Penicillium citrinum DHZ-2 with high yield of cellulase and hemicellulase and application thereof

Abstract

The invention belongs to the field of microorganisms and application thereof, and particularly relates to penicillium citrinum DHZ-2 with high yield of cellulase and hemicellulase and application of the penicillium citrinum DHZ-2 to degradation of rice straw. The invention provides Penicillium citrinum DHZ-2, and the preservation number of the Penicillium citrinum DHZ-2 is CCTCC (China Center For Type Culture Collection) NO: M20241130. The invention further provides application of the penicillium citrinum DHZ-2. The penicillium citrinum DHZ-2 is fermented in a fermentation culture medium to produce enzyme, so that cellulase and hemicellulase are obtained. The penicillium citrinum DHZ-2 disclosed by the invention can be used for degrading rice straws.

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 rice straw degradation. Background Art

[0002] Crop straw is a rich renewable resource, and its high-value utilization has become a research hotspot, mainly including fertilizer, energy, raw material, feed and base material. In the process of "five transformations", biomass degradation technology plays a key role.

[0003] Traditional methods of straw degradation are mainly physical and chemical. The physical method usually destroys the physical structure of the straw by mechanical crushing, but this method can only change the shape of the straw and cannot effectively degrade it; the chemical method uses chemical reagents such as strong acids and strong alkalis to crack the straw. Although the degradation efficiency is high, there are problems such as serious environmental pollution. In contrast, the biodegradation method using microorganisms and their secreted enzymes has the advantages of high efficiency, environmental protection, and economy, and has become an important research direction for the resource utilization of straw. There are many types of microorganisms used for straw biodegradation, among which fungi have strong enzymatic hydrolysis ability and play an important role in straw degradation. The cellulase secreted by fungi mainly includes endoglucanase (EC 3.2.1.4), exoglucanase (EC 3.2.1.91) and β-glucosidase (EC 3.2.1.21), which can synergistically degrade cellulose into glucose; while hemicellulase mainly refers to β-1,4-xylanase (EC 3.2.1.8), which can degrade hemicellulose into monosaccharides such as xylose. These enzymes can cut the cellulose and hemicellulose molecular chains in the straw, degrading them into small molecules such as glucose, xylose, etc., which can be further utilized to realize the resource utilization of straw.

[0004] At present, screening microbial strains with high production of (hemi) cellulase from special environments is a research hotspot in the field of microbial application. For example, the marine bacterial strain Bacillus marine sediment isolated from Gopur Beach in Odisha, India ( Bacillus oceanisediminis )、Breibacterium halodurans( 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.) 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 (hemiceliulase) in specific environments.

[0005] Penicillium citrinum Penicillium citrinum) is a filamentous fungus of the genus Penicillium that can secrete cellulases and hemicellulases for the hydrolysis of straw lignocellulose. At the same time, Penicillium also has the characteristics of short growth cycle, simple culture conditions, and rapid biomass accumulation, which can greatly reduce the time and economic cost of large-scale production. However, the Penicillium citrinum strains reported in current studies often only have one or a few cellulase or hemicellulase activities, which limits its practical use in straw degradation.

[0006] For example, Penicillium citrinum NAF5 can produce endoglucanase with an enzyme production of 3.13 U / mL (Saini et al., 2020), and can also produce β-glucosidase, with a yield of 2.48 U / mL after conditions optimization (Saini et al., 2024). Penicillium citrinum YS40-5 can produce high β-glucosidase under rice bran solid-state fermentation conditions, 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 was relatively high, and its maximum enzyme activity reached 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 activity of Avicel enzyme (crystalline cellulase) is 14.3±0.8U / gds, the activity of CMC enzyme (carboxymethyl cellulase) is 267.7±38.6U / gds, the activity of pNPG enzyme (p-nitrophenyl-α-D-pyranoglucopyranoside cellulase) is 103.7±4.5U / gds, the activity of pNPC enzyme (p-nitrophenyl-β-D-cellobioside cellulase) is 10.2±0.8U / gds, and the activity of xylanase 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 kind of 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 the use of the above 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] As a further improvement of the use of Penicillium citrinum DHZ-2 of the present invention: The fermentation medium is 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 is 4.5-5.5; The culture temperature (fermentation temperature) is 25~30℃; the culture time is 8~10 days.

[0016] As a further improvement of the use of Penicillium citrinum DHZ-2 of the present invention: For endoglucanase: culture temperature 25°C, culture time 8 days, rice straw powder 15g (rice straw powder addition ratio 1.5%), peptone 1.5g (peptone addition ratio 0.15%), pH 5; 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; 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; 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.

[0017] The present invention also provides a method for degrading rice straw using the above-mentioned Penicillium citrinum DHZ-2, comprising the following steps: The rice straw was mixed with Hutchinson inorganic salt medium and Penicillium citrinum DHZ-2 and then fermented.

[0018] As an improvement of the method for degrading rice straw by Penicillium citrinum DHZ-2 of the present invention: Rice straw was mixed with Hutcheson's inorganic salt medium and Penicillium citrinum DHZ-2 according to the ratio of 2±0.5 g rice straw (dry weight) to 100 mL Hutcheson's inorganic salt medium and 0.5 cm Penicillium citrinum DHZ-2 cake, and fermented at 30±2℃ constant temperature shaking incubator (200±20rpm / min) for 10-30 d.

[0019] As a further improvement of the method for degrading rice straw by Penicillium citrinum DHZ-2 of the present invention: Hutcheson 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, pH 7.2.

[0020] The pH can be adjusted by conventional methods such as 1 mol / L NaOH.

[0021] As a special group that feeds on soil, soil-feeding termites account for more than one-third of known termite species and have unique research value. Coptotermes sinensis is a soil-feeding termite that mainly feeds on organic matter in the soil and cellulose in decaying plants. Given the feeding characteristics of Coptotermes sinensis, it is likely that there are potential microbial strains that produce (hemi) cellulase in their intestines, which provides a new direction for the study of straw biodegradation.

[0022] The Penicillium citrinum DHZ-2 provided by the present invention is isolated from the Chinese twisted termite ( Sinocapritermes sp.) and was identified by morphological and ITS sequence comparison with the strain Penicillium citrinum ( Penicillium citrinum ) have an ITS sequence similarity of up to 100%.

[0023] The present invention has the following beneficial effects: 1) The present invention screened out a strain of Penicillium citrinum DHZ-2 from the intestine of the termite Pseudomonas sinensis, which can simultaneously secrete cellulase and hemicellulase, wherein the cellulase includes endoglucanase, exoglucanase and β-glucosidase; and the hemicellulase includes xylanase. Therefore, Penicillium citrinum DHZ-2 has great application potential in rice straw degradation.

[0024] 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 with the conventional MSM solid culture medium.

[0025] 3) By 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.

[0026] 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.

[0027] 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.

[0028] In summary, the present invention screened out a strain of Penicillium citrinum DHZ-2 that can produce a high amount of (semi) cellulase from the intestine of Coptis chinensis. 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-producing 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

[0029] The specific implementation modes of the present invention are further described in detail below with reference to the accompanying drawings.

[0030] Figure 1 This is a Congo red decolorization circle diagram of Penicillium citrinum DHZ-2 on a CMC-Na plate.

[0031] Figure 2 The colony morphology (left) and microscopic examination (right) of Penicillium citrinum DHZ-2 on the PDA plate.

[0032] Figure 3 This is a phylogenetic tree constructed based on the ITS sequence of Penicillium citrinum DHZ-2.

[0033] Figure 4 This is the effect of culture time on the (hemicellulase) activity produced by Penicillium citrinum DHZ-2.

[0034] Figure 5 This study is the effect of culture temperature on the (hemicellulase) activity produced by Penicillium citrinum DHZ-2.

[0035] Figure 6 This is the effect of initial pH on the (hemicellulase) activity produced by Penicillium citrinum DHZ-2.

[0036] Figure 7 This is the effect of carbon source type and addition ratio on the (hemi)cellulase activity produced by Penicillium citrinum DHZ-2.

[0037] Figure 8 This study is the effect of nitrogen source type and addition ratio on the (hemi)cellulase activity produced by Penicillium citrinum DHZ-2.

[0038] Fig. 9 This is a Pareto diagram showing the effect of each single factor on the production of (hemi)cellulase by Penicillium citrinum DHZ-2.

[0039] Fig.10 This is a 3D response surface diagram of the effects of pairwise factors on the production of (hemi)cellulase by Penicillium citrinum DHZ-2.

[0040] Figure 9~Figure 10 In: A stands for endoglucanase, B stands for exoglucanase, C stands for β-glucosidase, and D stands for xylanase.

[0041] Fig.11 is the degradation rate of rice straw in liquid fermentation mode from 0 to 30 days.

[0042] Fig.12 It is the sugar content of rice straw fermented in liquid fermentation mode for 0-30 days. DETAILED DESCRIPTION

[0043] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the present invention.

[0044] The culture medium and reagent preparation methods involved in the following examples are: 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. The pH was initially set to 3-13.

[0045] 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.

[0046] CMC-Na solid medium (g / L): CMC-Na 10.0 g, NaNO3 2.0 g, NH4Cl 2.0 g, KH2PO4 1.0 g, K2HPO4 1.0 g, peptone 1.0 g, MgSO4·7H2O 0.5 g, agar 20.0 g, distilled water 1 L. Natural pH.

[0047] 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.

[0048] 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. pH natural.

[0049] PDA liquid medium (g / L): potato dextrose broth powder 25 g, distilled water 1 L. pH natural.

[0050] PDA solid medium (g / L): potato dextrose agar powder 46.0 g, distilled water 1 L. Natural pH.

[0051] Preparation of DNS solution: Dissolve 3,5-dinitrosalicylic acid (3.15 g) in 500 mL of deionized water, stir and dissolve at 45°C, then add 100 mL (5 mol / L) sodium hydroxide solution and stir to mix evenly. Add 91.0 g of potassium sodium tartrate, 2.5 g of phenol, and 2.5 g of anhydrous sodium sulfite in sequence, stir and dissolve, cool to room temperature, dilute to 1000 mL with a 1000 mL volumetric flask (i.e., dilute to 1000 mL with deionized water), filter, and store the filtrate in a brown bottle, store in the dark for 7 days for later use.

[0052] The enzyme activity determination methods involved in the following embodiments are all conventional techniques, which can be as follows: Preparation of crude enzyme solution: Penicillium citrinum DHZ-2 was fermented on the culture medium, and the resulting bacterial solution was centrifuged (4°C, 8,000 rpm / min for 5 min). The supernatant was collected, which is the crude enzyme solution and can be used to determine the enzyme activity.

[0053] ① Endoglucanase The activity of endoglucanase was determined by the DNS method. 1% (1g / 100mL) CMC-Na (sodium carboxymethyl cellulose) was prepared as a substrate using 50mM sodium citrate buffer (pH5) as a solvent. Each 200μL crude enzyme solution and 800μL substrate 1% CMC-Na were placed in a 50℃ water bath for 60 min. The enzymatic reaction was terminated by adding 1mL DNS solution, and the mixture was placed in a boiling water bath for 10 min. After cooling, 3mL of distilled water was added, and the value at 540nm was measured using an ELISA reader. Three replicates were set, and the measured values ​​were substituted into the regression equation to obtain the amount of reducing sugar generated.

[0054] ②Exoglucanase The test method is the same as that of endoglucanase, except that the substrate is replaced with 1% Avicel (microcrystalline cellulose MCC) prepared with 50 mM sodium citrate buffer (pH 5) as the solvent.

[0055] ③β-glucosidase At 10mmol / L p NPG (4-nitrobenzene-β-D-pyranoglucoside) was used as the substrate. 200 μL of substrate was mixed with 100 μL of crude enzyme solution and placed in a 50°C water bath for 30 min. After the reaction, 2 mL of 1 mol / L Na2CO3 was added to terminate the test. The absorbance at 410 nm was measured on a microplate reader. Three replicates were set and the amount of p-nitrophenol generated was obtained by substituting into the regression equation.

[0056] ④ Xylanase Xylanase is the main hemicellulose degrading enzyme, so xylanase is used as a representative of hemicellulose. The DNS method was used to determine the xylanase activity. 1% beech xylan was prepared as a substrate with 50mM sodium citrate buffer (pH5) as the solvent. Each 200μL crude enzyme solution and 800μL 1% beech xylan solution were water bathed at 50℃ for 60 min. The enzymatic reaction was terminated by adding 1mL DNS solution, and the solution was boiled in water for 10 min. After cooling, 3mL of distilled water was added, and the value at 540nm was measured by an ELISA instrument. Three replicates were set, and the measured values ​​were substituted into the regression equation to obtain the amount of xylose generated.

[0057] ⑤ Enzyme activity calculation One unit of enzyme activity (U) is defined as the number of micromoles of glucose, p-nitrophenol, and xylose catalytically generated per minute under certain reaction conditions. The specific activity can be expressed as U / mL.

[0058] Enzyme activity (U / mL) = (product concentration × final reaction volume) / (product molar mass × crude enzyme solution volume × reaction time) × dilution factor × 1000.

[0059] Hutchinson inorganic salt medium (g / L): KH2PO4 1.0g, NaCl 0.1g, MgSO4▪7H2O 0.3g, NaNO3 2.5g, FeCl3 0.01g, CaCl2 0.1g, distilled water 1L.

[0060] Rice straw-inorganic salt liquid fermentation medium: 2 g rice straw segments, 100 mL Hutcheson inorganic salt medium.

[0061] Example 1: Isolation and identification of Penicillium citrinum DHZ-2 1) Strain isolation and initial screening Take 20 Chinese twisted termite workers for dissection. First, rinse the termite body surface with sterile water several times to rinse off 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 (pH7) 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 to 1 mL, and obtain the intestinal sample stock solution. Dilute the intestinal sample stock solution to 10 with PBS buffer. -3 -10 -6 , 50 μL of diluted intestinal fluid was spread on MSM medium, cultured at 30°C for 3-7 days, and the growth of the colonies was observed. Typical colonies were picked on the plate, streaked and purified to obtain single colonies, and the obtained single colonies were inoculated in LB and PDA medium for better growth. The purified strain obtained by the above operation was inoculated on the CMC-Na agar plate, inverted and cultured at 30°C, and an appropriate amount of 1 mg / mL Congo red dye was poured into the plate for staining for 30 minutes, and then the dye was poured out, and 1 mol / L NaCl solution was used for decolorization for 30 minutes to obtain strain DHZ-2, which formed a hydrolysis circle on the CMC-Na plate, as shown in 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.

[0062] 2) Morphology and molecular identification of strains Morphological analysis: Figure 2As shown, the morphological structure of the plate colony was observed. In the early stage, it grew on the PDA plate as white velvet, and later it became yellow-green due to spore production. The strain had radial grooves. When spores were produced in large quantities, the surface of the colony was covered with powdery particles. Under the microscope, the conidiophores were upright, with smooth walls and broom-like branching structures; the conidia were spherical and smooth in surface, and were preliminarily identified as Penicillium.

[0063] Molecular identification analysis: Scrape the hyphae on the PDA plate and quickly grind them into powder in liquid nitrogen. Extract the genomic DNA of the fungus according to the Shanghai Shenggong Fungal Genomic DNA Rapid Extraction Kit. Use ITS universal primers ITS1 and ITS4 to amplify the ITS sequence. The PCR amplification system is 50μL: GreenTaq Mix 25 μL, ITS1 2μL, ITS4 2μL, DNA template 1μL, and sterile deionized water to make up 20μL. The primers are as follows: ITS1: 5'-TCCGTAGGTGAACCTGCGG-3' (such as sequence number [ID] 1 in the sequence listing); ITS4: 5'-TCCTCCGCTTATTGATATGC-3' (such as sequence number [ID] 2 in the sequence listing).

[0064] The amplification reaction was carried out on a PCR instrument, and the amplification program was: 94℃ 3min; 95℃ 3min, 55℃ 30s, 72℃ 1min, 33 cycles, 72℃ 5min, and the termination temperature was 16℃. The ITS-PCR amplification products were detected by 1% agarose gel electrophoresis. The sequencing results were Blast aligned in NCBI, and the ITS sequences with high similarity were obtained in the GenBank database, and homology analysis was performed to construct the strain phylogenetic tree.

[0065] Phylogenetic tree Figure 3 As shown, strain DHZ-2 and Penicillium citrinum ( Penicillium citrinum ) had an ITS sequence similarity of up to 100%. Based on the colony morphology, microscopic observation and molecular identification results, the strain was identified as Penicillium citrinum ( Penicillium citrinum ), which was named Penicillium citrinum ( Penicillium citrinum )DHZ-2.

[0066] The deposit information of DHZ-2 is as follows: Deposit name: Penicillium citrinum DHZ-2 Penicillium citrinum DHZ-2, deposit unit: China Center for Type Culture Collection, deposit address: Wuhan University, Wuhan, China, deposit number: CCTCC NO:M 20241130, deposit date June 3, 2024.

[0067] Example 2: Determination of the ability of Penicillium citrinum DHZ-2 to produce (hemi) cellulase The specific steps are as follows: A single factor experiment was used to investigate the effects of enzyme production conditions on endoglucanase, exoglucanase, β-glucosidase and xylanase of the strains, including five types of fermentation conditions: culture time of the strain, culture temperature, initial pH, carbon source type and addition ratio, nitrogen source type and addition ratio.

[0068] CMC-Na liquid culture medium was used, with a culture medium content of 10 mL in each group. 0.5 cm bacterial cakes of Penicillium citrinum DHZ-2 were inoculated into the CMC-Na liquid culture medium as an enzyme production culture system.

[0069] ① Different culture time conditions The culture temperature was maintained at 30°C and the culture was carried out 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.

[0070] ② Different temperatures The culture temperature was set at 20℃, 25℃, 30℃, 35℃ and 40℃ respectively, and the culture was carried out at each temperature for 6 days. The effects of different temperatures on the (hemicellulase) production capacity of Penicillium citrinum DHZ-2 were measured to determine the optimal enzyme production temperature.

[0071] ③Cultivation at different initial pH The culture temperature was maintained at 30°C and the speed was 200 rpm / min in a shaker. The initial pH of the enzyme production culture system (CMC-Na liquid culture medium) was adjusted to 3, 5, 7, 9, 11, and 13, respectively, and the culture was kept at constant temperature for 6 days. The effect of different initial pH conditions on the ability of Penicillium citrinum DHZ-2 to produce (hemi) cellulase was determined to determine the optimal enzyme production pH.

[0072] ④Different carbon sources The carbon sources were rice straw powder, sodium carboxymethyl cellulose, sucrose, glucose, and soluble starch; 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 culture medium) was replaced with different carbon sources: rice straw powder, sodium carboxymethyl cellulose (CMC-Na), sucrose, glucose, and soluble starch, and cultured at constant temperature for 6 days to determine the effect of different carbon sources on the ability of Penicillium citrinum DHZ-2 to produce (hemi) cellulase and to determine the optimal carbon source for enzyme production.

[0073] Based on the best carbon source screened out, 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.

[0074] The preparation process of rice straw powder is as follows: collect fresh rice straw, wash away impurities and dry at 80°C to constant weight, then use a grinder to crush and sieve to obtain 100-mesh rice straw powder.

[0075] ⑤ Different nitrogen sources The types of nitrogen sources are beef extract, ammonium sulfate, protein, yeast powder, and urea; 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 culture medium) was replaced with different nitrogen sources: beef extract, ammonium sulfate, peptone, yeast powder, and urea, and cultured at constant temperature for 6 days to determine the effect 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.

[0076] Based on the best 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.

[0077] The results of the single factor experiment are as follows Figure 4 , Figure 5 , Figure 6 , Figure 7 , Figure 8 shown.

[0078] from Figure 4 It can be seen that the activities of the four enzymes changed with the extension of time. 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.

[0079] from Figure 5 It can be seen that the strain produces the highest endoglucanase at 25℃ (1.504U / mL); the strain produces the highest exoglucanase at 30℃ (1.638U / mL); the strain produces the highest β-glucosidase at 30℃ (1.279U / mL); the strain produces the highest xylanase at 30℃ (7.167U / mL), indicating that the four enzyme activities show the highest values ​​at 25℃ and 30℃ respectively.

[0080] from Figure 6It 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.

[0081] from Figure 7 From A to D, we can see that: through the effect of different carbon sources on the enzyme production of Penicillium citrinum DHZ-2, it was found that when rice straw powder was used as the only carbon source, the enzyme production could reach the highest value. The endoglucanase production could reach 1.883 U / mL, the exoglucanase production could reach 1.680 U / mL, the β-glucosidase production could reach 2.796 U / mL, and the xylanase production could reach 9.349 U / mL, indicating that rice straw powder was the best carbon source for the activities of these four enzymes.

[0082] from Figure 7 From E~F, we can know the effects of six ratios of rice straw powder (0.5%, 0.75%, 1.0%, 1.25%, 1.5%, 1.75%) on the activities of each enzyme; the endoglucanase showed a maximum value of 2.293U / mL when the ratio was 1.5%; the exoglucanase activity was the highest at 1.973U / mL when the ratio was 1.25%, and the β-glucosidase activity was the highest at 5.429U / mL when the ratio was 1.5%; the xylanase reached the maximum enzyme production of 10.481U / mL when the ratio was 1.25%, indicating that the four enzyme activities showed the best activity when the rice straw powder ratio was 1.25% and 1.5%, respectively.

[0083] from Figure 8 From A~D, we can know that when peptone is used as nitrogen source, the production of 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 8 From E~F, we can know the enzyme production at different peptone ratios (0.05%, 0.075%, 0.1%, 0.125%, 0.15%, 0.175%). The 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 0.150% peptone ratio, indicating that the four enzyme activities can reach the maximum enzyme production value when the peptone ratio is 0.150%.

[0084] Example 3: Response surface optimization method for (hemi)cellulase production by Penicillium citrinum DHZ-2 1) Screening of key factors affecting enzyme production ①Plackett-Burman Experimental Factors and Level Settings 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 depends on the left and right points of the optimal point of the single factor result, which are used 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.

[0085] Table 1 Plackett-Burman design of endoglucanase

[0086] Table 2 Plackett-Burman design of exoglucanase

[0087] Table 3 Plackett-Burman design of β-glucosidase

[0088] Table 4 Plackett-Burman design of xylanase

[0089] ②Plackett-Burman design results According to the setting of high and low levels of each factor, the experiment was carried out 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 diagram of the standardized effect of factors is shown in Fig. 9 As shown, Fig. 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.

[0090] Table 5 Plackett-Burman design results of endoglucanase

[0091] From Table 5, it can be seen that the endoglucanase had the best result in the 11th experimental design, that is, on the 6th day, when the pH was 7, the culture temperature was 30°C, 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. Fig. 9 A found that the three factors that have the most significant impact on endoglucanase are culture temperature, pH, and carbon source addition ratio.

[0092] Table 6 Exoglucanase Plackett-Burman test design and results

[0093] The data in Table 6 show that the exoglucanase achieved the best result 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. Fig. 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.

[0094] Table 7 β-glucosidase Plackett-Burman test design and results

[0095] According to the data shown in Table 7, it can be observed that β-glucosidase has the best effect in the 11th experimental design. Specifically, under the conditions of culturing to the 6th day, 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 2.042U / mL. Similarly, Fig. 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.

[0096] Table 8 Xylanase Plackett-Burman test design and results

[0097] From the data in Table 8, it can be seen that the xylanase yield was the 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 yield was 10.024 U / mL. Fig. 9In 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.

[0098] ③ Results of significance analysis of the Plackett-Burman model The Plackett-Burman model of endoglucanase shows P =0.0013, the model is significant as a whole. 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.

[0099] Similarly, the Plackett-Burman model for exoglucanases was overall significant ( P =0.0014), the experimental design is effective. R after model evaluation parameter adjustment 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, which is greater than 4, indicating that the model has high credibility and accuracy.

[0100] The Plackett-Burman model of β-glucosidase showed that the model was significant overall. P =0.0017, predicted R 2 The value is 0.7365, and the adjusted R² value is 0.8792. The difference between the two is less than 0.2, indicating that the model has reasonable consistency. CV%=8.39<10, the signal-to-noise ratio is 13.8018, and when the ratio is greater than 4, it indicates that the model has a good enough signal.

[0101] The Plackett-Burman model of xylanase was 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 accuracy and signal-to-noise ratio are at an ideal level.

[0102] 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.

[0103] 2) Optimize enzyme production conditions ①Box-Behnken experimental factors and level settings On the basis of the Plackett-Burman experimental results, according to the three significantly influencing factors screened out, the low (-1), medium (0), and high (1) levels of each factor were set. 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. With endoglucanase, exoglucanase, β-glucosidase, and xylanase as response values, the response surface experimental analysis of each enzyme activity was carried out. The experimental design is shown in Tables 9, 10, 11, and 12.

[0104] Table 9 Box-Behnken design of endoglucanase

[0105] Table 10 Box-Behnken design of exoglucanase

[0106] Table 11 Box-Behnken design of β-glucosidase

[0107] Table 12 Box-Behnken design of xylanase

[0108] ②Box-Behnken response surface design results The software Design-Expert 13 was used to perform multivariate fitting analysis on the experimental results. The experimental results are shown in Tables 13, 14, 15, and 16.

[0109] Table 13 Box-Behnke response surface design results of endoglucanase

[0110] The response value of endoglucanase varied from 1.413 U / mL to 3.890 U / mL. The endoglucanase activity of group 9 was the highest (3.890 U / mL), and the fermentation conditions were culture temperature of 25℃, pH of 5, and rice straw powder ratio of 1.5%. In the 10th group of experiments, when the culture temperature was 20℃, pH of 5, and the rice straw powder ratio was 1.25%, the response value was the lowest at 1.413 U / mL.

[0111] Table 14 Box-Behnke response surface design results of exoglucanase

[0112] The exoglucanase had the best response value of 4.641 U / mL under the fourth group of 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%.

[0113] Table 15 Box-Behnke response surface design results for β-glucosidase

[0114] 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.

[0115] Table 16 Box-Behnke response surface design results for xylanase

[0116] In the 14th group of experiments, when the culture temperature was 30℃, the proportion of rice straw powder was 1.5%, and the proportion of peptone was 0.175%, the maximum xylanase response value was 13.526 U / mL. In the 13th group of experiments, when the culture temperature was 35℃, the proportion of rice straw powder was 1%, and the proportion of peptone was 0.15%, the minimum xylanase response value was 5.989 U / mL.

[0117] According to the experimental results, the multiple regression equations of endoglucanase, exoglucanase, β-glucosidase and xylanase were obtained: Y (endoglucanase) =3.65+0.8484A-0.0879B-0.031C+0.0008AB-0.127AC+0.062BC-1.17A 2-0.172B 2 -0.1218C 2 ; Y (exoglucanase) =4.40+0.3331A+0.2375B+0.4029C+0.1313AB-0.1070AC-0.0668BC-1.92A 2 -0.2608B 2 -0.3330C 2 ; Y (β-glucosidase) =6.46+0.5178A+1.29B+0.2329C-0.0180AB+0.1020AC-0.3258BC-0.0208A 2 -1.96B 2 +0.4150C 2 ; Y (xylanase) =11.12-1.35A+1.47B+0.5598C+0.0915AB+0.1868AC+0.8088BC-2.19A 2 -0.3646B 2 -0.0058C 2 ; According to the regression equation, the response surface 3D graph of the regression equation is obtained, and the results are as follows Fig.10 shown.

[0118] First, the fermentation process of the strain of the present invention is initially set as follows: The optimized fermentation medium (g / L) is: 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.

[0119] The culture temperature is 26~31℃ and the culture time is 8~10 days; A 0.5 cm bacterial cake of Penicillium citrinum DHZ-2 (0.5 cm bacterial cake after 5 days of growth on PDA plate) was inoculated into 10 mL of optimized fermentation medium; 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: Endoglucanase: culture temperature 26.924℃, pH 4.342, carbon source (rice straw powder) ratio 1.397% (i.e., 13.97g); the other two factors are the optimal conditions of single factors: culture time is 8 days, nitrogen source (peptone) ratio is 0.15%; enzyme activity is 3.853 U / mL.

[0120] Exoglucanase: The culture temperature was 30.428℃, the proportion of carbon source (rice straw powder) was 1.325%, the proportion of nitrogen source (peptone) was 0.164%, and the other two factors were the optimal conditions of single factors: pH was 5, the culture time was 8 days; the enzyme activity was 4.572 U / mL.

[0121] β-Glucosidase: culture time 10 d, pH 5.503, nitrogen source (peptone) ratio 0.175%, the other two factors are the optimal conditions of single factors: culture temperature 30℃, carbon source (rice straw powder) ratio 1.5%; enzyme activity 7.829 U / mL.

[0122] Xylanase: The culture temperature was 28.449℃, the proportion of carbon source (rice straw powder) was 1.498%, the proportion of nitrogen source (peptone) was 0.175%, and the other two factors were the optimal conditions of single factors: pH was 5, and the culture time was 8 days; the enzyme activity was 13.680 U / mL.

[0123] Compared with the initial enzyme-producing CMC-Na medium before optimization at 8 days of cultivation, the endoglucanase (initial enzyme activity was 1.822U / mL) increased by 2.1 times, the exoglucanase (initial enzyme activity was 1.714U / mL) increased by 2.7 times, the β-glucosidase (initial enzyme activity was 1.899U / mL) increased by 4.12 times, and the xylanase (initial enzyme activity was 7.937U / mL) increased by 1.72 times.

[0124] 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: Endoglucanase: culture temperature 25℃, initial pH 5, rice straw powder addition ratio 1.5%; culture time 8 days, nitrogen source (peptone) ratio 0.15%; Exoglucanase: culture temperature 30°C, rice straw powder addition ratio 1.25%, peptone addition ratio 0.15%; initial pH 5, culture time 8 days; 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%; Xylanase: culture temperature 30℃, rice straw powder addition ratio 1.5%, peptone addition ratio 0.175%; initial pH 5, culture time 8 days.

[0125] ③ Box-Behnke model significance analysis results The Box-Behnke model of endoglucanase is overall significant ( P <0.01, among which 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 2 difference <0.2); CV%=7.27 (<10%), signal-to-noise ratio 13.36 (>4), indicating that the model is credible and has good predictive ability.

[0126] 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.

[0127] 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 model is generally reliable.

[0128] Similarly, the Box-Behnke model for 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 R2 =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.

[0129] Overall, all four models reached a very significant level ( P <0.01), CV% were all lower than 10%, and the signal-to-noise ratio was more than 4, indicating that the experimental design was reasonable and the model was reliable.

[0130] Example 4: Application of Penicillium citrinum DHZ-2 in rice straw degradation The preparation method of the Penicillium citrinum DHZ-2 bacterial cake is as follows: Penicillium citrinum ( Penicillium citrinum ) DHZ-2 was inoculated into 100 mL of PDA liquid medium using an inoculation loop and cultured at 30°C and 200 rpm / min shaking for 48 hours to obtain seed solution; 0.5 mL of seed solution was spread on a PDA plate and incubated for 5 days (the culture temperature was 30°C), and then a hole puncher was used to obtain a bacterial cake (0.5 cm) of Penicillium citrinum DHZ-2.

[0131] The specific steps of degradation are as follows: 1) Rice straw degradation rate (dry weight loss rate) 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).

[0132] Liquid fermentation: 2g of rice straw segments were placed in a 250 mL conical flask, and 100 mL of Hutcheson inorganic salt culture medium was sterilized (sterilized at 121℃ for 30 minutes). Three replicates were set for the treatment group and the control group. 0.5 cm of citrinum DHZ-2 cake was added to the treatment group, and the control group was a shake flask without cake. The flask was placed in a 30℃ constant temperature shaker and cultured at 200 rpm / min. The straw was taken out at four time points of 0, 10, 20, and 30 days, and washed and dried to a constant weight. The dry weight loss rate was measured. The results are as follows: Fig.11 shown.

[0133] The fermentation products obtained at four time points, 0, 10, 20, and 30 days, were treated as follows: ① coarse filtration: use a nylon filter bag (100 mesh) to filter and remove straw residues and mycelium; ② centrifugation: put the filtrate into a 50 mL centrifuge tube and centrifuge at 4°C and 8000 rpm / min for 10 minutes to obtain the fermentation supernatant.

[0134] from Fig.11It can be seen that after liquid fermentation, the degradation rate of rice straw in the treatment group was 28.93% on the 10th day, and the degradation rate increased to 43.95% on the 20th day, and further increased to 52.29% on the 30th day. The degradation rate of rice straw in the control group was only 28.68% on the 30th day.

[0135] This reflects the degradation efficiency and extent of rice straw by Penicillium citrinum DHZ-2 to a certain extent.

[0136] 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).

[0137] The dry weight is obtained by drying in an oven at 80-105°C to a constant weight.

[0138] 2) Cellulose and hemicellulose content of rice straw The rice straw after liquid fermentation for 0-30 days was sampled and dried (dried in an oven at 102°C), and the lignocellulose components were determined. The results are shown in Table 17. The main process is as follows: ① Air-dry the sample (the sample needs to be crushed to pass through a 1 mm sieve), weigh it and record it as M, and put it into the filter bag. Weigh the mass of the filter bag and record it as m1; at the same time, weigh the mass of the blank filter bag C1.

[0139] ② Neutral detergent fiber (NDF) determination: Add 20 g of sodium sulfite and 4.0 mL of thermostable α-amylase. Operate according to the NDF program of the ANKOM220 fiber analyzer. After completion, take out the sample and place it in a wide-mouth bottle, and perform the acetone washing step, that is, immerse the sample in acetone for 3-5 minutes, take it out, air-dry the sample in a 102°C oven, weigh it after cooling, and record it as m2. The blank filter bag is weighed and recorded as C2.

[0140] NDF (%) = [(m2-m1)+(C1-C2)]÷M×100.

[0141] ③ Determination of acid detergent fiber (ADF): Put the sample into the fiber analyzer and start the ADF program. After the program is finished, take out the sample and put it into a wide-mouth bottle with sufficient acetone. Wash it with acetone and measure the neutral detergent fiber (NDF). Dry the sample in an oven at 102℃ and weigh it, record it as m3. At the same time, weigh the mass of the blank filter bag and record it as C3.

[0142] ADF (%) = [(m3-m1)+(C1-C3)]÷M×100%.

[0143] ④ Determination of acid detergent lignin (ADL) and ash: After determining the ADF, immerse the sample in 72% sulfuric acid for 3 hours, and then rinse with plenty of water until neutral. Add acetone to cover the sample, soak for 3-5 minutes, take it out, dry the sample at 102°C and weigh it m4, and the weight of the blank filter bag is recorded as C4. Put the dried and weighed sample in a crucible (the weight of the crucible is recorded as m5), and the crucible with the blank sample is C5. Use a muffle furnace to burn the sample at 550°C, cool it to room temperature, weigh it and record it as m6, and the crucible with the blank sample and the ash weight are recorded as C6.

[0144] ADL (%) = [(m4+m5-m6)-(C4+C5-C6)]÷M×100 Ash content (%) = [(m6-m5)-(C6-C5)] ÷ M × 100.

[0145] ⑤ Calculation of analysis results Hemicellulose (%) = NDF (%) - ADF (%) Cellulose (%) = ADF (%) - [ADL (%) + ash (%)] 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); Degradation rate of cellulose and hemicellulose = (initial content - content after treatment) / initial content × 100%.

[0146] Table 17 Changes in cellulose and hemicellulose content in rice straw at different liquid fermentation times

[0147] Note: Different lowercase letters after the data in the same column indicate significant differences ( P <0.05) As shown in Table 17, with the increase of culture time, the cellulose and hemicellulose contents of rice straw changed significantly and gradually decreased. From 0 d to 30 d, it was found that the contents of cellulose and hemicellulose decreased to 19.32% and 16.50%, respectively, and the degradation rates were 34.51% and 47.01%, respectively. This shows that the degradation of cellulose and hemicellulose in rice straw is relatively easy for Penicillium citrinum DHZ-2, and the reduction of cellulose content may be due to the activity of endoglucanase, exoglucanase and β-glucosidase contained in Penicillium citrinum DHZ-2 itself; similarly, the high activity of xylanase contained in Penicillium citrinum DHZ-2 itself led to the reduction of hemicellulose content in rice straw after fermentation.

[0148] 3) Monosaccharide content produced by fermentation Liquid fermentation: Take 400 μL of fermentation supernatant corresponding to the fermentation time points of 0, 10, 20, and 30 days, respectively, and transfer them to 5 mL stoppered test tubes. Subsequently, add 400 μL of PMP methanol solution (PMP concentration is 0.5 mol / L) to each tube and vortex mix, and place it in a 70°C water bath for 2 hours of reaction. After the reaction is completed, remove the stoppered test tube from the water bath and place it at room temperature to cool naturally. After the test tube cools to room temperature, add 400 μL of 0.3 mol / L HCl to each test tube to neutralize to pH 6.0-7.0. After neutralization, add 1200 μL of water to the tube, and then add an equal volume (1200 μL) of chloroform, vortex mix, shake, let stand, and discard the chloroform phase. This extraction step is repeated twice to ensure sufficient extraction of metabolites. Finally, the aqueous phase was filtered using a 0.45 μm microporous membrane (water system), and the filtered aqueous phase samples were then used for high performance liquid chromatography (HPLC) injection analysis to analyze the monosaccharide content in the rice straw fermentation broth.

[0149] Fig.12 The figure shows the changes in the monosaccharide content of the liquid fermentation liquid of rice straw from 0 to 30 days. After Penicillium citrinum DHZ-2 degraded rice straw, the contents of mannose, rhamnose, galactose, xylose and arabinose increased significantly; hemicellulose is one of the main components of plant cell walls and is composed of various types of monosaccharides, including pentose (such as xylose and arabinose) and hexose (such as galactose, etc.). This shows that in the process of Penicillium citrinum DHZ-2 degrading rice straw, its xylanase played a certain degradation role, hydrolyzing the hemicellulose in rice straw into various monosaccharides. Overall, the content of glucose did not increase significantly, but showed a downward trend. It may be because after the 0-day rice straw was sterilized at high temperature, the cleavage of the plant cell wall components led to a relatively high glucose content. Due to the inorganic salt culture medium used in the fermentation process, the growth of the strain of Penicillium citrinum DHZ-2 itself requires more nutrient support. Cellulase hydrolyzes cellulose into monosaccharides such as glucose, but these glucose are then used by the strain for growth metabolism. Therefore, as the fermentation time increases, the glucose content in the fermentation broth will gradually decrease (the glucose content is 243.36 mg / L on day 0 and 108.28 mg / L on day 30). In the liquid fermentation broth, the galactose content increases from 39.18 mg / L to 115.85 mg / L, and the xylose content increases from 6.9 mg / L to 78.91 mg / L.

[0150] Description: Galactose is a six-carbon sugar found in hemicellulose of plant cell walls.

[0151] 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.

[0152] 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 0 days of fermentation.

[0153] After 30 days of fermentation of Penicillium citrinum NCIM-1398, the cellulose content and hemicellulose content were both higher than 25%, while after 30 days of fermentation of Penicillium citrinum DHZ-2 of the present invention, the cellulose content and hemicellulose content were 19.32% and 16.50%, respectively, and the degradation efficiency was better than that of Penicillium citrinum NCIM-1398.

[0154] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. Penicillium citrinum Penicillium citrinum )DHZ-2, characterized by: 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.

3. The use of Penicillium citrinum DHZ-2 according to claim 2, characterized in that: Cellulases include endoglucanases, exoglucanases, and β-glucosidases; Hemicellulases include xylanases.

4. The use of Penicillium citrinum DHZ-2 according to claim 2 or 3, characterized in that: The fermentation medium is 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 is 4.5-5.5; The culture temperature is 25~30℃; the culture time is 8~10 days.

5. The use of Penicillium citrinum DHZ-2 according to claim 4, 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°C, culture time 8 days, rice straw powder 12.5g, peptone 1.5g, pH 5; For β-glucosidase: culture temperature 30℃, culture time 10 days, rice straw powder 15g, peptone 1.75g, pH 5; For xylanase: culture temperature 30°C, culture time 8 days, rice straw powder 15g, peptone 1.75g, pH5.

6. The method for degrading rice straw using the 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 salt medium and Penicillium citrinum DHZ-2 and then fermented.

7. The method for degrading rice straw by Penicillium citrinum DHZ-2 according to claim 6, characterized in that: Rice straw was mixed with Hutcheson's inorganic salt medium and Penicillium citrinum DHZ-2 according to the ratio of 2±0.5g rice straw to 100mL Hutcheson's inorganic salt medium and 0.5cm Penicillium citrinum DHZ-2 cake, and fermented in a constant temperature shaking incubator at 30±2℃ for 10-30 days.

8. The method for degrading rice straw by Penicillium citrinum DHZ-2 according to claim 7, characterized in that: Hutcheson inorganic salt 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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