New bacterial strain nigella lutinosa for high yield of cellulase and application of new bacterial strain nigella lutinosa

Abstract

The invention discloses a new strain of black shell fungus for high yield of cellulase and application thereof, a filamentous fungus new strain Preussia sp.N-5 is obtained by separating, screening and identifying from a soil sample in Turpan region in Xinjiang, and the strain is subjected to stress resistance experiment and metabolic fermentation to obtain the cellulase. The new strain is verified through the characteristics of cellulase production, acid and alkali resistance, functional enzyme experiments, antibacterial performance and the like, the new strain is identified to be Preussia sp.N-5 in the field of black shell fungi, good alkali resistance and antibacterial characteristics are achieved, the activity of cellulase in fermentation liquor is 32.09 U / mL, and the new strain Preussia sp.N-5 shows good application prospects and has good application prospects. The feed additive can be widely applied to the field of animal feed.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to the technical field of a new strain of Pseudomonas aeruginosa with high cellulase production and application thereof. Background Art

[0002] Cellulase is a complex enzyme that degrades cellulose to produce glucose, mainly composed of endoglucanase, exoglucanase and β-glucosidase. Cellulose is the most abundant and widely distributed polysaccharide in nature and has a high utilization value. However, due to its complex chemical structure, most cellulose resources are currently discarded or incinerated, which not only wastes precious resources but also causes serious pollution to the environment. Therefore, finding cellulases with high activity and good stability has always been one of the research focuses.

[0003] The sources of cellulase are very wide. Organisms such as microorganisms, plants and animals can produce cellulase, but the production cost of animal and plant cellulase is high and the enzyme production is low. At present, the main organisms studied for producing cellulose-degrading enzymes are microorganisms. Due to the reasonable structure of the enzyme system of fungi and their high enzyme production efficiency, the extracellular enzymes produced by fungi can directly combine with cellulose for enzymatic degradation, thereby improving the degradation efficiency. They have become the main production strains of cellulase, such as Trichoderma, Penicillium, Aspergillus, etc. In recent years, with the excavation of special environmental microbial resources, the functional enzymes of their metabolism often show excellent functions, and have also become one of the important sources for the screening of cellulase-producing strains. Existing reports show that extremely hot and arid areas are rich in microbial resources, and the cellulases produced by them usually have the characteristics of strong degradation ability and wide adaptability. However, there are few reports on the excavation and development of related cellulase strains from extremely hot and arid areas at home and abroad. It is necessary to continuously explore and find better strains or strains with better properties and applications. Summary of the invention

[0004] Aiming at the technical problems of low cellulase activity, poor stability, and few existing strains for producing cellulase. The present invention aims to provide a new strain of high-yield cellulase, Preussia sp., and its application. The present invention isolates and screens a high-yield cellulase filamentous fungus from soil samples in Turpan, Xinjiang. After a series of identifications, the new strain is Preussia sp. N-5 in the field of Preussia sp., and is verified by optimizing the cellulase production conditions, functional enzyme experiments, stress resistance experiments, and antibacterial properties of the fermentation broth of the strain. The strain has cellulase activity, the cellulase produced is acidic cellulase, and has good alkali resistance and antibacterial properties. The fermentation broth of Preussia sp. N-5 has a cellulase activity of 32.09 U / mL, indicating that the new strain Preussia sp. N-5 and its fermentation broth provided by the present invention have good application prospects in the feed industry.

[0005] In order to achieve the above technical effects, the present invention is implemented through the following technical solutions.

[0006] The present invention provides a new strain of high-yield cellulase, which is Preussia sp. N-5 in the field of light black shell fungus. The Preussia sp. N-5 is preserved in China Center for Type Culture Collection (CCTCC) with a preservation number of CCTCC No: M20242344.

[0007] The gene sequence of the Preussia sp. N-5 strain is shown in SEQ ID NO: 1.

[0008] In the present invention, the culture medium used for separating Preussia sp. N-5 is a sodium carboxymethyl cellulose (CMC-Na) culture medium comprising: CMC-Na 10.0 g / L, KH2PO4 2.0 g / L, MgSO4·7H2O 0.5 g / L, (NH4)2SO4 2.0 g / L, agar 14.0 g / L, distilled water 1 L, pH 7.4-7.6.

[0009] In the present invention, the culture medium for preserving Preussia sp. N-5 is a potato dextrose agar (PDA) culture medium comprising: 6.0 g / L potato extract powder, 20.0 g / L glucose, 20.0 g / L agar, 0.1 g / L chloramphenicol, 1 L distilled water, and pH 6.0-6.5.

[0010] In the present invention, the fermentation enzyme production medium of the Preussia sp. N-5 is: CMC-Na 10.0 g / L, KH2PO4 2.0 g / L, MgSO4·7H2O 0.5 g / L, (NH4)2SO4 2.0 g / L, peptone 2.0 g / L, distilled water 1 L, pH 7.4-7.6.

[0011] The Preussia sp.N-5 used in the present invention belongs to a new strain in the field of Preussia sp. in filamentous fungi. Preussia sp.N-5 belongs to a new strain in the genus Preussia sp. in filamentous fungi, has distinct common characteristics of Preussia sp., and is verified in multiple aspects such as ITS gene sequence homology comparison analysis and morphological identification comparison. The strain numbered N-5 is clearly different from the standard model strain, and is consistent with the typicality analysis conclusion of the strain identification in the field. There are aerial hyphae on the PDA culture medium, which is earthy black. It is determined that the strain numbered N-5 is a new strain in the genus Preussia sp. in filamentous fungi, and has the significant characteristics of a new strain in the same genus Preussia sp.

[0012] The invention provides a method for preparing a fermentation liquid of a novel strain Preussia sp. N-5 with high cellulase production. The fermentation liquid is prepared by the following steps: rinsing spores of Preussia sp. N-5 with sterile water to prepare a suspension for standby use; inoculating the spore suspension into a fermentation enzyme production medium at an inoculation amount of 5% by volume, and culturing at 30 DEG C and 160 r / min for 6 days to obtain the Preussia sp. N-5 fermentation liquid.

[0013] Furthermore, the present invention provides a new strain Preussia sp. N-5 with high cellulase production and its application in animal feed.

[0014] Furthermore, the present invention provides a method for preparing fermentation liquid of a new strain Preussia sp. N-5 with high cellulase production, and application of the fermentation liquid obtained in animal feed.

[0015] By implementing the above technical solution provided by the present invention, the present invention obtains the following technical effects:

[0016] (1) The present invention provides a new strain that has been systematically and scientifically identified as Preussia sp. N-5 in the field of filamentous fungi, which belongs to a new species in the genus Preussia. The strain has cellulase activity, and the cellulase produced can degrade cellulose, and has good alkali resistance and antibacterial properties. The cellulase activity in the fermentation broth of the strain is 32.09 U / mL, which is an acidic cellulase. The fermentation broth prepared by the strain is applied to the feed industry with good application prospects.

[0017] (2) The cellulase produced by the new filamentous fungus strain Preussia sp. N-5 provided by the present invention still exhibits a high relative enzyme activity after being incubated at a temperature of 30 to 50°C and a pH of 3.0 to 8.0 for 1 hour. 2+ It can significantly improve enzyme activity, while SDS has a certain inhibitory effect on enzyme activity.

[0018] (3) The new filamentous fungus strain Preussia sp. N-5 provided by the present invention can grow well under acidic pH conditions, has good lipase and catalase activities and strong antibacterial ability, and can effectively inhibit pathogenic bacteria such as Staphylococcus aureus, Escherichia coli, Bacillus subtilis and Salmonella. Therefore, the present invention can not only be applied to animal feed, but also inhibit pathogenic bacteria. It can be seen that the cellulase produced by the new strain Preussia sp. N-5 provided in the present application is safer and more reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Shown is the phylogenetic tree of the new strain Preussia sp. N-5 constructed based on the ITS gene sequence.

[0020] Figure 2 Shown is the morphological characteristics of the new strain Preussia sp.N-5.

[0021] Figure 3 Shown is the cellulase production curve of the new strain Preussia sp.N-5.

[0022] Figure 4 Shown is the effect of temperature on the cellulase activity produced by the new strain Preussia sp.N-5.

[0023] Figure 5 It shows the temperature stability of cellulase produced by the new strain Preussia sp.N-5.

[0024] Figure 6 Shown is the effect of pH on the activity of cellulase produced by the new strain Preussia sp. N-5.

[0025] Figure 7 It shows the pH stability of cellulase produced by the new strain Preussia sp.N-5.

[0026] Figure 8 Shown is the effect of metal ions on the cellulase activity produced by the new strain Preussia sp.N-5.

[0027] Fig. 9 Shown is the effect of enzyme inhibitors on the cellulase activity produced by the new strain Preussia sp.N-5. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. In the absence of conflict, the following embodiments and the features in the embodiments can be combined with each other. If not specifically stated, the technical means used in the embodiments are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0029] The sodium carboxymethyl cellulose (CMC-Na) culture medium used in the present invention is: CMC-Na 10.0 g / L, KH2PO4 2.0 g / L, MgSO4·7H2O 0.5 g / L, (NH4)2SO4 2.0 g / L, agar 14.0 g / L, distilled water 1 L, pH 7.4-7.6.

[0030] The potato dextrose agar (PDA) culture medium adopted by the invention comprises: 6.0 g / L potato powder, 20.0 g / L glucose, 20.0 g / L agar, 0.1 g / L chloramphenicol, 1 L distilled water, and pH 6.0-6.5.

[0031] The fermentation enzyme production medium used in the present invention is: CMC-Na 10.0g / L, KH2PO4 2.0g / L, MgSO4·7H2O0.5g / L, (NH4)2SO4 2.0g / L, peptone 2.0g / L, distilled water 1L, pH 7.4-7.6.

[0032] The protein skimmed milk powder culture medium adopted by the invention comprises: 12 g / L skimmed milk powder, 4.0 g / L peptone, 3.0 g / L yeast extract powder, 20 g / L agar, and pH 7.0-7.4.

[0033] The starch culture medium used in the invention comprises: 10 g / L starch, 3 g / L beef extract powder, 10 g / L peptone, 5 g / L NaCl, 20 g / L agar, and a pH value of 7.0-7.4.

[0034] The lipase culture medium used in the present invention is: potato glucose agar culture medium added with 1% tributyrin and 0.05% rhodamine B, with a pH value of 7.0-7.4.

[0035] Example 1: Isolation, purification and identification of the new strain Preussia sp. N-5

[0036] (I) Separation and purification

[0037] The plate dilution method was used to separate filamentous fungi. Soil samples collected from Turpan, Xinjiang, were separated and purified by gradient dilution. 5 g of soil sample was weighed and placed in 45 mL of sterile water. Sterilized glass beads were added and shaken at 160 rpm and 30 °C for 30 min to fully disperse the sample. Standard gradient dilution was used to dilute 10 -1 ~10 -3 Concentration dilution; take 100 μL of each concentration dilution, spread it on a carboxymethyl cellulose sodium (CMC-Na) medium plate by conventional coating method, and place it in a 30℃ constant temperature incubator for culture; after one week, when colonies grow on the plate, pick a single colony on the plate for purification culture. The purified single colony is transferred to a PDA medium slant for storage.

[0038] (II) ITS gene identification

[0039] 1. Extraction of PCR Template DNA

[0040] The purified strain numbered N-5 was inoculated into potato glucose liquid medium and cultured in a shaking incubator at 30°C for 6 days. The bacteria were collected and the total genomic DNA was extracted using a fungal genomic DNA extraction kit.

[0041] 2. PCR Amplification

[0042] ITS gene sequence primers:

[0043] ITS1: 5'-GGTCATTTAGAGGAAGTAA-3';

[0044] ITS4: 5'-AGCCTSSCCTTANTDATATGC-3';

[0045] The total volume of the PCR reaction system was 25 μL, and the PCR amplification conditions were: pre-denaturation at 95°C for 5 min; denaturation at 94°C for 45 s, annealing at 50°C for 30 s, and extension at 72°C for 45 s, for 30 cycles; extension at 72°C for another 1 min, and cooling to 4°C for pre-storage.

[0046] 3. Sequence Determination

[0047] The PCR amplification product was sequenced after electrophoresis detection and purification, and the sequence length was 507 bp. The test results are shown in SEQ ID No: 1. BLAST homologous sequence search was performed on NCBI, and the phylogenetic tree was established by the Neighbor-Joining method using MEGA11.0 software commonly used in the field (repeated sampling 1000 times). The results are shown in the attached Figure 1As shown, the obtained sequence was compared and analyzed on the NCBI website and it was found that the strain numbered N-5 was similar to the standard model bacteria of the genus Preussia procaviicola CBS146981 T The highest homology was 93.73%, and it was similar to other adjacent type strains Preussia procaviae CBS146827 T The homology is 93.36%. In the phylogenetic tree constructed with ITS gene sequences, the strain numbered N-5 is similar to Preussia procaviae CBS146827 T Through the molecular level identification of the strain system well known in the art, it was confirmed that the strain numbered N-5 is a typical new strain in the genus Preussia and a new strain in the field of light black shell fungi in filamentous fungi.

[0048] Example 2: Morphological characteristics of the new strain Preussia sp. N-5

[0049] (I) Colony morphology characteristics of the new strain Preussia sp. N-5

[0050] Inoculate the strain numbered N-5 to be observed on the PDA plate and culture it at 30℃ for 7 days. Observe the characteristics of the colony and record and take photos for preservation. The results are recorded in the attached Figure 2 shown.

[0051] By the attached Figure 2 The results show that the strain numbered N-5 provided by the present invention grows slowly on the PDA medium, and the colony diameter reaches 20-30 mm after culturing for 7 days at 30°C. The colony is white to gray, with dark brown on the back, sparse aerial hyphae, and cracks.

[0052] (II) Characteristics of the new strain Preussia sp. N-5

[0053] The strain numbered N-5 was inoculated on PDA medium, and the pH range, protease, amylase, lipase, catalase, antibacterial properties were tested. From the results in Table 1, it can be seen that the strain Preussia sp. N-5 can grow at pH 5.0-13.0, and its metabolic lipase and catalase activities are both positive. From the results in Table 2, it can be seen that the strain Preussia sp. N-5 can effectively inhibit pathogenic bacteria such as Staphylococcus aureus, Escherichia coli, Bacillus subtilis and Salmonella.

[0054] Table 1: pH tolerance and enzyme production characteristics of strain Preussia sp. N-5

[0055]

[0056] Table 2: Antibacterial properties of strain Preussia sp. N-5

[0057]

[0058] According to the ITS gene sequence homology comparison analysis and morphological identification comparison provided in Example 1, the strain numbered N-5 is clearly different from the standard model strain, which is consistent with the typical analysis conclusion of the new strain identification in this field. The strain numbered N-5 is determined to be a new strain in the genus Preussia in a filamentous fungus, and has the significant characteristics of a new strain in the same genus Preussia, so it is tentatively named Preussia sp. N-5. The strain has been deposited in the Budapest Treaty International Microbiological Depository: China Type Culture Collection (CCTCC), address: Wuhan University, Wuhan, China, postal code: 430061, deposit date: October 25, 2024, and deposit number: CCTCCNo: M 20242344.

[0059] Example 3: Preparation of fermentation broth of new strain Preussia sp. N-5

[0060] In this embodiment, based on Example 1-2, the fermentation liquid of Preussia sp. N-5 is obtained by the following steps: spores of Preussia sp. N-5 are rinsed with sterile water to prepare a suspension for standby use; the spore suspension is inoculated into a fermentation enzyme production medium at an inoculation amount of 5% by volume, and cultured at 30° C. and 160 r / min for 6 days to obtain the fermentation liquid of Preussia sp. N-5.

[0061] Example 4: Application of fermentation broth of new strain Preussia sp. N-5

[0062] Based on Examples 1-3, this Example tests the application of a new strain Preussia sp. N-5 and its fermentation liquid.

[0063] (I) Cellulase activity of the fermentation broth of Preussia sp. N-5

[0064] The DNS method was used to determine the cellulase activity in the fermentation broth of Preussia sp. N-5. Take 0.5 mL of the fermentation broth of Preussia sp. N-5 in a 10 mL colorimetric tube, add 1.5 mL of 0.5% CMC-Na citric acid-sodium citrate buffer (pH 4.8), mix well, place the colorimetric tube in a 50°C water bath for 0.5 h, add 2 mL of DNS reagent, boil in a water bath for 5 min, quickly cool with running water and make up to 10 mL, mix well, and measure the absorbance at 540 nm. The crude enzyme solution inactivated at 100°C for 10 min was used as a control. The definition of cellulase activity is: at 50°C and pH 4.8, 1 mL of crude enzyme solution hydrolyzes the substrate to produce 1 μg of glucose per minute, which corresponds to one enzyme activity unit (U / mL).

[0065] By the attached Figure 3 The results show that the Preussia sp. N-5 provided by the present invention has good cellulase activity, and the cellulase activity of the fermentation liquid of Preussia sp. N-5 is calculated to be 32.09 U / mL.

[0066] (II) Determination of cellulase activity in fermentation broth of Preussia sp. N-5

[0067] (1) Optimal action temperature of cellulase

[0068] Take 0.5mL of the fermentation broth of Preussia sp. N-5 in a 10mL colorimetric tube, add 1.5mL of citric acid-sodium citrate buffer (pH 4.8) containing 0.5% CMC-Na, mix well, place the colorimetric tube in a water bath at 30℃, 40℃, 50℃, 60℃, 70℃, and 80℃ for 0.5h, add 2mL of DNS reagent, boil in a water bath for 5min, quickly cool with running water and make up to 10mL, mix well, and measure the absorbance at 540nm. The fermentation broth inactivated at 100℃ for 10min was used as a control. Calculate the cellulase activity of the bacterial broth, and investigate the effect of temperature change on the cellulase produced by the fermentation broth of Preussia sp. N-5. For specific results, see the attached Figure 4 shown.

[0069] By the attached Figure 4 The data show that the relative activity of cellulase in the fermentation broth of Preussia sp. N-5 provided by the present invention increases first and then decreases with increasing temperature, and the optimum action temperature is 50°C. When the temperature is higher than 50°C, the cellulase activity begins to decrease.

[0070] (2) Temperature stability of cellulase

[0071] Before the enzyme activity reaction, the fermentation liquid of Preussia sp. N-5 was placed in a water bath at 30℃, 40℃, 50℃, 60℃, 70℃, and 80℃ for 1 hour, and then the cellulase activity was measured at the optimal temperature after rapid cooling. At the same time, the temperature tolerance of cellulase was investigated with the enzyme activity of the untreated fermentation liquid as 100%. The specific results are shown in the attached Figure 5 shown.

[0072] By the attached Figure 5 The data show that the cellulase in the fermentation liquid of Preussia sp. N-5 provided by the present invention is relatively stable in the range of 30-50°C, and the relative enzyme activity is maintained at more than 80%. When the reaction temperature is higher than 50°C, the enzyme activity begins to decrease rapidly, and at 80°C, the relative enzyme activity of the cellulase is only 30.00%.

[0073] (3) Optimal pH for cellulase

[0074] Prepare buffer systems with different pH values ​​(3, 4, 5, 6, 7, 8), take 1.5mL of buffer containing 0.5% CMC-Na, add 0.5mL of Preussia sp. N-5 fermentation broth and mix evenly, place in a 50℃ water bath for 0.5h, calculate the cellulase activity of the bacterial broth, and investigate the effect of buffer pH on the cellulase activity of Preussia sp. N-5 fermentation broth. For specific results, see the attached Figure 6 shown.

[0075] By the attached Figure 6 The data show that the pH value of the enzyme reaction has a significant effect on the relative activity of cellulase. The pH affects the enzymatic efficiency by affecting the groups on the enzyme-related active sites. Too high or too low a pH value will inactivate the cellulase. The optimal pH for the cellulase in the fermentation broth of Preussia sp. N-5 is 4.0, and it has a wide pH range. It shows good enzyme activity at pH 4.0-6.0, but when the pH further decreases or increases, its enzyme activity decreases rapidly. At pH 3.0 and pH 8.0, its enzyme activity is only about 45%.

[0076] (4) pH stability of cellulase

[0077] Before the enzyme activity reaction, the fermentation liquid of Preussia sp. N-5 was placed in buffer systems with different pH values ​​(3, 4, 5, 6, 7, 8) and kept at 4°C for 1 hour before measuring the cellulase activity. At the same time, the enzyme activity of the untreated crude enzyme liquid was taken as 100% to investigate the tolerance of cellulase to pH. For specific results, see the attached Figure 7 shown.

[0078] By the attached Figure 7 The data show that the cellulase in the fermentation broth of Preussia sp. N-5 provided by the present invention is relatively stable at pH 3.0-8.0, and can still maintain more than 60% of the enzyme activity after being placed for 1 hour.

[0079] (5) Effects of metal ions on cellulase

[0080] When the ion concentration was 1mmol / L, the effects of different metal ions on the cellulase activity of the fermentation broth of Preussia sp. N-5 were investigated. The metal ions were K + Mg 2+ , Mn 2+ , Fe 3+ , Cu 2+ , Ca 2+ The fermentation broth of Preussia sp. N-5 and metal ions were placed in a water bath at 50°C for 30 min, and the enzyme activity of the fermentation broth without metal ions was used as a control. The specific statistical results are shown in the attached Figure 8 shown.

[0081] By the attached Figure 8 The data show that when the fermentation liquid of Preussia sp. N-5 was treated with different metal ions at the same concentration, the metal ion Cu 2+ It has a significant promoting effect on the cellulase activity produced by the fermentation broth of Preussia sp. N-5, and can be used as an activator of cellulase produced by the fermentation broth of Preussia sp. N-5; Mn 2+ It has a certain inhibitory effect on the cellulase produced by the fermentation broth of Preussia sp.N-5.

[0082] (6) Effects of enzyme inhibitors on cellulase

[0083] This experiment investigated the effects of different enzyme inhibitors on the cellulase activity of the fermentation broth of Preussia sp. N-5. Common enzyme inhibitors such as cetyltrimethylammonium bromide (CTAB), sodium dodecyl sulfonate (SDS), ethylenediaminetetraacetic acid (EDTA), and β-mercaptoethanol (2-Me) were added at a concentration of 1 mmol / L, respectively, and the mixture was in a water bath at 50°C for 30 min. The enzyme activity of the fermentation broth without adding inhibitors was used as the control. For specific statistical results, please refer to the attached Fig. 9 shown.

[0084] By the attached Fig. 9The data show that different enzyme inhibitors have a certain inhibitory effect on the cellulase activity of the fermentation broth of Preussia sp. N-5. Compared with the other three inhibitors, SDS has the most significant inhibitory effect, and its relative cellulase activity is 12%.

[0085] The above experiments show that the present invention separates and screens a new filamentous fungus strain Preussia sp. N-5 from soil samples in Turpan, Xinjiang. The strain and its fermentation broth have cellulase ability and good alkali resistance and antibacterial properties. The cellulase activity of the fermentation broth of the new strain Preussia sp. N-5 is 32.09 U / mL, and the cellulase produced is acidic cellulase, indicating that the new filamentous fungus Preussia sp. N-5 and its fermentation broth provided by the present invention are applied to the feed industry and have good application prospects.

[0086] The above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the present invention.

Claims

1. A new strain Preussia sp. N-5 with high cellulase production, characterized in that: The new strain Preussia sp. N-5 is deposited in China Center for Type Culture Collection (CCTCC) with a deposit number of CCTCC No: M 20242344. The gene sequence of the new strain Preussia sp. N-5 is shown in SEQ ID NO:

1.

2. A culture medium for isolating a novel strain Preussia sp. N-5 with high cellulase production as claimed in claim 1, characterized in that: The isolation medium of the new strain Preussia sp. N-5 is a sodium carboxymethyl cellulose medium comprising: CMC-Na 10.0 g / L, KH2PO4 2.0 g / L, MgSO4·7H2O0.5 g / L, (NH4)2SO4 2.0 g / L, agar 14.0 g / L, distilled water 1 L, pH 7.4-7.

6.

3. A culture medium for preserving the novel strain Preussia sp. N-5 with high cellulase production as claimed in claim 1, characterized in that: The preservation medium of the new strain Preussia sp. N-5 is a potato glucose agar (PDA) medium comprising 6.0 g / L potato extract powder, 20.0 g / L glucose, 20.0 g / L agar, 0.1 g / L chloramphenicol, 1 L distilled water, and a pH of 6.0-6.

5.

4. A culture medium for fermenting enzymes according to claim 1, wherein the culture medium comprises a novel strain Preussia sp. N-5 having a high cellulase production rate, The fermentation enzyme production medium of the new strain Preussia sp. N-5 is: CMC-Na 10.0g / L, KH2PO4 2.0g / L, MgSO4·7H2O 0.5g / L, (NH4)2SO4 2.0g / L, peptone 2.0g / L, distilled water 1L, pH 7.4-7.

6.

5. A method for preparing fermentation broth of a new strain Preussia sp. N-5 with high cellulase production, characterized in that: The fermentation liquid is prepared by the following steps: spores of the new strain Preussia sp. N-5 are washed with sterile water to prepare a suspension for standby use; the spore suspension is inoculated into a fermentation enzyme production medium according to an inoculation amount of 5% by volume, and cultured at 30° C. and 160 r / min for 6 days to obtain the fermentation liquid of the new strain Preussia sp. N-5.

6. Use of the novel strain Preussia sp. N-5 with high cellulase production as claimed in claim 1 in animal feed.

7. Use of the fermentation liquid obtained by the method for preparing the fermentation liquid of the novel strain Preussia sp. N-5 with high cellulase production as claimed in claim 5 in animal feed.

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