Penicillium citrinum DHZ-2 with high production of cellulase and hemicellulase and its application

Abstract

The present invention belongs to the field of microorganisms and their applications, and specifically relates to a Penicillium citrinum DHZ-2 with high cellulase and hemicellulase production and its application in the degradation of rice straw. Penicillium citrinum ) DHZ-2, whose deposit number is CCTCCNO: M20241130. The present invention also provides a use of the above-mentioned Penicillium citrinum DHZ-2: Penicillium citrinum DHZ-2 is fermented in a fermentation medium to produce enzymes, thereby obtaining cellulases and hemicellulases. Penicillium citrinum DHZ-2 of the present invention can be used to degrade rice straw.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms and their applications, and particularly relates to Penicillium citrinum DHZ-2 with high cellulase and hemicellulase production and its application in degrading rice straw. Background Art

[0002] Crop straw is a rich renewable resource, and its high-value utilization has become a research hotspot. This includes fertilizer, energy, raw material, feed, and base material. Biomass degradation technology plays a key role in these five processes.

[0003] Traditional straw degradation methods mainly include physical and chemical methods. Physical methods typically involve mechanical crushing to destroy the physical structure of straw, but this method only changes the straw's morphology and is ineffective in degradation. Chemical methods, which use strong acids and bases to crack straw, offer high degradation efficiency but also pose serious environmental risks. In contrast, biodegradation methods that utilize microorganisms and their secreted enzymes offer advantages such as high efficiency, environmental friendliness, and cost-effectiveness, and have become a key research direction for straw resource utilization. A wide variety of microorganisms are used for straw biodegradation, among which fungi possess powerful enzymatic capabilities and play a significant role in straw degradation. Fungal cellulase enzymes primarily include endoglucanases (EC 3.2.1.4), exoglucanases (EC 3.2.1.91), and β-glucosidases (EC 3.2.1.21), which work synergistically to degrade cellulose into glucose. Hemicellulases, primarily β-1,4-xylanases (EC 3.2.1.8), break down hemicellulose into monosaccharides such as xylose. These enzymes can cut the cellulose and hemicellulose molecular chains in straw, degrading them into small molecular substances such as glucose, xylose, etc., which can be further utilized to realize the resource utilization of straw.

[0004] At present, screening of microbial strains with high (hemi) cellulase production from special environments is a research hotspot in the field of microbial applications. For example, the marine bacterial strain Bacillus marine sediments isolated from the Gopur Beach in Odisha, India ( Bacillus oceanisediminis ), Haloperidol Brevibacterium ( Brevibacteriumhalotolerans ) and fast-growing psychrophiles ( Psychrobacterceler ), can produce salt-tolerant cellulase and has the ability to pre-treat rice straw; the study found that from wood-eating termites ( Microcerotermes sp.) was isolated from the intestine of a new type of Streptomyces ( Streptomyces sp.) strain MS-S2 can produce xylanase and cellulase and has been used to degrade wheat straw. These studies reveal that there are abundant microbial resources that produce (hemi)cellulase in specific environments.

[0005] Penicillium citrinum ( Penicillium citrinumPenicillium citrinum is a filamentous fungus that secretes cellulases and hemicellulases, which are used to hydrolyze straw lignocellulose. Penicillium also has a short growth cycle, simple culture conditions, and rapid biomass accumulation, significantly reducing the time and economic costs of large-scale production. However, the strains of Penicillium citrinum reported in current research often possess only one or a few cellulase or hemicellulase activities, limiting their practical use in straw degradation.

[0006] For example, Penicillium citrinum NAF5 can produce endoglucanase at a yield of 3.13 U / mL (Saini et al., 2020) and β-glucosidase, with a yield of up to 2.48 U / mL after optimized conditions (Saini et al., 2024). Penicillium citrinum YS40-5 can produce high levels of β-glucosidase under solid-state fermentation conditions using rice bran, with an activity of 159.1 U / g (Ng et al., 2010). Penicillium citrinum NCIM-1398 can co-produce endo-β-1,4-glucanase, xylanase, and amylase (Biswas et al., 2019).

[0007] The invention of CN114657073A "A strain of Penicillium citrinum with high cellobiase production and its application" discloses a strain of Penicillium citrinum with high cellobiase production ( Penicillium citrinum ) strain CB21, the activity of cellobiase (exoglucanase and endoglucanase) in the crude enzyme solution of the strain is relatively high, and its maximum enzyme activity reaches 342.5U / ml.

[0008] The invention of CN113046248A "A strain of Penicillium citrinum XZH-16 and its application" discloses Penicillium citrinum ( Penicillium citrinum ) Application of XZH-16 in the production of cellulase and / or lytic polysaccharide monooxygenase (LPMO).

[0009] The invention of CN106047730A "Oxalic acid Penicillium EU2101 and its application in preparing cellulase preparation and degrading cellulose" discloses Oxalic acid Penicillium ( Penicillium oxalicum ) EU2101, the filter paper enzyme activity of the cellulase preparation prepared therefrom is 41.7±1.0U / gds, the Avicel enzyme (crystalline cellulase) activity is 14.3±0.8U / gds, the CMC enzyme (carboxymethyl cellulase) activity is 267.7±38.6U / gds, the pNPG enzyme (p-nitrophenyl-α-D-pyranoglucopyranoside cellulase) activity is 103.7±4.5U / gds, the pNPC enzyme (p-nitrophenyl-β-D-cellobioside cellulase) activity is 10.2±0.8U / gds, and the xylanase activity is 1257.1±102.1U / gds.

[0010] Therefore, there is currently a lack of Penicillium citrinum that can simultaneously secrete multiple enzymes such as endoglucanase, exoglucanase, β-glucosidase and xylanase. Penicillium citrinum ). Summary of the Invention

[0011] The technical problem to be solved by the present invention is to provide a Penicillium citrinum DHZ-2 strain which has high yield of cellulase and hemicellulase, good application potential for cellulose and hemicellulose degradation, and can promote efficient biodegradation of rice straw.

[0012] In order to solve the above technical problems, the present invention provides a Penicillium citrinum ( Penicillium citrinum )DHZ-2, its deposit number is CCTCCNO: M20241130, and the deposit date is June 3, 2024.

[0013] The present invention also provides a use of the above-mentioned Penicillium citrinum DHZ-2: Penicillium citrinum DHZ-2 is fermented in a fermentation medium (optimized fermentation medium) to produce enzymes, thereby obtaining cellulase and hemicellulase.

[0014] As an improvement on the use of Penicillium citrinum DHZ-2 of the present invention: the cellulase includes endoglucanase, exoglucanase and β-glucosidase; and the hemicellulase includes xylanase.

[0015] Further improvements to the use of Penicillium citrinum DHZ-2 of the present invention:

[0016] The fermentation medium consisted of 12.5-15 g rice straw powder, 2.0 g NaNO₃, 2.0 g NH₄Cl, 1.0 g KH₂PO₄, 1.0 g K₂HPO₄, 1.5-1.75 g peptone, 0.5 g MgSO₄·7H₂O, and 1 L distilled water; the pH was 4.5-5.5.

[0017] The culture temperature (fermentation temperature) is 25~30℃; the culture time is 8~10 days.

[0018] Further improvements to the use of Penicillium citrinum DHZ-2 of the present invention:

[0019] For endoglucanase: culture temperature 25°C, culture time 8 days, rice straw powder 15g (rice straw powder addition ratio is 1.5%), peptone 1.5g (peptone addition ratio is 0.15%), pH 5;

[0020] For exoglucanase: culture temperature 30°C, culture time 8 days, rice straw powder 12.5g (rice straw powder addition ratio 1.25%), peptone 1.5g (peptone addition ratio 0.15%), pH 5;

[0021] For β-glucosidase: culture temperature 30°C, culture time 10 days, rice straw powder 15g (rice straw powder addition ratio is 1.5%), peptone 1.75g ​​(peptone addition ratio is 0.175%), pH 5;

[0022] For xylanase: culture temperature 30℃, culture time 8 days, rice straw powder 15g (rice straw powder addition ratio 1.5%), peptone 1.75g ​​(peptone addition ratio 0.175%), pH 5.

[0023] The present invention also provides a method for degrading rice straw using the above-mentioned Penicillium citrinum DHZ-2, comprising the following steps:

[0024] The rice straw was mixed with Hutchinson's inorganic salt medium and Penicillium citrinum DHZ-2 and then fermented.

[0025] As an improvement of the method for degrading rice straw by Penicillium citrinum DHZ-2 of the present invention:

[0026] Rice straw was mixed with Hutcheson's medium and Penicillium citrinum DHZ-2 at a ratio of 2 ± 0.5 g rice straw (dry weight) to 100 mL Hutcheson's medium and 0.5 cm thick Penicillium citrinum DHZ-2 cake. The mixture was then fermented at 30 ± 2°C in a constant temperature shaker (200 ± 20 rpm / min) for 10–30 d.

[0027] As a further improvement of the method for degrading rice straw by Penicillium citrinum DHZ-2 of the present invention:

[0028] Hutcheson's inorganic salts medium (g / L): KH2PO4 1.0 g, NaCl 0.1 g, MgSO4▪7H2O 0.3 g, NaNO3 2.5 g, FeCl3 0.01 g, CaCl2 0.1 g, distilled water 1 L, pH 7.2.

[0029] The pH can be adjusted using conventional methods such as 1 mol / L NaOH.

[0030] Geophagous termites, a specialized group that feeds on soil, make up over one-third of known termite species and hold unique research value. The Chinese termite, Coptotermes sinensis, is a type of geophagous termite that primarily feeds on organic matter in the soil and lignocellulose from decaying plants. Given this feeding habit, it is likely that their guts harbor promising strains of hemicellulase-producing microorganisms, offering new avenues for straw biodegradation research.

[0031] The present invention provides Penicillium citrinum DHZ-2 from the Chinese twist termite ( Sinocapritermessp.) and was screened from the intestine. The strain was identified by morphology and ITS sequence comparison with Penicillium citrinum ( Penicillium citrinum ) have an ITS sequence similarity of up to 100%.

[0032] The present invention has the following beneficial effects:

[0033] 1) This study isolated a strain of Penicillium citrinum DHZ-2 from the gut of the termite C. sinensis. It simultaneously secretes cellulases and hemicellulases, including endoglucanases, exoglucanases, and β-glucosidases; and hemicellulases, including xylanases. Therefore, Penicillium citrinum DHZ-2 has great potential for rice straw degradation.

[0034] 2) Using the specific culture medium (optimized fermentation medium) and fermentation culture conditions of the present invention, the enzyme activities of endoglucanase, exoglucanase, β-glucosidase and xylanase were increased by 2.1 times, 2.7 times, 4.12 times and 1.72 times respectively compared to conventional MSM solid culture medium.

[0035] 3) Using the Penicillium citrinum DHZ-2 of the present invention, the degradation rate of rice straw reached 52.29%, and the cellulose and hemicellulose contents in the rice straw decreased by 34.51% and 47.01%, respectively.

[0036] 4) The galactose content in the rice straw product degraded by Penicillium citrinum DHZ-2 reached 115.85 mg / L, and the xylose content reached 78.91 mg / L.

[0037] The products of rice straw degradation by Penicillium citrinum DHZ-2 include galactose and xylose, indicating that its hemicellulase activity is significant and can simultaneously release multiple fermentable sugars; while the degradation products of Penicillium oxalicum EU2101 mainly focus on glucose production, and do not involve hemicellulase degradation to produce sugar.

[0038] In summary, the present invention screened out a strain of Penicillium citrinum DHZ-2 that can produce a high amount of (hemi)cellulase from the intestine of the Chinese termite Coptotermes sinensis. The strain can simultaneously secrete multiple enzymes such as endoglucanase, exoglucanase, β-glucosidase and xylanase, showing that the strain has the characteristics of a multi-enzyme system; at the same time, the present invention optimized the four enzyme production activities of Penicillium citrinum DHZ-2 through response surface experiments and studied its effect on rice straw degradation, which has important practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The specific embodiments of the present invention are further described in detail below with reference to the accompanying drawings.

[0040] Figure 1 This is a Congo red decolorization circle diagram of Penicillium citrinum DHZ-2 on a CMC-Na plate.

[0041] Figure 2 The colony morphology (left) and microscopic examination (right) of Penicillium citrinum DHZ-2 on PDA plates.

[0042] Figure 3 This is a phylogenetic tree constructed based on the ITS sequence of Penicillium citrinum DHZ-2.

[0043] Figure 4 This is the effect of culture time on the (hemi)cellulase activity produced by Penicillium citrinum DHZ-2.

[0044] Figure 5 This study is about the effect of culture temperature on the (hemi)cellulase activity produced by Penicillium citrinum DHZ-2.

[0045] Figure 6 This is the effect of initial pH on the (hemi)cellulase activity produced by Penicillium citrinum DHZ-2.

[0046] Figure 7 This is the effect of carbon source type and addition ratio on the (hemi)cellulase activity produced by Penicillium citrinum DHZ-2.

[0047] Figure 8 This is the effect of nitrogen source type and addition ratio on the (hemi)cellulase activity produced by Penicillium citrinum DHZ-2.

[0048] Figure 9 This is a Pareto diagram showing the effect of each single factor on the (hemi)cellulase production of Penicillium citrinum DHZ-2.

[0049] Figure 10 This is a 3D response surface diagram of the effects of two factors on the (hemi)cellulase production of Penicillium citrinum DHZ-2.

[0050] Figures 9 and 10 In the figure: A represents endoglucanase, B represents exoglucanase, C represents β-glucosidase, and D represents xylanase.

[0051] Figure 11 is the degradation rate of rice straw in liquid fermentation mode from 0 to 30 days.

[0052] Figure 12 The sugar content of rice straw fermented in liquid fermentation mode for 0-30 days. DETAILED DESCRIPTION

[0053] The present invention will be further described below with reference to specific examples. It should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the present invention.

[0054] The culture medium and reagent preparation methods involved in the following examples are:

[0055] MSM solid medium (g / L): KH2PO4 1.0 g, K2HPO4 1.0 g, (NH4)SO4 2.0 g, MgSO4 0.2 g, CaCl2 0.1 g, FeSO4 0.05 g, MnSO4 0.02 g, CuSO4 0.001 g, ZnSO4 0.001 g, peptone 0.05 g, agar powder 15 g, alkali lignin 1.0 g, distilled water 1 L. Initially set the pH to 3-13.

[0056] Note: As common sense, alkali lignin needs to be pretreated, that is, first dissolve 1.0g of alkali lignin with 1 mol / L NaOH, and then adjust to neutral pH with 1 mol / L HCl.

[0057] CMC-Na solid medium (g / L): CMC-Na 10.0 g, NaNO 3 2.0 g, NH 4 Cl 2.0 g, KH 2 PO 4 1.0 g, K 2 H PO 4 1.0 g, peptone 1.0 g, MgSO 4 · 7H 2 O 0.5 g, agar 20.0 g, distilled water 1 L. Natural pH.

[0058] CMC-Na liquid medium (g / L) as the original medium for enzyme production: the formula is the same as the solid medium, but the agar is removed.

[0059] LB solid medium (g / L): peptone 10.0 g, yeast powder 5.0 g, NaCl 10.0 g, agar powder 15.0 g, distilled water 1 L. Natural pH.

[0060] PDA liquid medium (g / L): 25 g potato dextrose broth powder, 1 L distilled water. Natural pH.

[0061] PDA solid medium (g / L): 46.0 g potato dextrose agar powder, 1 L distilled water. Natural pH.

[0062] Preparation of DNS solution: Dissolve 3,5-dinitrosalicylic acid (3.15 g) in 500 mL of deionized water and stir at 45°C to dissolve. Then, add 100 mL of 5 mol / L sodium hydroxide solution and stir to mix thoroughly. Add 91.0 g of potassium sodium tartrate, 2.5 g of phenol, and 2.5 g of anhydrous sodium sulfite in that order and stir to dissolve. Cool to room temperature and dilute to 1000 mL in a 1000 mL volumetric flask (i.e., dilute to 1000 mL with deionized water). Filter and store the filtrate in a brown bottle in the dark for 7 days until ready for use.

[0063] The enzyme activity determination methods involved in the following examples are all conventional techniques, which can be as follows:

[0064] Preparation of crude enzyme solution: Ferment Penicillium citrinum DHZ-2 on culture medium, centrifuge the resulting bacterial solution (4°C, 8,000 rpm / min for 5 min), and collect the supernatant, which is the crude enzyme solution, which can be used to determine enzyme activity.

[0065] ① Endoglucanase

[0066] Endoglucanase activity was determined using the DNS method. 1% (1g / 100mL) CMC-Na (sodium carboxymethyl cellulose) was prepared as a substrate in 50mM sodium citrate buffer (pH 5). 200μL of crude enzyme solution was mixed with 800μL of 1% CMC-Na in a 50°C water bath for 60 minutes. The enzymatic reaction was terminated by adding 1mL of DNS solution. The reaction was then boiled in a water bath for 10 minutes. After cooling, 3mL of distilled water was added and the concentration at 540nm was measured using a microplate reader. Three replicates were set up, and the measured values ​​were substituted into the regression equation to determine the amount of reducing sugars produced.

[0067] ② Exoglucanase

[0068] The test method is the same as that of endoglucanase, except that the substrate is replaced with 1% Avicel (microcrystalline cellulose MCC) prepared in 50 mM sodium citrate buffer (pH 5).

[0069] ③β-glucosidase

[0070] At 10mmol / L p NPG (4-nitrophenyl-β-D-glucopyranoside) was used as the substrate. 200 μL of substrate was mixed with 100 μL of crude enzyme solution and incubated in a 50°C water bath for 30 minutes. After the reaction, 2 mL of 1 mol / L Na₂CO₃ was added to terminate the assay. The absorbance at 410 nm was measured on a microplate reader in triplicate. The amount of p-nitrophenol generated was calculated by substituting the results into a regression equation.

[0071] ④ Xylanase

[0072] Xylanase is the primary hemicellulose-degrading enzyme and was therefore used as a representative hemicellulase. Xylanase activity was determined using the DNS method. A 1% beechwood xylan substrate was prepared in 50 mM sodium citrate buffer (pH 5). 200 μL of crude enzyme solution was mixed with 800 μL of a 1% beechwood xylan solution in a 50°C waterbath for 60 minutes. The enzymatic reaction was terminated by adding 1 mL of DNS solution, followed by boiling in a waterbath for 10 minutes. After cooling, 3 mL of distilled water was added. The activity at 540 nm was measured using a microplate reader with three replicates. The measured values ​​were then substituted into the regression equation to determine the amount of xylose produced.

[0073] ⑤ Calculation of enzyme activity

[0074] One unit of enzyme activity (U) is defined as the number of micromoles of glucose, p-nitrophenol, and xylose catalyzed per minute under certain reaction conditions. The specific activity can be expressed as U / mL.

[0075] Enzyme activity (U / mL) = (product concentration × final reaction volume) / (product molar mass × crude enzyme solution volume × reaction time) × dilution factor × 1000.

[0076] Hutchinson inorganic salt medium (g / L): KH2PO4 1.0 g, NaCl 0.1 g, MgSO4▪7H2O 0.3 g, NaNO3 2.5 g, FeCl3 0.01 g, CaCl2 0.1 g, distilled water 1 L.

[0077] Rice straw-inorganic salt liquid fermentation medium: 2 g rice straw segments, 100 mL Hutcheson inorganic salt medium.

[0078] Example 1: Isolation and identification of Penicillium citrinum DHZ-2

[0079] 1) Strain isolation and initial screening

[0080] Take 20 Chinese twisted termite workers for dissection. First, rinse the termite body surface several times with sterile water to wash away the soil residue on the body surface as much as possible, then disinfect the termite cuticle with 75% alcohol for 1 minute, and finally rinse with sterile PBS buffer (pH 7) 1-2 times. Dissect the disinfected Chinese twisted termite workers under sterile conditions, and use dissecting forceps to dissect the termite intestine. Place the separated termite intestinal tissue in a 1.5mL centrifuge tube containing 200μL PBS buffer and store it in an ice bath. Use a grinding rod to grind the intestinal sample into a homogenate in an ice bath, add PBS buffer to make the volume 1 mL, and obtain the intestinal sample stock solution. Dilute the intestinal sample stock solution to 10 with PBS buffer. -3 -10 -6 , take 50 μL of diluted intestinal fluid and spread it on MSM culture medium, culture it at 30℃ for 3-7 days, observe the growth of the colony, pick typical colonies on the plate, streak and purify to obtain single colonies, inoculate the obtained single colonies in LB and PDA culture medium to make them grow better. The purified strain obtained by the above operation was inoculated on CMC-Na agar plate, cultured inverted at 30℃, poured an appropriate amount of 1 mg / mL Congo red dye solution into the plate for staining for 30 minutes, then poured off the dye solution, and decolorized with 1 mol / L NaCl solution for 30 minutes to obtain strain DHZ-2, which formed a hydrolysis circle on the CMC-Na plate, as shown in the figure. Figure 1 As shown, the diameter of the transparent zone (D) of the tested strain was 5 cm, and the diameter of the colony (d) was 3.4 cm.

[0081] 2) Morphological and molecular identification of strains

[0082] Morphological analysis: Figure 2 As shown, observation of the colony morphology on the plate revealed a white, velvety appearance on the PDA plate. Later, due to spore production, the strain exhibited radial grooves. When spores were produced in large quantities, the colony surface was covered with powdery particles. Microscopic observation revealed upright, smooth-walled conidiophores with broom-like branching structures. The conidia were spherical and smooth, indicating preliminary identification as Penicillium.

[0083] Molecular Identification Analysis: Mycelia were scraped from a PDA plate and quickly ground into a powder in liquid nitrogen. Genomic DNA was extracted using the Shanghai Shenggong Fungal Genomic DNA Rapid Extraction Kit. ITS sequences were amplified using the universal primers ITS1 and ITS4. The PCR amplification system in 50 μL was: 25 μL GreenTaq Mix, 2 μL ITS1, 2 μL ITS4, 1 μL DNA template, and sterile deionized water to 20 μL. Primers were as follows:

[0084] ITS1: 5'-TCCGTAGGTGAACCTGCGG-3' (SEQ ID NO. [ID] 1 in the sequence listing);

[0085] ITS4: 5'-TCCTCCGCTTATTGATATGC-3' (e.g., sequence number [ID] 2 in the sequence listing).

[0086] Amplification reactions were performed in a PCR instrument using the following cycle: 94°C for 3 minutes, 95°C for 3 minutes, 55°C for 30 seconds, and 72°C for 1 minute, followed by 33 cycles at 72°C for 5 minutes, with a termination temperature of 16°C. ITS-PCR products were detected by 1% agarose gel electrophoresis. Sequencing results were then Blast-aligned at NCBI. ITS sequences with high similarity were identified in the GenBank database, and homology analysis was performed to construct a phylogenetic tree of the strains.

[0087] Phylogenetic tree Figure 3 As shown, strain DHZ-2 and Penicillium citrinum ( Penicillium citrinum ) with an ITS sequence similarity of up to 100%. Based on the results of colony morphology, microscopic observation and molecular identification, the strain was identified as Penicillium citrinum ( Penicillium citrinum ), which was named Penicillium citrinum ( Penicillium citrinum )DHZ-2.

[0088] The deposit information of DHZ-2 is as follows:

[0089] Deposit name: Penicillium citrinum DHZ-2 Penicillium citrinumDHZ-2, deposited in China Center for Type Culture Collection, deposited at Wuhan University, Wuhan, China, deposited on June 3, 2024.

[0090] Example 2: Determination of the (hemi)cellulase production capacity of Penicillium citrinum DHZ-2

[0091] The specific steps are as follows:

[0092] A single-factor experiment was used to investigate the effects of enzyme production conditions on the strain's endoglucanase, exoglucanase, β-glucosidase, and xylanase, including five types of fermentation conditions: culture time, culture temperature, initial pH, carbon source type and addition ratio, and nitrogen source type and addition ratio.

[0093] CMC-Na liquid culture medium was used, with a culture medium content of 10 mL per group. 0.5 cm2 cakes of Penicillium citrinum DHZ-2 were inoculated into the CMC-Na liquid culture medium as an enzyme production culture system.

[0094] ① Different culture time conditions

[0095] The culture temperature was maintained at 30°C and the culture was cultured in a shaker at a speed of 200 rpm / min. The culture time was set to 2-10 days. The effects of different culture times on the (hemi)cellulase production capacity of Penicillium citrinum DHZ-2 were measured every 2 days, that is, the culture time was set to 2, 4, 6, 8, and 10 days.

[0096] ② Different temperatures

[0097] The culture temperatures were set at 20℃, 25℃, 30℃, 35℃, and 40℃, respectively. The cultures were cultured at various temperatures for 6 days. The effects of different temperatures on the (hemi)cellulase production capacity of Penicillium citrinum DHZ-2 were measured to determine the optimal enzyme production temperature.

[0098] ③ Culture at different initial pH

[0099] The culture was maintained at 30°C and shaker speed of 200 rpm / min. The initial pH of the enzyme production culture system (CMC-Na liquid medium) was adjusted to 3, 5, 7, 9, 11, and 13, respectively. The culture was then cultured at this constant temperature for 6 days. The effects of different initial pH conditions on the (hemi)cellulase production capacity of Penicillium citrinum DHZ-2 were determined to determine the optimal pH for enzyme production.

[0100] ④ Different carbon sources

[0101] The carbon sources were rice straw powder, sodium carboxymethyl cellulose, sucrose, glucose, and soluble starch;

[0102] Under natural pH conditions, the culture temperature was maintained at 30°C and the rotation speed was 200 rpm / min. The CMC-Na in the enzyme production culture system (CMC-Na liquid medium) was replaced with different carbon sources: rice straw powder, sodium carboxymethyl cellulose (CMC-Na), sucrose, glucose, and soluble starch. Culture was continued at this constant temperature for 6 days to determine the effects of different carbon sources on the (hemi)cellulase production capacity of Penicillium citrinum DHZ-2 and to determine the optimal carbon source for enzyme production.

[0103] Based on the best carbon source screened, the carbon source ratio of the culture medium was adjusted to 0.5%, 0.75%, 1.0%, 1.25%, 1.5%, and 1.75%, respectively, to determine the optimal addition ratio of the carbon source and to determine the optimal carbon source ratio for enzyme production.

[0104] The preparation process of rice straw powder is as follows: fresh rice straw is collected, impurities are washed off, and then dried at 80°C to constant weight, and then crushed and sieved using a grinder to obtain 100-mesh rice straw powder.

[0105] ⑤ Different nitrogen sources

[0106] The types of nitrogen sources include beef extract, ammonium sulfate, protein, yeast powder, and urea;

[0107] Under natural pH conditions, the culture temperature was maintained at 30°C and the rotation speed was 200 rpm / min. The peptone in the enzyme production culture system (CMC-Na liquid medium) was replaced with different nitrogen sources: beef extract, ammonium sulfate, peptone, yeast powder, and urea. The culture was then cultured at this constant temperature for 6 days to determine the effects of different nitrogen sources on the (hemi)cellulase production capacity of Penicillium citrinum DHZ-2 and to determine the optimal nitrogen source for enzyme production.

[0108] Based on the optimal nitrogen source screened out, the nitrogen source ratio of the culture medium was adjusted to 0.05%, 0.075%, 0.1%, 0.125%, 0.15%, and 0.175%, respectively, to determine the optimal addition ratio of the nitrogen source and to determine the optimal nitrogen source ratio for enzyme production.

[0109] The results of the single-factor experiment are as follows Figure 4 、 Figure 5 、 Figure 6 、 Figure 7 、 Figure 8 shown.

[0110] from Figure 4 It can be seen that the activities of the four enzymes changed with time. The activities of endoglucanase, exoglucanase, β-glucosidase and xylanase all reached the highest enzyme production on the 8th day, reaching 1.822U / mL, 1.714U / mL, 1.899U / mL and 7.937U / mL respectively.

[0111] from Figure 5It can be seen that the strain produces the highest endoglucanase at 25°C (1.504U / mL); the strain produces the highest exoglucanase at 30°C (1.638U / mL); the strain produces the highest β-glucosidase at 30°C (1.279U / mL); the strain produces the highest xylanase at 30°C (7.167U / mL), indicating that the four enzyme activities show the highest values ​​at 25°C and 30°C respectively.

[0112] from Figure 6 It can be seen that the activities of the four enzymes all reached the highest value at pH 5, with endoglucanase reaching 1.508U / mL, exoglucanase reaching 1.760U / mL, and β-glucosidase reaching 1.549U / mL. Compared with the above three cellulases, xylanase has a higher activity, reaching 6.954U / mL.

[0113] from Figure 7 As shown in A-D, the effects of different carbon sources on enzyme production by Penicillium citrinum DHZ-2 were observed. When rice straw powder was used as the sole carbon source, enzyme production reached its highest values. Enzyme production reached 1.883 U / mL for endoglucanase, 1.680 U / mL for exoglucanase, 2.796 U / mL for β-glucosidase, and 9.349 U / mL for xylanase, indicating that rice straw powder is the optimal carbon source for these four enzyme activities.

[0114] from Figure 7 E~F show the effects of six rice straw powder ratios (0.5%, 0.75%, 1.0%, 1.25%, 1.5%, 1.75%) on the activities of various enzymes; endoglucanase showed a maximum value of 2.293U / mL at a ratio of 1.5%; exoglucanase had a maximum enzyme activity of 1.973U / mL at a ratio of 1.25%, and β-glucosidase had a maximum enzyme activity of 5.429U / mL at a ratio of 1.5%; xylanase reached a maximum enzyme production of 10.481U / mL at a ratio of 1.25%, indicating that the four enzyme activities showed optimal activity at rice straw powder ratios of 1.25% and 1.5%, respectively.

[0115] from Figure 8 From A to D, we can see that when peptone is used as nitrogen source, the production of the four enzymes is the best: endoglucanase is 1.415U / mL, exoglucanase is 1.20U / mL, β-glucosidase is 1.374U / mL, and xylanase is 7.922U / mL. Figure 8E~F can be used to know the enzyme production conditions at different peptone ratios (0.05%, 0.075%, 0.1%, 0.125%, 0.15%, 0.175%). The activities of endoglucanase, exoglucanase and β-glucosidase reached the maximum values ​​of 1.605U / mL, 2.302U / mL and 2.158U / mL respectively when the peptone ratio was 0.150%. The activity of xylanase reached 10.515U / mL at a peptone ratio of 0.150%. This shows that the activities of the four enzymes can reach the highest enzyme production value when the peptone ratio is 0.150%.

[0116] Example 3: Response surface optimization method for (hemi)cellulase production by Penicillium citrinum DHZ-2

[0117] 1) Screening of key factors affecting enzyme production

[0118] ① Plackett-Burman experimental factors and level settings

[0119] Based on the results of the single-factor experiment, Design Expert 13 software was used to design the factors affecting enzyme production of endoglucanase (Table 1), exoglucanase (Table 2), β-glucosidase (Table 3), and xylanase (Table 4) according to the Plackett-Burman experimental design principle. Each factor was set at a low level (-1) and a high level (1). The setting of the low / high level was determined by the two points on the left and right of the optimal point of the single-factor result, which served as the high and low levels of the experimental design. Three key factors with a significant impact on enzyme production were screened out through 12 groups of experiments designed by DesignExpert 13.

[0120] Table 1 Plackett-Burman design of endoglucanase

[0121]

[0122] Table 2 Plackett-Burman design of exoglucanase

[0123]

[0124] Table 3 Plackett-Burman design of β-glucosidase

[0125]

[0126] Table 4 Plackett-Burman design of xylanase

[0127]

[0128] ②Plackett-Burman design results

[0129] Based on the setting of high and low levels of each factor, the experiment was conducted with endoglucanase (Table 5), exoglucanase (Table 6), β-glucosidase (Table 7), and xylanase (Table 8) as the response values. The experimental design and results are shown in Tables 5 to 8 below. The Pareto chart of the standardized effects of the factors is shown in Figure 9 As shown, Figure 9 A, B, C, and D represent endoglucanase, exoglucanase, β-glucosidase, and xylanase, respectively. The orange bar graph represents a positive effect, that is, when the factor level increases, the response value increases accordingly. The blue bar graph represents a negative effect, that is, when the factor level increases, the response value decreases instead.

[0130] Table 5 Plackett-Burman design results of endoglucanase

[0131]

[0132] From Table 5, it can be seen that the endoglucanase showed the best results in the 11th experimental design, that is, on the 6th day, when the pH was 7, the culture temperature was 30℃, the carbon source (rice straw powder) ratio was 1.25%, and the nitrogen source (peptone) ratio was 0.175%, the endoglucanase was 1.451 U / mL. Figure 9 A found that the three factors that have the most significant impact on endoglucanase are culture temperature, pH, and carbon source addition ratio.

[0133] Table 6 Exoglucanase Plackett-Burman test design and results

[0134]

[0135] The data in Table 6 show that the exoglucanase achieved the best results in the third experimental design, specifically: on the 10th day of culture, the pH value was 7, the culture temperature was 25°C, the carbon source (rice straw powder) ratio was 1.5%, and the nitrogen source (peptone) ratio was 0.175%. At this time, the exoglucanase activity was 2.312 U / mL. Figure 9 B shows that the factors that have the greatest impact on exoglucanase are culture temperature, carbon source addition ratio, and nitrogen source addition ratio.

[0136] Table 7 β-glucosidase Plackett-Burman test design and results

[0137]

[0138] According to the data shown in Table 7, it can be observed that β-glucosidase had the best effect in the 11th experimental design. Specifically, under the conditions of culture to day 6, initial pH value of 7, culture temperature at 35°C, carbon source (rice straw powder) ratio of 1.25%, and nitrogen source (peptone) ratio of 0.175%, the activity of β-glucosidase reached the highest value of 2.042 U / mL. Similarly, Figure 9 It can be seen from the C that pH, culture time and nitrogen source addition ratio have the most significant effects on β-glucosidase.

[0139] Table 8 Xylanase Plackett-Burman test design and results

[0140]

[0141] From the data in Table 8, it can be seen that the xylanase activity was best in the fifth experimental design, i.e., on the 10th day, when the pH was 7, the culture temperature was 25°C, the carbon source (rice straw powder) ratio was 1.5%, and the nitrogen source (peptone) ratio was 0.175%, the xylanase activity was 10.024 U / mL. Figure 9 Among the D, the top three factors are culture temperature, nitrogen source addition ratio, and carbon source addition ratio, indicating that these three factors have the most significant effects on xylanase in the PBD experiment.

[0142] ③ Results of significance analysis of the Plackett-Burman model

[0143] The Plackett-Burman model of endoglucanase shows P =0.0013, the model is significant as a whole. And the overall evaluation of the model found that the adjusted R 2 =0.8910, predicted R 2 =0.7623, predicted R 2 The difference between the value and the adjusted R² value is less than 0.2, indicating that the model fits well; the coefficient of variation CV%=4.93<10, and the signal-to-noise ratio is 12.3554 greater than 4, indicating the reliability of the model.

[0144] Likewise, the Plackett-Burman model for exoglucanases was significantly positive overall ( P =0.0014), the experimental design is effective. Model evaluation parameter adjustment R 2 =0.8873, predicted R 2 =0.7540, the difference between the two is less than 0.2, and the model fits well; CV%=6.98<10, and the signal-to-noise ratio is 12.3439 greater than 4, indicating that the model has high credibility and accuracy.

[0145] The Plackett-Burman model of β-glucosidase showed that the model was significant overall. P =0.0017, predicted R 2 The value of 0.7365 and the adjusted R² value of 0.8792 are less than 0.2, indicating that the model has reasonable consistency. The CV% is 8.39 < 10, and the signal-to-noise ratio is 13.8018. When the ratio is greater than 4, it indicates that the model has a good enough signal.

[0146] The Plackett-Burman model of xylanase is highly significant overall ( P <0.0001), predicted R 2 The value (0.9328) and the adjusted R 2 The difference between the values ​​(0.9651) is less than 0.2; CV%=9.11<10, and the signal-to-noise ratio (22.8637) is greater than 4, indicating that the experimental precision and signal-to-noise ratio are at an ideal level.

[0147] In summary, the Plackett-Burman models of all the above enzymes passed the significance test ( P <0.05), and the model evaluation parameters showed that the experimental design was reasonable, providing a basis for the subsequent Box-Behnken response surface optimization experimental design.

[0148] 2) Optimize enzyme production conditions

[0149] ① Box-Behnken experimental factors and level settings

[0150] On the basis of the Plackett-Burman experimental results, the low (-1), medium (0), and high (1) levels of each factor were set according to the three significantly influencing factors screened out. The 3-factor 3-level experimental scheme was designed using Design Expert 13 software according to the Box-Behnken experimental design principle. The remaining two factors were the optimal conditions for the single-factor experiment. The response surface experimental analysis of each enzyme activity was carried out with endoglucanase, exoglucanase, β-glucosidase, and xylanase as the response values. The experimental design is shown in Tables 9, 10, 11, and 12.

[0151] Table 9 Box-Behnken design of endoglucanase

[0152]

[0153] Table 10 Box-Behnken design of exoglucanase

[0154]

[0155] Table 11 Box-Behnken design of β-glucosidase

[0156]

[0157] Table 12 Box-Behnken design of xylanase

[0158]

[0159] ②Box-Behnken response surface design results

[0160] The experimental results were subjected to multivariate fitting analysis using the software Design-Expert 13. The experimental results are shown in Tables 13, 14, 15, and 16.

[0161] Table 13 Box-Behnke response surface design results for endoglucanase

[0162]

[0163] The endoglucanase response values ​​ranged from 1.413 U / mL to 3.890 U / mL, with the highest endoglucanase activity (3.890 U / mL) observed in Group 9, which achieved fermentation at 25°C, a pH of 5, and a 1.5% rice straw powder ratio. In Group 10, the lowest response value, 1.413 U / mL, was achieved at 20°C, a pH of 5, and a 1.25% rice straw powder ratio.

[0164] Table 14 Box-Behnke response surface design results for exoglucanase

[0165]

[0166] The exoglucanase had the best response value of 4.641 U / mL under the following fermentation conditions: culture temperature of 30℃, rice straw powder ratio of 1.25%, and peptone ratio of 0.15%. The lowest response value was 1.412 U / mL when the culture temperature was 25℃, rice straw powder ratio of 1.25%, and peptone ratio of 0.125%.

[0167] Table 15 Box-Behnke response surface design results for β-glucosidase

[0168]

[0169] Under the 10th experimental design, that is, on the 10th day, when the pH was 5 and the peptone ratio was 0.175%, the β-glucosidase reached a maximum response value of 7.946 U / mL; while on the 6th day, when the pH was 3 and the peptone ratio was 0.15%, the minimum response value was 2.862 U / mL.

[0170] Table 16 Box-Behnke response surface design results for xylanase

[0171]

[0172] In experiment 14, the maximum xylanase response was 13.526 U / mL when the culture temperature was 30°C, the rice straw powder ratio was 1.5%, and the peptone ratio was 0.175%. In experiment 13, the minimum xylanase response was 5.989 U / mL when the culture temperature was 35°C, the rice straw powder ratio was 1%, and the peptone ratio was 0.15%.

[0173] According to the experimental results, the multiple regression equations of endoglucanase, exoglucanase, β-glucosidase and xylanase were obtained:

[0174] Y (endoglucanase)

[0175] =3.65+0.8484A-0.0879B-0.031C+0.0008AB-0.127AC+0.062BC-1.17A 2 -0.172B 2 -0.1218C 2 ;

[0176] Y (exoglucanase)

[0177] =4.40+0.3331A+0.2375B+0.4029C+0.1313AB-0.1070AC-0.0668BC-1.92A 2 -0.2608B 2 -0.3330C 2 ;

[0178] Y (β-glucosidase)

[0179] =6.46+0.5178A+1.29B+0.2329C-0.0180AB+0.1020AC-0.3258BC-0.0208A 2 -1.96B 2 +0.4150C 2 ;

[0180] Y (xylanase)

[0181] =11.12-1.35A+1.47B+0.5598C+0.0915AB+0.1868AC+0.8088BC-2.19A 2 -0.3646B 2 -0.0058C 2 ;

[0182] According to the regression equation, the response surface 3D graph of the regression equation is obtained, and the results are as follows Figure 10 shown.

[0183] First, the fermentation process of the strain of the present invention is initially set as follows:

[0184] The optimized fermentation medium (g / L) is:

[0185] Rice straw powder 12.5-15 g, NaNO3 2.0 g, NH4Cl 2.0 g, KH2PO4 1.0 g, K2HPO4 1.0 g, peptone 1.5-1.75 g, MgSO4·7H2O 0.5 g, distilled water 1 L; pH 4-5.6.

[0186] The culture temperature is 26-31°C and the culture time is 8-10 days;

[0187] A 0.5 cm cake of Penicillium citrinum DHZ-2 (0.5 cm cake after 5 days of growth on PDA plates) was inoculated into 10 mL of optimized fermentation medium;

[0188] Secondly, combining the 3D graph and regression equation, we can know the theoretical optimal enzyme production conditions and results of the four enzyme activities are as follows:

[0189] Endoglucanase: culture temperature 26.924℃, pH 4.342, carbon source (rice straw powder) ratio 1.397% (i.e., 13.97g); the other two factors were the optimal conditions of single factors: culture time 8 days, nitrogen source (peptone) ratio 0.15%; enzyme activity 3.853 U / mL.

[0190] Exoglucanase: The optimal conditions for the culture temperature, carbon source (rice straw powder) ratio, nitrogen source (peptone) ratio, and other two factors were 30.428℃, 1.325%, 0.164%, and 5.5% pH, respectively. The culture time was 8 days. The enzyme activity was 4.572 U / mL.

[0191] β-glucosidase: The optimal conditions for the culture time were 10 days, the pH was 5.503, the nitrogen source (peptone) ratio was 0.175%, and the other two factors were single-factor optimal conditions: the culture temperature was 30°C, and the carbon source (rice straw powder) ratio was 1.5%; the enzyme activity was 7.829 U / mL.

[0192] Xylanase: The optimal conditions for culture temperature and nitrogen source (rice straw powder) were 28.449°C, 1.498% and 0.175% respectively. The pH value was 5 and the culture time was 8 days. The enzyme activity was 13.680 U / mL.

[0193] Compared with the initial enzyme-producing CMC-Na medium before optimization after culturing for 8 days, the endoglucanase activity (initial enzyme activity was 1.822 U / mL) increased by 2.1 times, the exoglucanase activity (initial enzyme activity was 1.714 U / mL) increased by 2.7 times, the β-glucosidase activity (initial enzyme activity was 1.899 U / mL) increased by 4.12 times, and the xylanase activity (initial enzyme activity was 7.937 U / mL) increased by 1.72 times.

[0194] In summary, after adjusting the data of the Box-Behnk experiment, the optimal enzyme production conditions for Penicillium citrinum DHZ-2 were obtained as follows:

[0195] Endoglucanase: culture temperature 25°C, initial pH 5, rice straw powder addition ratio 1.5%; culture time 8 days, nitrogen source (peptone) ratio 0.15%;

[0196] Exoglucanase: culture temperature 30°C, rice straw powder addition ratio 1.25%, peptone addition ratio 0.15%; initial pH 5, culture time 8 days;

[0197] β-glucosidase: culture time 10 days, initial pH 5, peptone addition ratio 0.175%; culture temperature 30°C, carbon source (rice straw powder) ratio 1.5%;

[0198] Xylanase: culture temperature 30℃, rice straw powder addition ratio 1.5%, peptone addition ratio 0.175%; initial pH 5, culture time 8 days.

[0199] ③ Box-Behnke model significance analysis results

[0200] The Box-Behnke model of endoglucanase is significant overall ( P <0.01), among which the temperature (℃) had a significant effect on enzyme activity ( P < 0.0001). The model fit is high, R 2 =0.9805, adjusted R 2 =0.9455, predicted R 2 =0.9217 (Adjusted R 2 and predict R 2difference <0.2); CV%=7.27 (<10%), signal-to-noise ratio 13.36 (>4), indicating that the model is reliable and has good predictive ability.

[0201] The Box-Behnke model of exoglucanase is significant ( P <0.01), temperature (℃), carbon source ratio (%), and nitrogen source ratio (%) significantly affected enzyme activity ( P <0.05), adjusted R 2 =0.9671, predicted R 2 =0.9146, the difference between the two is less than 0.2; the R 2 =0.9882, CV%=6.48 (<10%), and signal-to-noise ratio 19.11 (>4), indicating that the model accuracy and signal strength are excellent.

[0202] The Box-Behnke model of β-glucosidase was significant ( P <0.01), pH had a significant effect on enzyme activity ( P <0.0001), time (days) was significant ( P =0.0064). Adjust R 2 and predict R 2 are 0.9541 and 0.7953 respectively, with a difference of less than 0.2; R 2 =0.9836, CV%=5.79 (<10%), signal-to-noise ratio 18.88 (>4), and the overall model is reliable.

[0203] Similarly, the Box-Behnke model of xylanase is significant ( P <0.01), among which temperature (℃), carbon source ratio (%), and nitrogen source ratio (%) significantly affected enzyme activity ( P <0.05). Adjusted R 2 =0.9309, predicted R 2 =0.7558, the difference is less than 0.2; R 2 =0.9753, CV%=5.45 (<10%), signal-to-noise ratio 18.39 (>4), and the model fit was good.

[0204] Overall, all four models reached extremely significant levels ( P <0.01), CV% were all lower than 10%, and the signal-to-noise ratios were all over 4, indicating that the experimental design was reasonable and the model was reliable.

[0205] Example 4: Application of Penicillium citrinum DHZ-2 in rice straw degradation

[0206] The preparation method of Penicillium citrinum DHZ-2 cake is as follows:

[0207] Penicillium citrinum ( Penicillium citrinum DHZ-2 was inoculated into 100 mL of PDA liquid medium using an inoculation loop and cultured at 30°C with a shaker at 200 rpm / min for 48 hours to obtain seed solution.

[0208] 0.5 mL of seed liquid was spread on a PDA plate and cultured for 5 days (culture temperature was 30°C), and then a hole punch was used to obtain a Penicillium citrinum DHZ-2 cake (0.5 cm).

[0209] The specific steps of degradation are as follows:

[0210] 1) Rice straw degradation rate (dry weight loss rate)

[0211] Rice straw segments: Select clean and unrotten rice straw and cut it into short segments of about 2 cm in length. Clean the attached dust and impurities and place it in an oven at 80°C for drying (drying to constant weight).

[0212] Liquid fermentation: 2g of rice straw segments were placed in a 250mL conical flask and sterilized with 100mL of Hutcheson's inorganic salt culture medium (sterilized at 121℃ for 30 minutes). Three replicates were set up for each treatment group and control group. A 0.5cm thick cake of Penicillium citrinum DHZ-2 was added to the treatment group, while the control group was a shake flask without cake. The flask was placed in a constant temperature shaker at 30℃ and 200 rpm / min for incubation. The straw was removed at four time points (0, 10, 20, and 30 days), washed, and dried to a constant weight. The dry weight loss rate was measured. The results are as follows: Figure 11 shown.

[0213] The fermentation products obtained at the four time points of 0, 10, 20, and 30 days were treated as follows: ① Coarse filtration: Use a nylon filter bag (100 mesh) to filter to remove straw residues and mycelium; ② Centrifugation: The filtrate was placed in a 50 mL centrifuge tube and centrifuged at 4°C and 8000 rpm / min for 10 minutes to obtain the fermentation supernatant.

[0214] from Figure 11 As can be seen from the figure, after liquid fermentation, the degradation rate of rice straw in the treatment group was 28.93% on the 10th day, rising to 43.95% on the 20th day, and further increasing to 52.29% on the 30th day. In contrast, the degradation rate of rice straw in the control group was only 28.68% on the 30th day.

[0215] This reflects the degradation efficiency and extent of rice straw by Penicillium citrinum DHZ-2 to a certain extent.

[0216] Degradation rate = (m-m1) / m×100%, where m is the initial weight of rice straw (dry weight) and m1 is the weight of rice straw after liquid fermentation (dry weight).

[0217] The dry weight is obtained by drying in an oven at 80-105° C. to a constant weight.

[0218] 2) Cellulose and hemicellulose content of rice straw

[0219] Rice straw samples were taken from 0-30 days after liquid fermentation and dried (dried in an oven at 102°C) to determine the lignocellulose components. The results are shown in Table 17. The main process is as follows:

[0220] ① Air-dry the sample (the sample must be crushed to pass through a 1 mm sieve), weigh it, record it as M, and place it into a filter bag. Weigh the mass of the filter bag, record it as m1; also weigh the mass of the blank filter bag, C1.

[0221] ② Neutral detergent fiber (NDF) determination:

[0222] Add 20 g of sodium sulfite and 4.0 mL of thermostable α-amylase. Follow the NDF procedure on the ANKOM220 fiber analyzer. After completion, remove the sample and place it in a wide-mouth bottle. Perform the acetone wash step: submerge the sample in acetone for 3-5 minutes, remove it, and air-dry it in a 102°C oven. After cooling, weigh it and record it as m2. The weight of the blank filter bag is recorded as C2.

[0223] NDF (%) = [(m2-m1)+(C1-C2)]÷M×100.

[0224] ③Determination of acid detergent fiber (ADF):

[0225] Place the sample into the fiber analyzer and start the ADF program. After the program is completed, remove the sample and place it in a wide-mouth bottle with sufficient acetone. Wash with acetone and measure the neutral detergent fiber (NDF). Dry the sample in a 102°C oven and weigh it, recording it as m3. At the same time, weigh the mass of the blank filter bag and record it as C3.

[0226] ADF (%) = [(m3-m1)+(C1-C3)]÷M×100%.

[0227] ④ Determination of acid detergent lignin (ADL) and ash:

[0228] After measuring the ADF, immerse the sample in 72% sulfuric acid for 3 hours. Rinse with plenty of water until neutral. Add acetone to cover the sample, soak for 3-5 minutes, remove, and dry at 102°C. The sample is weighed (m4). The weight of the blank filter bag is recorded as C4. The dried, weighed sample is placed in a crucible (the weight of the crucible is recorded as m5). The crucible containing the blank sample is recorded as C5. Incinerate the sample in a muffle furnace at 550°C, cool to room temperature, and weigh (m6). The weight of the crucible containing the blank sample and the ash is recorded as C6.

[0229] ADL (%) = [(m4+m5-m6)-(C4+C5-C6)]÷M×100

[0230] Ash content (%) = [(m6-m5)-(C6-C5)] ÷ M × 100.

[0231] ⑤ Calculation of analysis results

[0232] Hemicellulose (%) = NDF (%) - ADF (%)

[0233] Cellulose (%) = ADF (%) - [ADL (%) + ash (%)]

[0234] After the cellulose and hemicellulose contents of rice straw were determined, the cellulose and hemicellulose degradation rates were calculated for the initial content (0 days) and the content after treatment (30 days);

[0235] Degradation rate of cellulose and hemicellulose = (initial content - content after treatment) / initial content × 100%.

[0236] Table 17 Changes in cellulose and hemicellulose content in rice straw at different liquid fermentation times

[0237]

[0238] Note: Different lowercase letters after the data in the same column indicate significant differences ( P <0.05)

[0239] As shown in Table 17, the cellulose and hemicellulose contents of rice straw changed significantly with increasing incubation time, both gradually decreasing. From 0 to 30 days, the cellulose and hemicellulose contents decreased to 19.32% and 16.50%, respectively, with degradation rates reaching 34.51% and 47.01%, respectively. This indicates that Penicillium citrinum DHZ-2 readily degrades cellulose and hemicellulose in rice straw. The reduction in cellulose content may be due to the endoglucanase, exoglucanase, and β-glucosidase activities inherent in Penicillium citrinum DHZ-2. Similarly, the high xylanase activity inherent in Penicillium citrinum DHZ-2 contributes to the reduction in hemicellulose content in rice straw after fermentation.

[0240] 3) Monosaccharide content produced by fermentation

[0241] Liquid fermentation: 400 μL of fermentation supernatant corresponding to fermentation time points 0, 10, 20, and 30 days were transferred to 5 mL stoppered tubes. Subsequently, 400 μL of PMP methanol solution (PMP concentration 0.5 mol / L) was added to each tube and vortexed to mix thoroughly. The tubes were then placed in a 70°C water bath for 2 h. After the reaction was complete, the stoppered tubes were removed from the water bath and allowed to cool naturally at room temperature. After the tubes cooled to room temperature, 400 μL of 0.3 mol / L HCl was added to each tube to neutralize the mixture to a pH of 6.0-7.0. After neutralization, 1200 μL of water was added to the tubes, followed by an equal volume (1200 μL) of chloroform. The tubes were vortexed and shaken, allowed to stand, and the chloroform phase was discarded. This extraction step was repeated twice to ensure complete extraction of the metabolites. Finally, the aqueous phase was filtered using a 0.45 μm microporous membrane (water system), and the filtered aqueous phase sample was then used for high performance liquid chromatography (HPLC) injection analysis to analyze the monosaccharide content in the rice straw fermentation broth.

[0242] Figure 12 The data show the changes in monosaccharide content in the rice straw fermentation broth from day 0 to 30. After Penicillium citrinum DHZ-2 degraded rice straw, the contents of mannose, rhamnose, galactose, xylose, and arabinose increased significantly. Hemicellulose, a major component of plant cell walls, is composed of various monosaccharides, including pentoses (such as xylose and arabinose) and hexoses (such as galactose). This suggests that during the rice straw degradation process, the xylanase enzyme in Penicillium citrinum DHZ-2 played a role in hydrolyzing the hemicellulose in rice straw into various monosaccharides. Overall, the glucose content did not increase significantly, but instead showed a downward trend. This may be because the high-temperature sterilization of the 0-day-old rice straw resulted in the breakdown of plant cell wall components, resulting in a relatively high glucose content. The inorganic salt medium used during the fermentation process required more nutrients for the growth of Penicillium citrinum DHZ-2 itself. Cellulase hydrolyzes cellulose into monosaccharides such as glucose, which is then used by the strain for growth and metabolism. Therefore, as fermentation time increased, the glucose content in the fermentation broth gradually decreased (the glucose content was 243.36 mg / L on day 0 and 108.28 mg / L on day 30). In the liquid fermentation broth, the galactose content increased from 39.18 mg / L to 115.85 mg / L, and the xylose content increased from 6.9 mg / L to 78.91 mg / L.

[0243] Description: Galactose is a six-carbon sugar found in hemicellulose in plant cell walls.

[0244] Control: According to the enzyme activity detection method of the present invention, Penicillium citrinum NAF5, Penicillium citrinum YS40-5, strain CB21, and Penicillium citrinum XZH-16 do not have xylanase activity, while Penicillium citrinum NCIM-1398 does not have endoglucanase or exoglucanase activity.

[0245] Referring to the rice straw degradation experiment of the present invention, it was found that after 30 days of fermentation using Penicillium citrinum NAF5, Penicillium citrinum YS40-5, strain CB21, and Penicillium citrinum XZH-16, the hemicellulose content did not decrease significantly compared to that on day 0 of fermentation.

[0246] After 30 days of fermentation, the cellulose content and hemicellulose content of Penicillium citrinum NCIM-1398 were both higher than 25%, while after 30 days of fermentation, the cellulose content and hemicellulose content of Penicillium citrinum DHZ-2 of the present invention were 19.32% and 16.50%, respectively, and the degradation efficiency was better than that of Penicillium citrinum NCIM-1398.

[0247] Finally, it should be noted that the above examples are merely specific embodiments of the present invention. Obviously, the present invention is not limited to the above examples and is subject to numerous variations. All variations that can be directly derived or conceived by a person of ordinary skill in the art from the disclosure of the present invention are considered to be within the scope of protection of the present invention.

Claims

1. Penicillium citrinum DHZ-2, characterized in that: The deposit number is CCTCCNO: M20241130, and the deposit date is June 3, 2024.

2. The use of Penicillium citrinum DHZ-2 according to claim 1, characterized in that: Penicillium citrinum DHZ-2 is fermented in a fermentation medium to produce enzymes, thereby obtaining cellulase and hemicellulase; Cellulases include endoglucanases, exoglucanases, and β-glucosidases; Hemicellulases include xylanases; The fermentation medium is rice straw powder 12.5-15g, NaNO3 2.0g, NH4Cl 2.0g, KH2PO4 1.0g, K2HPO4 1.0 g, peptone 1.5-1.75 g, MgSO4·7H2O 0.5 g, distilled water 1 L; pH 4.5-5.5; The culture temperature is 25-30°C; the culture time is 8-10 days.

3. The use of Penicillium citrinum DHZ-2 according to claim 2, characterized in that: For endoglucanase: culture temperature 25°C, culture time 8 days, rice straw powder 15g, peptone 1.5g, pH 5; For exoglucanase: culture temperature 30℃, culture time 8 days, rice straw powder 12.5g, peptone 1.5g, pH 5; For β-glucosidase: culture temperature 30°C, culture time 10 days, rice straw powder 15g, peptone 1.75g, pH 5; For xylanase: culture temperature 30℃, culture time 8 days, rice straw powder 15g, peptone 1.75g, pH 5.

4. The method for degrading rice straw using Penicillium citrinum DHZ-2 according to claim 1, characterized in that The following steps are involved: The rice straw was mixed with Hutcheson's inorganic salts medium and Penicillium citrinum DHZ-2 at a ratio of 2±0.5 g rice straw to 100 mL Hutcheson's inorganic salts medium and 0.5 cm Penicillium citrinum DHZ-2 cake, and fermented in a constant temperature shaker at 30±2°C for 10 to 30 days. After Penicillium citrinum DHZ-2 degraded rice straw, the contents of mannose, rhamnose, galactose, xylose and arabinose increased significantly.

5. The method for degrading rice straw using Penicillium citrinum DHZ-2 according to claim 4, characterized in that: Hutcheson's inorganic salts medium: KH2PO4 1.0 g, NaCl 0.1 g, MgSO4·7H2O 0.3 g, NaNO3 2.5 g, FeCl3 0.01 g, CaCl2 0.1 g, distilled water 1 L, pH 7.2.

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