Enzymatically deconstructed wood powder, method for preparing the same and use thereof

Abstract

The present application belongs to the field of bioengineering technology, and particularly relates to a kind of enzymatic broussonetia papyrifera powder and its preparation method and application.The present application uses Aspergillus niger WZ001 as a starting strain, constructs and selects a strain of high-yield Aspergillus niger tannase, and then uses the recombinant tannase obtained by fermentation to enzymatically hydrolyze tannin in broussonetia papyrifera powder, which can convert broussonetia papyrifera powder tannin into gallic acid, which can effectively improve the palatability of feed using broussonetia papyrifera powder as one of the raw materials.On this basis, since the Aspergillus niger host has a rich enzyme system, amylase, glucoamylase, protease, cellulase, etc., the Aspergillus niger tannase of the present application can play the role of a complex enzyme, so it can enzymatically hydrolyze broussonetia papyrifera powder more comprehensively, and the product obtained after enzymatic hydrolysis has a higher nutritional value, which reflects a high economic value and social benefit.In addition, the Aspergillus niger tannase constructed in the present application can meet the requirements of food-grade enzyme preparation, so the safety of the enzymatically hydrolyzed broussonetia papyrifera powder is guaranteed.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of bioengineering technology, and particularly relates to a kind of enzymatic broussonetia papyrifera powder and its preparation method and application. BACKGROUND

[0002] Broussonetia papyrifera has other names such as broussonetia papyrifera, valley pulp tree, and false waxberry, and is widely distributed in North China, Central China, South China, Southwest China, and Northwest China. In China, people use the leaves of broussonetia papyrifera as feed raw materials for livestock. As livestock feed, the advantages of broussonetia papyrifera leaves are very prominent: the protein content is as high as 20% to 30%, and the nutritional components such as amino acids, vitamins, and trace elements are also very rich; the feed made by fermenting broussonetia papyrifera leaves as the main raw material is green and environmentally friendly, does not contain pesticides or hormones, and has a fresh and pleasant smell; after eating, the livestock grows fast, has strong disease resistance, and has a short feeding cycle.

[0003] Because of the presence of tannin (about 1.6% content) in broussonetia papyrifera powder, the feed tastes bitter, and tannin easily forms a complex with protein, affecting the nutrient intake of animals. Additional sweeteners (saccharin) need to be added to improve the taste of the feed, increase the palatability of the feed, and increase the intake of the feed by animals. Experimental data shows that tannic acid is not absorbed in the small intestine of piglets, but is partially decomposed by microorganisms in the colon and absorbed as gallic acid. Generally speaking, 70-80% of the tannic acid ingested by piglets is excreted through feces. For adult pigs, at least 30% of the tannic acid is excreted with feces.

[0004] Tannase is an enzyme that is studied more in the process of tannin degradation, and can hydrolyze the ester bonds in tannin. Tannase is an induced enzyme that can be induced in the presence of tannic acid and tannin. Tannase is widely present in various fungi and bacteria. The commonly used tannase-producing bacteria in production are mainly fungi such as Aspergillus oryzae, etc.

[0005] Aspergillus niger has a long history. As early as in ancient China, people used Aspergillus niger to make sauces, soy sauce, rice wine, etc. Because Aspergillus niger grows vigorously, has a short fermentation period, and does not produce toxins, it is certified by the US FDA as a safe strain. Aspergillus niger has strong protein expression, secretion, and modification capabilities, and the recombinants have high genetic stability. With more and more proteins successfully expressed in Aspergillus niger and proven to have high yield and activity, Aspergillus niger has become an important enzyme protein expression system and gradually an important industrial enzyme preparation and organic acid production strain. Industrial enzymes produced by Aspergillus niger have played a huge role in the fields of starch processing, fermentation, brewing, beverages, animal feed, and papermaking industries, etc.

[0006] Patent CN100467586C discloses a kind of simultaneously high yield naringinase and hesperidinase, and can realize industrial production Aspergillus niger WZ001 (Aspergillus niger WZ001) and its application. Specifically, the enzyme yield of the strain is high, when using liquid fermentation culture, the enzyme yield of naringinase and hesperidinase can reach 14000 U / g or more; when using solid-state fermentation culture, the enzyme yield of naringinase and hesperidinase can reach 6000 U / g or more. Therefore, to solve the technical problem of high tannin content in the feed using broussonetia papyrifera powder as raw material, which leads to poor palatability of feed, further application research and development is carried out on Aspergillus niger WZ001 (Aspergillus niger WZ001). SUMMARY

[0007] The present application uses Aspergillus niger WZ001 as the starting strain to prepare a new type of Aspergillus niger tannase, which can enzymatically hydrolyze tannin in broussonetia papyrifera powder, thereby solving the technical problem of high tannin content in the feed using broussonetia papyrifera powder as raw material, which leads to poor palatability of feed. The following technical solutions are used to achieve the above-mentioned technical problem:

[0008] A preparation method of enzymatically hydrolyzed broussonetia papyrifera powder, comprising the following steps:

[0009] S1, using wild-type Aspergillus niger as the starting strain, knocking out pyrG gene to obtain Aspergillus niger pyrG gene-deficient strain; the wild-type Aspergillus niger has the preservation number CCTCC NO.206047; the pyrG gene is complemented in the form of tannase gene plus pyrG gene, and an Aspergillus niger high-tannase-producing strain is screened; the Aspergillus niger high-tannase-producing strain is cultured in a fermenter to obtain Aspergillus niger tannase;

[0010] S2, using the Aspergillus niger tannase to enzymatically hydrolyze broussonetia papyrifera powder to obtain enzymatically hydrolyzed broussonetia papyrifera powder.

[0011] As a preferred embodiment, step S2 comprises the following steps:

[0012] S2.1, dissolving broussonetia papyrifera powder raw material in water to obtain pretreated raw material;

[0013] S2.2, adding enzyme solution of the Aspergillus niger tannase to the pretreated raw material to perform enzymatic hydrolysis, and obtaining enzymatic hydrolysis product;

[0014] S2.3, inactivating the enzymatic hydrolysis product to obtain enzymatically hydrolyzed broussonetia papyrifera powder.

[0015] As a preferred embodiment, in step S2.1, the mass-to-volume ratio of broussonetia papyrifera powder raw material to water is 1:3.

[0016] As preferred, the volume-mass ratio of the enzyme solution added in step S2.2 to the amount of Broussonetia papyrifera powder added in the pretreated raw material is 1% to 10%.

[0017] As preferred, the enzymolysis conditions in step S2.2 are: temperature 25 to 60℃, and time 1 to 6h.

[0018] As preferred, the inactivation treatment in step S2.3 is: heating the enzymolysis product to boiling, and then cooling to room temperature to obtain the enzymolysis Broussonetia papyrifera powder.

[0019] An enzymolysis Broussonetia papyrifera powder prepared by any of the above preparation methods.

[0020] A feed containing the above enzymolysis Broussonetia papyrifera powder.

[0021] Compared with the prior art, the present application has the following beneficial effects:

[0022] The present application uses Aspergillus niger WZ001 as the starting strain, constructs and screens a high-yield Aspergillus niger tannase strain, and then uses the recombinant tannase obtained by fermentation to enzymolysis tannin in Broussonetia papyrifera powder, which can convert Broussonetia papyrifera powder tannin into gallic acid, which can effectively improve the palatability of the feed using Broussonetia papyrifera powder as one of the raw materials. On this basis, since the Aspergillus niger host has a rich enzyme system, amylase, glucoamylase, protease, cellulase, xylanase, etc., the Aspergillus niger tannase of the present application can play the role of a complex enzyme, so it can enzymolysis Broussonetia papyrifera powder more comprehensively, and the product obtained after enzymolysis has higher nutritional value, which reflects higher economic value and social benefits. In addition, the Aspergillus niger tannase constructed in the present application can meet the requirements of food-grade enzyme preparations, so the safety of the enzymolysis Broussonetia papyrifera powder obtained is guaranteed. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 : Knockout Aspergillus niger pyrG recombinant plasmid map;

[0024] Figure 2 : Verification of knockout Aspergillus niger pyrG recombinant plasmid colony PCR product gel electrophoresis map;

[0025] Figure 3 : Tannase gene PCR product gel electrophoresis map;

[0026] Figure 4 : Aspergillus niger tannase recombinant plasmid map;

[0027] Figure 5 : Standard tannic acid HPLC map;

[0028] Figure 6 : Standard gallic acid HPLC map. DETAILED DESCRIPTION

[0029] The application will be further described below with reference to the specific embodiments of the specification. Those skilled in the art will be able to implement the application based on these descriptions. In addition, the embodiments of the application involved in the following description are generally only a part of the embodiments of the application, not all the embodiments. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments in the application without creative labor should be within the scope of protection of the application.

[0030] In the following examples, the specific formula of the culture medium is as follows:

[0031] (1) LB liquid medium: 0.5% yeast powder, 1% NaCl, 1% peptone, pH 7.0, 121℃, sterilization for 20 min.

[0032] (2) LB solid medium: based on the LB liquid medium, add 2% agar powder, 121℃, sterilization for 20 min.

[0033] (3) DPY medium: 1% peptone, 0.5% yeast powder, 2% glucose, 0.5% KH2PO4, 0.05% MgSO4·7H2O, pH natural, 115℃, sterilization for 30 min.

[0034] (4) PDA solid medium composition (W / V): take 200g peeled potatoes, fully boil, filter the obtained filtrate, add 20g glucose, cool, add water to 1L, add 2% agar powder, pH natural, 115℃, sterilization for 30 min.

[0035] (5) High osmotic 2% CD solid medium: 35% sucrose, 0.3% NaNO3, 0.2% KCl, 0.1% KH2PO4, 0.05% MgSO4·7H2O, 0.001% FeSO4·7H2O, add 2% agar powder, 121℃, sterilization for 20 min.

[0036] (6) High osmotic 0.5% CD soft agar medium: same solid formula, only agar powder is changed to 0.5%, 121℃, sterilization for 20 min.

[0037] (7) Seed liquid medium composition (W / V): 2% glucose, 0.5% peptone, 0.2% yeast extract powder, 0.1% K2HPO4, 0.05% MgSO4, solvent is water, adjust pH to 6.8;

[0038] (8) Fermentation medium composition (W / V): 4.2% corn steep liquor, 3% soybean meal, 2% glucose, 1.5% K2HPO4, 1.5% (NH4)2SO4, 0.3% citric acid, 0.25% yeast extract, 0.25% yeast extract powder, 0.02% CaCl2, 0.5% MgSO4, solvent is water, pH natural.

[0039] The pH natural means that the pH of the fermentation medium is not specially adjusted, and the pH value after adding the medium components is natural.

[0040] (9) Uracil solution: weigh 2.442 g of uracil, dissolve in 10 mL of sterile ddH2O, filter sterilized with 0.22 μm filter membrane and aliquot, store at -20℃.

[0041] (10) 5-Fluoroorotic acid (5-FOA): weigh 1 g of 5-FOA powder, dissolve in 10 mL of dimethyl sulfoxide, filter sterilized with 0.22 μm filter membrane and aliquot, store at -20℃.

[0042] (11) 0.8M NaCl solution: weigh 46.8 g of NaCl, dissolve in ddH2O and dilute to 1 L.

[0043] (12) STC buffer: weigh 10.93 g of sorbitol, 0.303 g of Tris, 0.277 g of anhydrous CaCl2, dissolve in ddH2O and dilute to 50 mL, pH 7.5, filter sterilized with 0.22 μm filter membrane, store at 4℃.

[0044] (13) PEG buffer: weigh 30 g of PEG 6000, 0.061 g of Tris, 0.277 g of anhydrous CaCl2, dissolve in ddH2O and dilute to 50 mL, pH 7.5, sterilize at 121℃ for 20 min, store at 4℃ after cooling.

[0045] (14) 100 mM sodium phosphate buffer (pH 6.0): weigh 1.37 g of NaH2PO4·2H2O, 0.43 g of Na2HPO4·12H2O, dissolve in ddH2O and dilute to 100 mL, pH 6.0, store at 4℃.

[0046] (15) Enzymatic solution: weigh 0.2 g of cellulase, 0.1 g of snailase, 0.1 g of lyticase, 0.05 g of lysozyme, 0.468 g of NaCl, 1 mL of the above sodium phosphate buffer, add 9 mL of ddH2O to dissolve thoroughly, filter sterilized with 0.22 μm filter membrane, store at 4℃.

[0047] In the following examples, tannic acid ( Figure 5 ) and gallic acid ( Figure 6HPLC analysis method: the chromatographic column was CAPCELL PAK ADME column (250 mm x 4.6 mm I.D.), the mobile phase was 0.5% formic acid methanol solution (V / V). Detection method: flow rate was 0.4 mL / min, injection volume was 10 μL, detection wavelength was 280 nm, column temperature was 35 °C.

[0048] Example 1 Construction of Aspergillus niger pyrG gene-deficient strain

[0049] The wild-type Aspergillus niger (China Center for Type Culture Collection, CCTCC NO. 206047) preserved in the laboratory was used as the starting strain to construct the Aspergillus niger pyrG gene-deficient strain.

[0050] 1.1 Construction of pyrG knockout plasmid

[0051] First, the pyrG sequence gene (Gene ID: 4985706) derived from Aspergillus niger was searched from the NCBI database.

[0052] The genome of Aspergillus niger CCTCC NO. 206047 was used as the template, and the primers Up-pyrG-F / Up-pyrG-R and Do-pyrG-F / Do-pyrG-R (Table 1-1) were used to amplify the upstream and downstream gene fragments pyrGL and pyrGR with homologous arms, respectively. The pET-28a(+) plasmid was used as the template, and the primers 28ZT-F and 28ZT-R (Table 1-1) were used to amplify the vector fragment ZT-1. After PCR, the size of the amplified gene band was verified by 1.0% agarose gel electrophoresis. The correct PCR products were purified. The upstream fragment pyrGL and the vector fragment ZT-1 were subjected to one-step cloning, and the recombinant plasmid pET-28a(+)-pyrGL containing pyrGL was constructed by transforming the E. coli DH5α competent cells. The bacteria were cultured to extract the plasmid and verify whether it was correct. If correct, the plasmid was linearized and subjected to one-step cloning with the downstream fragment pyrGR, and the recombinant plasmid pET-28a(+)-pyrGLR containing pyrGLR was constructed by transforming the E. coli DH5α competent cells. Figure 1

[0053] Table 1-1 Primer names and sequences

[0054]

[0055] Note: The underlined part is the homologous sequence required for cloning.

[0056] ​The recombinant plasmid pET-28a(+)-pyrGLR constructed is used as a template for amplifying the target gene fragment, and the primers pyrG-1 and pyrG-2 (Table 1-1) are used to amplify the target gene expression fragment for the transformation of Aspergillus niger protoplasts. Agarose gel electrophoresis is used to verify the electrophoresis ( Figure 2

[0057] 1.2 Preparation and transformation of Aspergillus niger protoplasts

[0058] 1.2.1 Preparation of protoplasts

[0059] 1) Mycelium culture: Aspergillus niger strain preserved in a 4°C refrigerator was inoculated into an antibiotic-free PDA culture medium and cultured in a 30°C incubator for 4-5 days. Spores were washed off with 4mL sterile ddH2O to prepare a spore suspension, which was inoculated into 50mL DPY culture medium and cultured at 30°C, 200rpm for 24h.

[0060] 2) Collection of mycelium: After the culture solution was filtered out using a vacuum filtration pump, the mycelium was collected, washed with sterile ddH2O, and then washed with 0.8M NaCl solution. Filtration was performed until the mycelium was in a dry state. The mycelium was scraped with a sterile medicine spoon, and about 0.8g of mycelium was placed in a sterile conical flask and 10mL of enzyme solution was added.

[0061] 3) Enzymatic hydrolysis: The conical flask was placed in a water bath shaker and enzymatic hydrolysis was performed at 30°C, 100rpm for about 3h.

[0062] 4) Filtration: The mycelium after enzymatic hydrolysis in step 3) was filtered into a 50mL sterile centrifuge tube using a funnel with 4 layers of magic filter cloth. The centrifuge tube containing the protoplasts was centrifuged at 4°C, 1200rpm for 15min, and the supernatant was discarded. 3mL of STC buffer was added to the precipitate to wash and resuspend it, and it was centrifuged at 4°C, 1200rpm for 10min, and the supernatant was discarded. 1mL of STC buffer was added to the protoplast precipitate and it was resuspended by blowing with a blunt-ended syringe.

[0063] 1.2.2 Protoplast transformation

[0064] In order to ensure the accuracy of the transformation rate, the transformation rate is divided into positive group, negative group and transformation group.

[0065] Positive group: In a 2mL centrifuge tube, 160μL of protoplast suspension, 100μL of STC buffer and 60μL of PEG solution were added and mixed evenly;

[0066] ​​Negative group: In 2 mL centrifuge tube, 160 μL protoplast suspension, 100 μL STC buffer, 60 μL PEG solution, 1% uracil solution and 5-FOA were added respectively, and mixed evenly;

[0067] Transformation group: In 2 mL centrifuge tube, 160 μL protoplast suspension, 1% uracil solution and 5-FOA, 30-100 μL amplified fragment and 60 μL PEG solution were mixed;

[0068] The centrifuge tube was placed on ice, and 2-3 times of gentle inversion was performed every 10 min. After 30 min, 1.5 mL PEG was added to the transformation centrifuge tube, and mixed evenly by gentle inversion. The mixture was placed at room temperature for 25 min.

[0069] 1.2.3 Plate culture

[0070] The lower layer of the positive group was plated with 2% CD, and the lower layer of the negative group and the transformation group was plated with 2% CD containing 1% (W / V) uracil solution and 5-FOA.

[0071] Positive group: 3 mL 0.7% CD soft agar medium, 1.5 mL STC buffer, and the corresponding mixture obtained by transformation were mixed and evenly poured onto the high-osmotic 2% CD solid medium.

[0072] Negative group: 3 mL 0.7% CD soft agar medium, 1.5 mL STC buffer, and 1% (W / V) uracil solution and 5-FOA were mixed with the corresponding mixture obtained by transformation and evenly poured onto the high-osmotic 2% CD solid medium.

[0073] Transformation group: 3 mL 0.7% CD soft agar medium, 1.5 mL STC buffer, and 1% (W / V) uracil solution and 5-FOA were mixed with the corresponding mixture obtained by transformation and evenly poured onto the high-osmotic 2% CD solid medium containing 1% (W / V) uracil solution and 5-FOA.

[0074] The transformants growing on the 2% CD plate in the experimental group were inoculated into PDA medium containing 5-FOA and uracil solution, and incubated at 30°C in a constant temperature incubator for 3-5 days. The growth conditions were observed and compared.

[0075] The better growing Aspergillus niger transformants in the culture medium were selected for genomic PCR verification. The PCR products with correct bands were sent for sequencing. The target gene sequence and the sequencing results were compared, and the Aspergillus niger transformant with correct sequence was the pyrG-deficient strain, which was preserved in a 4°C refrigerator.

[0076] Case 2 Construction of Aspergillus niger high-tannase-producing strain and determination of tannase activity in fermentation supernatant

[0077] 2.1 Obtaining of tannase gene and construction of recombinant plasmid

[0078] NCBI search for tannase gene derived from Aspergillus niger (GenBank: XM_001401772). The wild-type Aspergillus niger CCTCC NO. 206047 genome was used as the template for PCR amplification with CHTan-F / CHTan-R primers (Table 2-1), and Primer Star MasterMix (Takara) high-fidelity enzyme was selected, with the conditions being pre-denaturation at 98°C for 3 min; amplification stage for 30 cycles at 98°C for 10 s, 60°C for 10 s, and 72°C for 20 s; and extension at 72°C for 10 min. The PCR product was digested and purified to obtain the purified fragment (target gene with homologous arms), which was denoted as fragment tan-1.

[0079] The plasmid pCAMBIA-tan (containing pyrG expression cassette) was used as the template for PCR amplification with ZTN1-F / ZTN1-R primers (see Table 2-1), and Primer Star MasterMix (Takara) high-fidelity enzyme was selected, with the conditions being pre-denaturation at 98°C for 3 min; amplification stage for 30 cycles at 98°C for 10 s, 55°C for 5 s, and 72°C for 90 s; and extension at 72°C for 10 min. The PCR product was digested and purified to obtain the linearized vector, which was denoted as fragment ZT-2.

[0080] Table 2-1 Primer names and sequences

[0081]

[0082] Note: The underlined part is the homologous sequence required for cloning.

[0083] The fragments tan-1 and ZT-2 were recombined into E. coli DH5α competent cells using a one-step cloning kit to construct the pCAMBIA-tan recombinant plasmid (pCAMBIA-tan). The bacteria were cultured to extract the plasmid, and the tannase gene expression cassette was obtained from the plasmid P, which was sent to Genscript for sequencing. After comparison, the recombinant plasmid was extracted, and the plasmid containing the tannase gene and pyrG expression cassette gene P was denoted as TAN-1 and used for Aspergillus protoplast transformation. Figure 3

[0084] 2.2 Protoplast preparation and transformation

[0085] ​Using the Aspergillus niger auxotrophic strain as the starting strain, the Aspergillus niger tannase strain was constructed (same as the above-mentioned implementation case 1.2 step)

[0086] Among them, the upper and lower layers of the positive control group were added with 1% (W / V) uracil solution and 5-FOA. The negative group and the experimental group were not added with 1% (W / V) uracil solution and 5-FOA.

[0087] After transformation, each strain was placed in a 30°C constant temperature mold incubator and cultured for 3-4 days to observe its growth condition in the cell.

[0088] 2.3 Screening of Aspergillus niger tannase strain

[0089] 2.3.1 Extraction of Aspergillus niger transformant genome

[0090] The spores of the Aspergillus niger transformant well grown on the 2% CD plate were inoculated into PDA medium and recorded as the first generation of positive transformant, which was cultured in a 30°C constant temperature mold incubator for 3-4 days. The mycelium grown out can be used for whole genome extraction.

[0091] 2.3.2 Verification of tannase transformant genome

[0092] Using the Aspergillus niger transformant genome as the template, the verification primer was used for genome PCR identification. The PCR product was detected by electrophoresis, and the transformant genome with correct band size was reserved. The PCR liquid was sent to the sequence, and the sequence was consistent, which was determined as the positive transformant of Aspergillus niger. The first generation of positive transformant was transferred to the corresponding PDA plate, which was regarded as the second generation of transformant. Then the first generation of positive transformant with good genetic stability was inoculated into PDA solid slant and preserved at 4°C.

[0093] The second generation of transformant was streaked on PDA plate, activated at 30°C for 5d, and inoculated into seed liquid culture medium to make spore suspension. The seed liquid was cultured at 30°C, 220rpm for 24h, and then transferred to fermentation medium and fermented at 30°C, 220rpm for 5d. The supernatant was collected by centrifugation at 12000rpm for 10min.

[0094] 2.3.3 Detection of tannase supernatant enzyme activity of Aspergillus niger

[0095] The tannase enzyme activity was detected by the pyrogallol method using pyrogallol. In this method, propyl gallate (PG) was used as the reaction substrate, and the gallic acid produced by the decomposition of PG by tannase could form a red complex with pyrogallol under alkaline conditions. The complex had maximum absorption at 520nm, and accordingly the amount of generated gallic acid could be calculated by measuring the change of A520, so as to calculate the enzyme activity.

[0096] The steps for detecting tannase enzyme activity are as follows:

[0097] (1) Reaction start, PG solution and enzyme solution to be tested in 30℃ water bath for 5-10 min;

[0098] (2) Take 4 clean test tubes, 1 empty tube, 3 test tubes. Add 0.25 mL PG solution to each tube, then add 0.25 mL enzyme solution to be tested to the test tube, and react for 5 min under 30℃ water bath;

[0099] (3) Add 0.3 mL methanol tannin solution (0.667%, W / V) to all test tubes, and incubate for 5 min;

[0100] (4) Add 4.2 ml KOH (0.5M) solution to all test tubes, and add 0.25 mL enzyme solution to the blank tube. After placing at 30℃ for 10 min, set the blank tube to zero, and measure the absorbance of each solution at 520 nm.

[0101] The different transformants of Aspergillus niger tannase strains obtained by construction were subjected to shake flask fermentation, and the enzyme activity was shown in Table 2-2. The results of Table 2-2 showed that the shake flask fermentation tannase enzyme activity of strain Tan-5 was the highest, and the enzyme activity was 5.92 U / mL.

[0102] Table 2-2 Enzyme activity of tannase fermentation of strains

[0103]

[0104] Example 3 Fermentation of Aspergillus niger tannase in 5L fermenter

[0105] The strain Tan-5 with high enzyme activity was selected for 5L fermenter culture. The strain Tan-5 was streaked on PDA plate, activated at 30℃ for 5d, and inoculated into seed liquid medium to form spore suspension, which was cultured at 30℃, 220rpm for 24h.

[0106] The 5L fermenter was sterilized in a high-pressure steam sterilization pot at 115℃ for 30min to complete the emptying.

[0107] Take 126 g of corn syrup, 90 g of soybean meal, 60 g of glucose, 45 g of K2HPO4, 45 g of (NH4)2SO4, 9 g of citric acid, 7.5 g of yeast extract, 7.5 g of yeast extract powder, 0.06 g of CaCl2, 15 g of MgSO4, add water to 3 L, mix uniformly, and then pour into the fermentation tank. At the same time, 0.05% defoaming agent needs to be added, check the airtightness of the fermentation tank and connect the air filter membrane, put in the pH electrode and dissolved oxygen electrode, and then sterilize in the high-pressure steam sterilization pot at 115°C for 30 min to complete the real sterilization. Connect the fermentation tank with the temperature probe, condensate water, air compressor, and feeding bottle, set the temperature and pH, and set the stirring speed to 600 rpm. The aeration rate is set to 1 VVM. After the fermentation tank parameters reach the set value and remain stable, inoculate the seed liquid. Use flame sealing method to inoculate the seed liquid into the fermentation tank at a 6% inoculation amount. Set the fermentation cycle at this time as 0h, so that sampling can be performed every 12h. Use phosphoric acid and 20% ammonia water to maintain the pH at the optimal value during fermentation. After 36h of fermentation, the cells enter the rapid enzyme production stage, and intermittent feeding is started, and the speed is adjusted to 400 rpm. After 72h, continuous feeding is started, and continuous fermentation is carried out for 5 days, until no feeding can be performed, and the fermentation is ended. The enzyme activity reaches 20.16 U / mL. Use gauze to filter out the Aspergillus niger cells, centrifuge at 10,000 rpm for 10 min, remove the remaining substances, obtain tannase enzyme liquid, and store in a refrigerator at 4°C for standby use.

[0108] Example 4 Process of Enzymatic Treatment of Broussonetia papyrifera Powder (Different Enzyme Addition Amounts)

[0109] (1) Pretreatment of Broussonetia papyrifera powder (tannin content 1.49%): Add Broussonetia papyrifera powder raw material to tap water (solid-liquid ratio 1:3), heat to 60°C, and stir for 3h;

[0110] (2) Add tannase enzyme liquid treatment: Cool the Broussonetia papyrifera powder material of step (1) to room temperature, add a certain amount of Aspergillus niger fermented tannase enzyme liquid prepared in Example 3, and perform enzymatic hydrolysis at a certain temperature (30°C) for a certain period of time (6h). During the process, the hydrolysis of tannin can be achieved;

[0111] (3) Inactivation of tannase: Heat the material of step (2) to boiling (20 min) to inactivate the tannase, and cool to room temperature to obtain the palatability-improved Broussonetia papyrifera powder feed raw material.

[0112] This example explores the effect of different enzyme addition amounts (v / w) on the enzymatic hydrolysis of Broussonetia papyrifera powder tannin in the fermentation liquid. In this example, the enzyme amounts are 1%, 2%, 3%, 4%, 5%, 8%, and 10%. The conversion rate of the enzymatic reaction is shown in Table 4-1. The results show that when the enzyme addition amount is higher than 5%, the tannin conversion rate can reach more than 80%.

[0113] Table 4-1 Effect of different enzyme addition amount (v / w) on enzymatic hydrolysis of tannin in fermented Broussonetia papyrifera powder

[0114]

[0115]

[0116] Example 5 Process of enzymatic treatment of Broussonetia papyrifera powder (different enzymatic hydrolysis temperature)

[0117] In this example, the effect of enzymatic hydrolysis temperature on tannin in fermented Broussonetia papyrifera powder was explored. According to the method of Example 4, the enzyme addition amount was 5%, the enzymatic hydrolysis temperature was 25℃, 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, respectively, and other conditions were unchanged. The enzymatic hydrolysis of hydrolyzable tannin in feed was carried out, and the conversion rate was shown in Table 5-1. The results showed that the tannin conversion rate was the highest at 30℃.

[0118] Table 5-1 Effect of different enzymatic hydrolysis temperature on enzymatic hydrolysis of tannin in fermented Broussonetia papyrifera powder

[0119]

[0120] Example 6 Process of enzymatic treatment of Broussonetia papyrifera powder (different enzymatic hydrolysis time)

[0121] In this example, the effect of enzymatic hydrolysis time on tannin in fermented Broussonetia papyrifera powder was explored. According to the method of Example 4, the enzyme addition amount was 5%, the enzymatic hydrolysis temperature was 30℃, and the enzymatic hydrolysis time was 1h, 2h, 3h, 4h, 5h, 6h, 15h, respectively, and other conditions were unchanged. The enzymatic hydrolysis of hydrolyzable tannin in feed was carried out, and the conversion rate was shown in Table 6-1. The results showed that the tannin conversion rate was more than 80% when the enzymatic hydrolysis time was more than 6h.

[0122] Table 6-1 Effect of different enzymatic hydrolysis time on enzymatic hydrolysis of tannin in fermented Broussonetia papyrifera powder

[0123]

[0124]

Claims

1. A method for preparing enzymatically structured flour, characterized in that, The method comprises the following steps: S1, knocking out pyrG gene of wild type Aspergillus niger to obtain Aspergillus niger pyrG gene deficient strain; the wild type Aspergillus niger has a preservation number of CCTCC NO.206047; the pyrG gene is back-supplemented in the form of tannase gene plus pyrG gene to obtain Aspergillus niger high-yield tannase strain; the Aspergillus niger high-yield tannase strain is cultured in a fermenter to obtain Aspergillus niger tannase; the nucleotide sequence of the tannase gene is GenBank:XM_001401772; the Gene ID of the pyrG gene is 4985706; S2, using the Aspergillus niger tannase to enzymolyze broussonetia papyrifera powder to obtain enzymolyzed broussonetia papyrifera powder.

2. The production method according to claim 1, characterized by, Step S2 comprises the following steps: S2.1, dissolving broussonetia papyrifera powder raw material in water to obtain pretreated raw material; S2.2, adding enzyme solution of the Aspergillus niger tannase to the pretreated raw material to perform enzymolysis to obtain enzymolysis product; S2.3, performing inactivation treatment on the enzymolysis product to obtain enzymolyzed broussonetia papyrifera powder.

3. The preparation method according to claim 2, characterized in that, In step S2.1, the mass-volume ratio of broussonetia papyrifera powder raw material to water is 1:

3.

4. The production method according to claim 2, characterized by, In step S2.2, the volume-mass ratio of enzyme solution addition amount to broussonetia papyrifera addition amount in pretreated raw material is 1% to 10%.

5. The preparation method according to claim 2, characterized in that, In step S2.2, the enzymolysis condition is: temperature 25 to 60 ℃, time 1 to 6 h.

6. The method of claim 2, wherein, In step S2.3, the inactivation treatment is: heating the enzymolysis product to boiling, and then cooling to room temperature to obtain enzymolyzed broussonetia papyrifera powder.

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