environments (such as high temperature and high substrate concentration). This application uses heavy ion mutagenesis to select and breed white-spore Aspergillus niger, which effectively overcomes the above problems.

[0006] Therefore, the white spores of Aspergillus niger in this application Aspergillus niger B704, or its active ingredient as a microbial agent, can be better applied in the degradation of cellulose and / or hemicellulose, improving the degradation rate of cellulose and / or hemicellulose; and based on Aspergillus niger (white spores)... Aspergillus niger B704 forms a method for degrading cellulose and / or hemicellulose.

[0007] The above-mentioned methods for degrading cellulose and / or hemicellulose include culturing the above-mentioned white-spore Aspergillus niger in a fermentation medium. Aspergillus niger B704; Preferably, the above-mentioned white spore Aspergillus niger. Aspergillus nigerSeed culture of B704 grown to the logarithmic growth phase was inoculated into the above-mentioned fermentation medium at an inoculum volume of 5%-10%. The fermentation medium consisted of: 70 g / L corn husks, 30 g / L wheat bran, 1.5 g / L ammonium sulfate, 1.0 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, and 0.1 g / L calcium chloride. The culture conditions were: 72 h culture time, 30℃ culture temperature, 180 r / min rotation speed, and an initial pH of 5.9-6.2. The above seed culture was obtained by culturing the white spores of Aspergillus niger on a seed culture medium, which included: sodium nitrate, dipotassium hydrogen phosphate, magnesium sulfate, potassium chloride, ferrous sulfate, sucrose, and agar. Specifically, the content of sodium nitrate was 3 g / L, the content of dipotassium hydrogen phosphate was 1 g / L, the content of magnesium sulfate was 0.5 g / L, the content of potassium chloride was 0.5 g / L, the content of ferrous sulfate was 0.01 g / L, the content of sucrose was 30 g / L, and the content of agar was 20 g / L.

[0008] The beneficial effects of this application are as follows: This application proposes a white-spore Aspergillus niger. Aspergillus niger B704 exhibits cellulose and hemicellulose degradation rates of 82% and 60%, respectively, significantly higher than existing Aspergillus niger species. This application relates to the Aspergillus niger species described in this application. Aspergillus niger B704 possesses the synergistic characteristics of "high enzyme production + white spores," achieving an effective combination of "morphological markers" and "functional specificity," making it applicable to agriculture (straw degradation), food industry (cellulose processing), and bioenergy (biomass conversion). Furthermore, this application simplifies breeding identification, reduces industrial costs, and promotes the large-scale utilization of biomass resources; it also reduces occupational exposure and pollution risks, making it suitable for the food and feed industries; and it enriches genetic resources, contributing to safe industrial development. Attached Figure Description

[0009] Figure 1 The figure shown is a heavy ion mutagenesis lethality curve of Aspergillus niger. Figure 2 The image shown is a colony diagram of Aspergillus niger, whose spores are white. Figure 3 The bar chart shown is a graph of the degradation rates of cellulose and hemicellulose of eight Aspergillus niger strains. Figure 4 The image shows white-sporeed Aspergillus niger. Aspergillus niger Figure showing the xylanase activity of B704; Figure 5 The image shows white-sporeed Aspergillus niger. Aspergillus niger Figure showing the endoglucanase activity of B704; Figure 6 The image shows white-sporeed Aspergillus niger. Aspergillus nigerFigure showing the exoglucanase activity of B704; Figure 7 The image shows white-sporeed Aspergillus niger. Aspergillus niger Image showing the enzyme activity of filter paper enzymes in B704; Figure 8 The image shows white-sporeed Aspergillus niger. Aspergillus niger Figure showing the laccase activity of B704; Figure 9 The image shows white-sporeed Aspergillus niger. Aspergillus niger Figure showing the genetic stability analysis results of heavy ion mutagenesis strain B704. Detailed Implementation

[0010] The following will provide a clear and complete description of the concept and technical effects of this application in conjunction with the embodiments and accompanying drawings, so as to fully understand the purpose, solution and effects of this application. It should be noted that, unless otherwise specified, the embodiments and features in the embodiments of this application can be combined with each other.

[0011] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0012] Example 1 1. Heavy ion irradiation-induced mutagenesis Aspergillus niger H001 (from bovine rumen fluid from the Xikouzi Slaughterhouse in Xincheng District, Hohhot, Inner Mongolia) was inoculated onto agar plates. After culturing for 3 days, the spores of the original strain H001 were washed off with sterile water and quickly dispersed. One mL of the spore suspension was placed in a 35 mm irradiation dish and subjected to a heavy ion beam with an energy of 80 MeV / u provided by the Lanzhou Heavy Ion Accelerator National Laboratory (CSR-HIRFL). 12 C 6+ The spore suspensions in the irradiation dish were irradiated with irradiation doses of 0 Gy, 40 Gy, 80 Gy, 120 Gy, 160 Gy, and 200 Gy, respectively. The lethality rate was calculated as follows: Lethality (%) = ((number of viable bacteria before mutation - number of viable bacteria after mutation) / number of viable bacteria before mutation) * 100%.

[0013] The results are as follows: Within the stable range of heavy ion beam control in this experiment, a lethality curve was plotted with relative irradiation dose as the x-axis and lethality rate as the y-axis. The results are as follows: Figure 1 As shown. By Figure 1 It can be seen that, 12 C 6+Within the heavy ion irradiation dose range of 0 Gy to 200 Gy, the lethality curve exhibited a "saddle-shaped" curve, first decreasing, then increasing, and then decreasing again, with increasing irradiation dose. At an irradiation dose of 160 Gy, the lethality was 82%, but at 190 Gy, the bacterial mortality briefly decreased, forming a noticeable bulge. Subsequently, with further increases in irradiation dose, the bacterial lethality decreased significantly again. This unique "saddle-shaped" curve is considered to be the result of the combined effects of damage caused by energy and momentum, and protection and stimulation caused by mass and charge. This indicates that the irradiation dose range selected in this experiment was reasonable. To improve screening efficiency, based on the higher positive mutation rate within a lethality range above 80%, bacterial suspensions irradiated at 160 Gy were plated on screening plates. Single colonies with rapid growth, white spores, and large spore rings were selected for further testing.

[0014] 2. Initial screening for Aspergillus niger The irradiated spore solution and the control group bacterial solution were diluted according to the required ratio. Since the optimal colony count in the culture dish during the initial screening process is 3-5 colonies, and the irradiated bacteria exhibit varying degrees of mortality, the dilution factors varied, with dilutions occurring sequentially at irradiation doses of 0 Gy, 40 Gy, 80 Gy, 120 Gy, 160 Gy, and 200 Gy. After dilution, the bacterial solution was thoroughly shaken, and 0.1 μL of the solution was pipetted onto an aesculin plate. The plate was sealed with sealing film and incubated at 30℃ for 5 days. Three parallel groups were established for each irradiation dose. Spore color and colony diameter were observed, and strains with white spores and large colony diameters were selected as the initial screening strains.

[0015] The results are as follows: Aspergillus niger was mutagenized by heavy ion beam irradiation, and the resulting bacterial suspensions were further mutagenized by ion beams of different energies and diluted to 10⁻⁶. -3 10 -3.5 10 -4 Take 0.1 mL of each of the three dilutions, add them dropwise onto a esculin solid medium plate, spread them evenly, invert the plate, and incubate for 2 days. Afterward, pick strains with obvious color zones on the esculin plates and observe the enzyme-producing color zones of each mutant strain. Transfer to PDA solid plates and incubate for 5 days. The colony diagram is shown below. Figure 2 As shown in Table 1, strains with larger mycelial circles were selected for secondary screening.

[0016] Table 1. HC values ​​of various bacterial colonies irradiated by heavy ion beam

[0017] As shown in Table 1, after heavy ion irradiation mutagenesis, a total of 41 mutant strains were obtained, numbered B701-B741. These strains were carefully streaked onto esculin agar to ensure the appearance of single colonies and discoloration zones, and incubated at 30 ℃ for 48 h. The ratio of the discoloration zone diameter to the Aspergillus niger colony diameter (HC value) is a direct indicator of the strain's performance. Aesculin can bind firmly to cellulose, a large polysaccharide. Cellulase degrades cellulose in the plate into smaller sugar molecules. Congo red cannot bind to these smaller sugar molecules and is thus eluted, resulting in a clear zone. Generally, a higher HC value indicates higher yield and performance; a lower HC value indicates lower yield and performance. Based on the HC value, dominant Aspergillus niger strains were selected to screen for mutant strains. Single colonies after irradiation mutagenesis were initially screened by plate culture, and the HC value of each colony was calculated. The results are shown in Table 1. The growth performance of the strains was greatly altered. Due to the uncertainty of mutations after heavy ion irradiation, the screened strains exhibited both positive and negative mutations. Among them, three strains had negative HC values, indicating significantly lower performance than the original strain. The remaining strains all had positive HC values, although some of these changes were not significant. A total of seven strains showed an increase in diameter compared to the original strain. Therefore, these seven strains were preserved for further research.

[0018] 3. Aspergillus niger rescreening The strains obtained from the initial screening were added to seed culture medium and cultured until the logarithmic growth phase to obtain seed solution (OD). 600 =0.8), 100 mL of fermentation medium was placed in a 250 mL Erlenmeyer flask, and the seed culture (OD) was... 600 =0.8) was inoculated into the fermentation medium at a 5% inoculum and cultured at a temperature of 30℃ and a shaking speed of 180 rpm for 72 h to obtain the fermentation sample, and its cellulose degradation rate was determined.

[0019] The seed culture medium consists of: sodium nitrate 3 g / L, dipotassium hydrogen phosphate 1 g / L, magnesium sulfate 0.5 g / L, potassium chloride 0.5 g / L, ferrous sulfate 0.01 g / L, sucrose 30 g / L, and agar 20 g / L. The fermentation culture medium consists of: corn husk 70 g / L, wheat bran 30 g / L, ammonium sulfate 1.5 g / L, potassium dihydrogen phosphate 1.0 g / L, magnesium sulfate heptahydrate 0.5 g / L, and calcium chloride 0.1 g / L.

[0020] The method for determining the cellulose degradation rate includes the following steps: (1) Centrifuge the fermentation sample at 8000 rpm for 10 min, collect the precipitate and dry it to constant weight, and measure its weight change. Three parallel samples are used for each group.

[0021] (2) Weigh 0.025 g of the dried sample, add 0.25 mL of 74% sulfuric acid, and keep the mixture at 30 °C for 60 min. Stir constantly during the process to ensure that the cellulose and hemicellulose are fully degraded. After keeping the mixture at 30 °C for 60 min, immediately place the mixture in an ice bath to stop the reaction, and add 7 mL of distilled water.

[0022] (3) Determination of sugar loss due to sulfuric acid: Accurately weigh 0.050 g of glucose and 0.050 g of xylose, three parallel samples, add 1 mL of sulfuric acid with a mass fraction of 74%, keep in a water bath at 30 ℃ for 60 min, then immediately put in an ice bath, add 28 mL of distilled water and place.

[0023] (4) Seal the roller tube containing the above sample and boil it in an autoclave for 60 min. To prevent sugar degradation, when the temperature drops to 100℃ (pressure is 0 Pa), immediately remove the sample, shake it well and let it stand.

[0024] (5) After the sample has cooled, centrifuge it, take the supernatant, filter it with a 0.22 μm water-based needle filter, and determine the glucose and xylose content using high performance liquid chromatography. Calculate the mass of cellulose and hemicellulose from the concentration of monosaccharides, and calculate the degradation rate of cellulose and hemicellulose.

[0025] The chromatographic conditions are as follows: Mobile phase: 0.05 mol / L H2SO4 (pH=2); Injection volume: 20 μL; Mobile phase flow rate: 0.5 mL / min; Column temperature: 50 ℃; Column: Aminex HPX-87H column (300 mm × 7.8 mm id, 9 μm) (Bio-Rad Laboratories, USA); RI differential detector; Chromatograph: 1515breeze Aminex high performance liquid chromatograph (Waters Corporation, USA).

[0026] The calculation methods for the degradation rate of cellulose and hemicellulose are as follows: Mass of sugar in the sample:

[0027] The mass of remaining dextran:

[0028] Mass of remaining xylan:

[0029] Cellulose degradation rate in the sample

[0030] Hemicellulose degradation rate in the sample

[0031] The degradation results of cellulose and hemicellulose are as follows: Figure 3 As shown, by Figure 3 It can be seen that, compared with the original strain H001, the cellulose degradation rate and hemicellulose degradation rate of all 7 strains were improved, among which the mutant strain Aspergillus niger showed the most significant improvement. Aspergillus niger B704 exhibited the highest cellulose and hemicellulose degradation rates, at 82% and 60%, respectively. Compared to H001, the cellulose degradation rate increased by 41.38%, and the enzyme activity also increased by 42.86%. This is likely because heavy ion mutagenesis altered genes related to cellulase production, leading to increased yield. Compared to the initial screening results, the H / C ratio showed a positive correlation with the cellulose degradation rate, indicating the reliability of the initial screening results.

[0032] In addition, the activities of xylanase, endoglucanase, exoglucanase, filter paper enzyme, and laccase were also measured. The measurement methods are as follows: (1) Xylanase activity assay The mutant strain Aspergillus niger with white spores Aspergillus niger B704 was added to seed culture medium (sodium nitrate 3 g / L, dipotassium hydrogen phosphate 1 g / L, magnesium sulfate 0.5 g / L, potassium chloride 0.5 g / L, ferrous sulfate 0.01 g / L, sucrose 30 g / L, agar 20 g / L) and cultured until the logarithmic growth phase to obtain the seed culture (OD). 600 =0.8), 100 mL of xylanase production medium was placed in a 250 mL Erlenmeyer flask, and the seed culture (OD) was added. 600 =0.8) was inoculated at a rate of 5% into the xylanase production medium and cultured at 30°C and 180 rpm for 72 h to obtain the fermentation sample. The xylanase production medium consisted of: 10 g / L birch xylan, 4 g / L ammonium sulfate (or 4 g / L yeast extract in other embodiments), and 0.8 g / L potassium dihydrogen phosphate (or 0.4 g / L magnesium sulfate in other embodiments).

[0033] The reaction system consisted of 50 μL of supernatant from the fermentation sample as crude enzyme solution, 100 μL of 1% xylan (beech) solution, mixed well, reacted at 50℃ for 30 min, 200 μL of DNS solution was added to terminate the reaction, boiled for 10 min, allowed to stand and cool, and then diluted to 1.25 mL with distilled water. The mixture was shaken well and the OD value was measured at a wavelength of 540 nm.

[0034] The results are as follows Figure 4 As shown, by Figure 4 It can be seen that the mutant strain *Aspergillus niger* with white spores... Aspergillus nigerThe xylanase activity of B704 gradually increased from 20 h to 50 h, reaching its maximum at 50 h. This indicates that the strain was in the logarithmic growth phase or the peak enzyme production phase during this period. The cells proliferated rapidly, and the expression of xylanase synthesis-related genes (such as xynA and xynB) was active. Metabolic resources were preferentially allocated to enzyme synthesis and secretion. At the same time, the carbon and nitrogen sources in the culture medium were sufficient, and there was no substrate limitation or metabolite inhibition. Therefore, the xylanase activity continued to increase and reached its peak at 50 h, reflecting that the enzyme production capacity of the strain reached its optimal state at this time. Between 50 and 100 hours, the xylanase activity of the mutant strain gradually decreased, indicating that the strain had entered a stationary or apoptotic phase. Nutrients in the culture medium were gradually depleted, cell growth slowed or even stopped, and the supply of precursors for xylanase synthesis (amino acids, ATP, etc.) was insufficient. Simultaneously, byproducts such as organic acids and ammonia accumulated during metabolism may inhibit the expression of enzyme-related genes or lead to enzyme protein degradation (e.g., protease hydrolysis). Furthermore, high concentrations of xylanase products may also reduce their own synthesis rate through feedback inhibition mechanisms, ultimately resulting in a gradual decrease in enzyme activity. In summary, these results clearly identify the mutant strain *Aspergillus niger*. Aspergillus niger The optimal harvest time for xylanase production from B704 is 50 h (the peak enzyme activity point). This conclusion can provide a key basis for subsequent fermentation process optimization (such as controlling the fermentation cycle and feeding strategies), avoid enzyme activity loss due to excessive fermentation time, and improve the efficiency and benefits of industrial production.

[0035] (2) Assay of endoglucanase activity The mutant strain Aspergillus niger with white spores Aspergillus niger B704 was added to seed culture medium (sodium nitrate 3 g / L, dipotassium hydrogen phosphate 1 g / L, magnesium sulfate 0.5 g / L, potassium chloride 0.5 g / L, ferrous sulfate 0.01 g / L, sucrose 30 g / L, agar 20 g / L) and cultured until the logarithmic growth phase to obtain the seed culture (OD). 600 =0.8), 100 mL of endoglucanase production medium was placed in a 250 mL Erlenmeyer flask, and the seed culture (OD) was added. 600 =0.8) was inoculated at a rate of 5% into the endoglucanase production medium and cultured at 30°C and 180 rpm for 72 h to obtain the fermentation sample. The endoglucanase production medium consisted of: sodium carboxymethyl cellulose 10 g / L, peptone 3 g / L (in other embodiments, yeast extract 5 g / L can also be selected), and potassium dihydrogen phosphate 1 g / L (in other embodiments, magnesium sulfate 0.5 g / L can also be selected).

[0036] Take 50 μL of the supernatant of the fermentation sample and add it to a centrifuge tube containing 150 μL of 0.5% sodium carboxymethyl cellulose (CMC-Na). React at 50℃ for 30 min. Add 50 μL of 1M NaOH solution and 150 μL of 3,5-dinitrosalicylic acid (DNS) solution to the centrifuge tube to terminate the reaction. Vortex to thoroughly mix the reaction solution, then boil in a water bath for 5 min. Terminate the reaction with an ice-water mixture. Adjust the volume of the reaction solution to 1250 μL and measure its absorbance at 540 nm. Calculate the enzyme activity based on the standard curve of endoglycanase activity.

[0037] The result is as follows Figure 5 As shown; by Figure 5 It can be seen that the mutant strain *Aspergillus niger* with white spores... Aspergillus niger The activity of B704 endoglucanase gradually increased from 20 to 60 hours, reaching its maximum at 60 hours. This indicates that the strain was in its vigorous growth phase (logarithmic growth phase to early stationary phase) during this period, with active cell metabolism, gradually increasing expression levels of genes related to endoglucanase synthesis (such as EGL family genes), and sufficient carbon and nitrogen sources in the culture medium, without substrate deficiency or metabolic inhibition, providing favorable conditions for enzyme synthesis and secretion. The peak activity at 60 hours signifies that the strain's ability to produce endoglucanase has reached its optimal state, at which point the enzyme synthesis rate and secretion efficiency are in balance. After 60 hours, the activity gradually decreases. After 60 hours, key nutrients such as carbon and nitrogen sources in the culture medium have been largely consumed, failing to provide sufficient precursors (such as amino acids and nucleotides) and energy (ATP) for the continuous synthesis of endoglucanase. The strain's metabolic focus shifts from "enzyme synthesis" to "maintaining cell survival," leading to a significant decrease in the enzyme synthesis rate, while existing enzymes continue to degrade (such as protease hydrolysis), ultimately resulting in reduced activity. Furthermore, in the later stages of fermentation, the strain accumulates metabolic byproducts such as organic acids (e.g., citric acid, acetic acid) and ammonia. These substances not only alter the pH of the fermentation system (deviating from the optimal pH for endoglucanase), but may also directly inhibit the expression of enzyme-related genes (egl family) or disrupt the spatial structure of the enzyme protein, leading to its inactivation and further exacerbating the decline in enzyme activity. In summary, this result further clarifies that the optimal harvest window for endoglucanase is 60 hours, and also points the way for optimizing the fermentation process, thereby extending the peak enzyme activity period and improving overall enzyme production efficiency.

[0038] (3) Determination of exoglucanase activity The mutant strain Aspergillus niger with white spores Aspergillus nigerB704 was added to seed culture medium (sodium nitrate 3 g / L, dipotassium hydrogen phosphate 1 g / L, magnesium sulfate 0.5 g / L, potassium chloride 0.5 g / L, ferrous sulfate 0.01 g / L, sucrose 30 g / L, agar 20 g / L) and cultured until the logarithmic growth phase to obtain the seed culture (OD). 600 =0.8), 100 mL of exoglucanase production medium was placed in a 250 mL Erlenmeyer flask, seed culture (OD) 600 =0.8) was inoculated at a rate of 5% into the exoglucanase production medium and cultured at 30°C and 180 rpm for 72 h to obtain the fermentation sample. The exoglucanase production medium consisted of: 8 g / L microcrystalline cellulose, 3 g / L ammonium sulfate (or 2 g / L yeast extract in other embodiments), and 1 g / L dipotassium hydrogen phosphate (or 0.3 g / L calcium chloride in other embodiments).

[0039] Take 500 μL of the supernatant from the fermentation sample as the crude enzyme solution, add 50 μL of the substrate p-nitrophenyl cellulose biglycoside (pNPC, 1 mg / mL), mix well, react at 50 ℃ for 30 min, and then add 150 μL of 10% Na2CO3 to terminate the reaction. Calculate the enzyme activity by measuring the amount of p-nitrophenol produced at a wavelength of 405 nm.

[0040] The result is as follows Figure 6 As shown; by Figure 6 It can be seen that the mutant strain *Aspergillus niger* with white spores... Aspergillus niger The exoglucanase activity of strain B704 gradually increased from 20 h to 60 h, reaching its maximum at 60 h. During the 20 h-60 h period, the strain was in a nutrient-rich, vigorous growth phase, with active expression of genes related to exoglucanase synthesis, and a continuous increase in the rate of enzyme synthesis and secretion. Enzyme production capacity reached its peak at 60 h. After 60 h, the exoglucanase activity of the strain decreased, indicating that key nutrients such as carbon sources (e.g., cellulose, inducers), nitrogen sources, and phosphorus sources in the culture medium had been largely depleted. On the one hand, the amino acids (enzyme protein precursors) and ATP (energy source) required for exoglucanase synthesis could not be continuously supplied due to nutrient scarcity, leading to a sharp decline in the rate of new enzyme synthesis. On the other hand, the metabolic focus of the strain shifted from "enzyme production" to "maintaining cell survival," reducing the priority of resource allocation and further inhibiting enzyme-related pathways.

[0041] (4) Determination of enzyme activity on filter paper The filter paper enzyme activity (FPU) was determined using the DNS colorimetric method. Using Whatman No. 1 standard filter paper as the substrate, the diluted enzyme solution (obtained after fermentation in the above-mentioned fermentation medium) was mixed with buffer and filter paper strips under the conditions of pH 4.8 and 50 ℃ and reacted for 60 min. The reducing sugar produced by enzymatic hydrolysis was developed by the DNS reagent in a boiling water bath, and the absorbance was measured at a wavelength of 540 nm. The concentration of reducing sugar was calculated by combining the glucose standard curve. Finally, the filter paper enzyme activity value was obtained according to the definition of "1 μmol of glucose produced per milliliter of enzyme solution per minute".

[0042] The result is as follows Figure 7 As shown; by Figure 7 It can be seen that the mutant strain *Aspergillus niger* with white spores... Aspergillus niger The filter paper enzyme activity of B704 gradually increased from 20 h to 60 h, indicating that the strain was in the late logarithmic growth phase to the early stationary phase. The culture medium had sufficient carbon sources (such as cellulose substrates) and nitrogen sources, and cell proliferation and metabolic activities were vigorous. On the one hand, sufficient nutrition provided the necessary energy (ATP) and precursor substances (amino acids) for the synthesis of various components of the cellulase system, ensuring the continuous translation and processing of endoglucanase, exoglucanase, and β-glucosidase. On the other hand, the strain had not entered the death phase, the cell structure was intact, and the secretory functions of organelles such as the endoplasmic reticulum and Golgi apparatus were highly efficient, which gradually increased the accumulation of extracellular cellulase system, ultimately manifested as a continuous increase in filter paper enzyme activity. After 60 hours, the activity of filter paper enzymes decreased, indicating that the nutrients (carbon and nitrogen sources) in the culture medium were depleted. The expression of enzyme-related genes (egl, cbh, bgl) was inhibited due to insufficient energy and precursors, leading to a significant decrease in the new synthesis rate of the three types of enzymes. This resulted in an inability to replenish the extracellular degraded or inactivated enzyme proteins, causing a gradual decrease in total enzyme activity.

[0043] (5) Laccase activity assay The mutant strain Aspergillus niger with white spores Aspergillus niger B704 was added to seed culture medium (sodium nitrate 3 g / L, dipotassium hydrogen phosphate 1 g / L, magnesium sulfate 0.5 g / L, potassium chloride 0.5 g / L, ferrous sulfate 0.01 g / L, sucrose 30 g / L, agar 20 g / L) and cultured until the logarithmic growth phase to obtain the seed culture (OD). 600 =0.8), 100 mL of laccase-producing medium was placed in a 250 mL Erlenmeyer flask, and the seed culture (OD) was added.600 =0.8) was inoculated into the laccase production medium at a 5% inoculum and cultured at 30°C and 180 rpm for 72 h to obtain the fermentation sample. The laccase production medium consisted of: 5 g / L dextran (in other embodiments, 0.5 g / L guaiacol (added after sterilization) can also be selected), 2 g / L ammonium sulfate (in other embodiments, 3 g / L yeast extract can also be selected), and 1 g / L potassium dihydrogen phosphate.

[0044] The oxidation of ABTS was measured by monitoring at 420 nm. A 3 mL reaction system was used at 37 °C. The reaction mixture contained 2 mL of 0.5 mmol / L ABTS (dissolved in 0.1 mmol / L, pH 5.0 acetate-sodium acetate buffer). 1 mL of supernatant from the fermentation sample was added to initiate the reaction, and the change in absorbance at 420 nm was measured after 3 min. One unit of enzyme activity (U) is defined as the amount of enzyme required to oxidize 1 μmol of ABTS per minute. (Distilled water was used as a control instead of the enzyme solution.) Enzyme activity (U / mL) =

[0045] ΔA is the change in absorbance; V1 is the total volume of the reaction solution (mL); V2 is the volume of the crude enzyme solution (mL); Δt is the reaction time.

[0046] The result is as follows Figure 8 As shown; by Figure 8 It can be seen that the mutant strain *Aspergillus niger* with white spores... Aspergillus niger The laccase activity of B704 gradually increased from 20 to 50 hours, indicating that during this period, the strain was in the early stationary phase of a nutrient-rich logarithmic growth phase. Carbon and nitrogen sources, as well as inducers (such as phenols) in the culture medium, were not significantly depleted, and cellular metabolic activity was vigorous. On the one hand, sufficient energy (ATP) and precursors (amino acids) ensured the translation and processing of laccase proteins; on the other hand, the expression of laccase synthesis-related genes (such as the lac gene) remained continuously activated, the synthesis rate of the enzyme protein exceeded the degradation rate, and the accumulation of extracellular laccase gradually increased, ultimately manifesting as a continuous increase in enzyme activity. After 50 hours, the laccase activity began to decrease, indicating that the carbon and nitrogen sources in the culture medium, as well as the inducers (such as phenols) required for laccase synthesis, had been largely depleted. The strain's metabolic energy (ATP) and the supply of precursors (amino acids) for enzyme protein synthesis were insufficient, and the transcription and translation efficiency of laccase-related genes (lac genes) decreased sharply. The synthesis rate of new enzymes was much lower than the degradation / inactivation rate of existing enzymes, leading to a cessation or even a decrease in the accumulation of extracellular laccase.

[0047] 4. Analysis of the genetic stability of heavy ion-mutated strains White spores of Aspergillus niger were screened again. Aspergillus niger Genetic stability analysis was performed on B704, and white-spore Aspergillus niger was included. Aspergillus niger B704 was subjected to continuous subculturing as follows: The mutagenized strain was designated as generation 0. The generation 0 bacterial culture was inoculated at 1% onto seed culture medium (sodium nitrate 3 g / L, dipotassium hydrogen phosphate 1 g / L, magnesium sulfate 0.5 g / L, potassium chloride 0.5 g / L, ferrous sulfate 0.01 g / L, sucrose 30 g / L, agar 20 g / L) and cultured at 30℃ and 180 rpm for 18 h to obtain generation 1 seed culture. The generation 1 seed culture was then inoculated at 1% onto seed culture medium and cultured at 30℃ and 180 rpm for 18 h to obtain generation 2 seed culture. This process was repeated for 12 generations, with the cellulose degradation rate measured every two generations.

[0048] The result is as follows Figure 9 As shown; by Figure 9 It can be seen that the white spores of Aspergillus niger Aspergillus niger After 12 generations, the cellulose and hemicellulose degradation rates of B704 did not differ significantly between generations, indicating that the mutagenized Aspergillus niger possesses stable heritable cellulose and hemicellulose degradation capabilities.

[0049] The above description is merely a preferred embodiment of this application. This application is not limited to the above-described embodiments. Any embodiment that achieves the technical effect of this application using the same means should fall within the protection scope of this application. Within the protection scope of this application, the technical solutions and / or implementation methods can have various modifications and variations.

Claims

1. A white-sporeed Aspergillus niger, characterized in that, The white-sporeed Aspergillus niger is classified as follows: Aspergillus niger The name is Aspergillus niger B704 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on January 6, 2025, with accession number GDMCC No: 65633.

2. A microbial agent, characterized in that, The active ingredient of the microbial agent includes Aspergillus niger as described in claim 1.

3. The use of the white spore Aspergillus niger according to claim 1 or the fungal agent according to claim 2 in improving the degradation rate of cellulose and / or hemicellulose.

4. A method for degrading cellulose and / or hemicellulose, characterized in that, The method includes using the white spore Aspergillus niger as described in claim 1 or the fungal agent as described in claim 2.

5. The method according to claim 4, characterized in that, This includes culturing the white spore Aspergillus niger using a fermentation medium.

6. The method according to claim 5, characterized in that, The fermentation medium comprises: 70 g / L corn husks, 30 g / L wheat bran, 1.5 g / L ammonium sulfate, 1.0 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, and 0.1 g / L calcium chloride.

7. The method according to claim 5, wherein the culture conditions are: culture time of 72 h, culture temperature of 30 °C, rotation speed of 180 r / min, and the initial pH value of the fermentation medium is 5.9-6.

2.

8. The method according to claim 5, characterized in that, The seed culture of the white-spore Aspergillus niger cultured to the logarithmic growth phase was inoculated into the fermentation medium at an inoculation rate of 5%-10%.

9. The method according to claim 8, characterized in that, The seed culture was obtained by culturing the white spore Aspergillus niger in a seed culture medium, which included: sodium nitrate, dipotassium hydrogen phosphate, magnesium sulfate, potassium chloride, ferrous sulfate, sucrose, and agar.

10. The method according to claim 9, characterized in that, The sodium nitrate content is 3 g / L, the dipotassium hydrogen phosphate content is 1 g / L, the magnesium sulfate content is 0.5 g / L, the potassium chloride content is 0.5 g / L, the ferrous sulfate content is 0.01 g / L, the sucrose content is 30 g / L, and the agar content is 20 g / L.

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