CN111549020B - Preparation of complex enzyme rich in acidic pectinase, strain and application thereof - Google Patents

Preparation of complex enzyme rich in acidic pectinase, strain and application thereof Download PDF

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CN111549020B
CN111549020B CN202010511065.8A CN202010511065A CN111549020B CN 111549020 B CN111549020 B CN 111549020B CN 202010511065 A CN202010511065 A CN 202010511065A CN 111549020 B CN111549020 B CN 111549020B
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pectinase
aspergillus niger
complex enzyme
rich
acidic
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CN111549020A (en
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吴勃
王云龙
徐永雷
王天珍
刘广晓
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Hangzhou Biocom Biological Technology Co ltd
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Hangzhou Biocom Biological Technology Co ltd
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    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
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Abstract

The invention provides a preparation method of a complex enzyme rich in acidic pectinase, a bacterial strain and application thereof; the cultured fermentation product contains pectinase, xylanase, mannase, acid protease, cellulase, amylase and beta-glucanase. Aspergillus niger (Aspergillus niger) named as Aspergillus niger BAK200345 with the preservation number of CGMCC No.19614. The complex enzyme rich in the acid pectinase provided by the fermentation product of the invention has rich pectinase types, and pectin esterase, pectin hydrolase, pectin lyase, polygalacturonase and rhamnogalacturonase exist, so that the complex enzyme is a complete pectinase system. The enzyme activity of the acidic pectinase is high and can reach 132960U/g at most; the compound enzyme can reduce the feed-egg ratio, improve the laying rate of laying hens, reduce the phenomenon of sticking feces, improve the intestinal health and has good application effect.

Description

Preparation of complex enzyme rich in acidic pectinase, bacterial strain and application thereof
Technical Field
The invention relates to the field of complex enzymes, in particular to preparation of a complex enzyme rich in acidic pectinase, a bacterial strain and application thereof.
Background
Pectin is a high-molecular polysaccharide compound, widely exists in plants such as fruits, vegetables, corn, soybean and the like, is a main component of plant cell walls, and is mixed in primary cell walls and intercellular spaces of the plants. At present, most of feed raw materials are plant raw materials, pectin serving as one of anti-nutritional factors exists in the plant feed raw materials, and the pectin can improve the viscosity of chyme after entering the digestive tract, so that the digestion and absorption of the animal on nutrient substances are influenced.
Pectins are mainly classified into pectins, pectic acids, and protopectics. Pectin is a long-chain high-molecular compound formed by connecting galacturonic acid ester and a small amount of galacturonic acid through alpha-1, 4-glycosidic bond; pectic acid is a straight chain of about 100 galacturonic acids connected by alpha-1, 4 bonds, mainly present in the middle layer; the molecular weight of protopectin is higher than that of pectic acid and pectin, the methyl esterification degree is between the two, the protopectin mainly exists in a primary wall, is insoluble in water, and is converted into soluble pectin under the action of dilute acid and protopectinase. Pectin in plant tissues can be combined with cellulose, hemicellulose, lignin, protein and other substances to show the inherent forms of tissues of various parts.
The cleavage of pectin requires a series of enzymes. Pectinase is a multienzyme composite system for decomposing pectin substances, which can break down plant cell walls and degrade macromolecular galacturonic acid into galacturonic acid and pectic acid micromolecule substances, thereby rapidly and thoroughly removing pectin. According to the mode of action on the substrate, it can be classified into polygalacturonase, pectin lyase and pectin esterase. Polygalacturonases can be further classified into exo-polygalacturonases, endo-polygalacturonases; pectate lyases are classified into endo-polygalacturonate lyase, exo-polygalacturonate lyase, endo-polymethylgalacturonate lyase, exo-polymethylgalacturonate lyase. They can be classified into acidic pectinase and alkaline pectinase according to their optimum pH. Acid pectinase is the main source of the current industrial enzyme preparations and is mainly produced by fungi; alkaline pectinase is mainly produced by bacteria and is suitable for alkaline environments.
As animal somatic cells have no cell wall structure and can not secrete to produce pectinase, the pectinase is generally added exogenously in livestock and poultry production. Many research test results show that the addition of pectinase with proper level in the feed can improve the production performance of animals and improve the quality of livestock and poultry products. Researches such as ginger xianxiao and the like find that pectinase is added into daily ration to degrade pectin of plant cell walls, so that cell wall cracking is caused, absorption and utilization of CF, NDF and AD in the daily ration are facilitated, and the effects of improving production performance, growth speed and leg muscle rate of broiler chickens, reducing abdominal fat rate and promoting digestion and absorption of nutrient substances are remarkable; studies of Zqian and the like find that the addition of a proper amount of pectinase preparation into daily ration can obviously improve the digestibility of goose on cellulose and promote the digestion and absorption of goose on calcium and phosphorus. In addition, researches find that the complete cracking of plant cell walls needs the synergistic action of pectinase, cellulase, hemicellulase, protease and the like to form a multi-enzyme composite system, so that the digestion in the digestive tract can be smoothly carried out. Tahir and other researches show that the pectinase has remarkable synergistic effect on cellulase and hemicellulase in the aspect of improving the digestibility of protein and dry matters; the release of monosaccharide is also similar; zyla et al have shown that the combination of pectinase and protease can improve the mineral and calcium-phosphorus retention of turkey bones.
At present, most of the application of pectinase is concentrated in food and other industries, and liquid fermentation is mostly adopted. The liquid fermentation product is single, and for plant feed raw materials with complex components, multiple enzymes are required to act synergistically. Therefore, further studies on complex enzyme systems are necessary.
Disclosure of Invention
The invention provides a preparation method of a compound enzyme rich in acidic pectinase, a bacterial strain and application thereof, and solves the problem of single enzyme system in a fermentation product in the prior art.
The technical scheme of the invention is realized as follows:
a preparation method of complex enzyme rich in acidic pectinase comprises the steps of,
the strain is Aspergillus niger BAK200345 with CGMCC No.19614;
the cultured fermentation product contains pectinase, xylanase, mannanase, acid protease, cellulase, amylase and beta-glucanase.
In some embodiments, the preparation of the complex enzyme comprises a solid fermentation step;
inoculating the seed liquid on a solid fermentation culture medium, uniformly stirring, and performing fermentation culture in a koji room;
the solid fermentation medium is prepared according to the following proportion: 80-90% of bran, 5-10% of corn flour, 2-5% of soybean meal, 1-2% of ammonium sulfate, 0.05-0.1% of magnesium sulfate, 0.2-0.3% of dipotassium phosphate and 60-70% of initial water, and sterilizing at 121 ℃ for 40min.
Further, the solid fermentation medium is replaced by the following formula:
80-85% of bran, 5-10% of soybean meal, 5-10% of corn flour, 1-2% of ammonium chloride, 0.05-0.1% of magnesium sulfate, 0.5-1% of calcium carbonate, 0.2-0.3% of dipotassium phosphate, 60-65% of initial moisture and sterilization at 121 ℃ for 40min;
or the like, or, alternatively,
80-90% of bran, 5-10% of soybean meal, 5-10% of palm meal, 1-2% of ammonium sulfate, 0.05-0.1% of magnesium sulfate, 0.01-0.02% of manganese sulfate, 1-2% of calcium carbonate, 0.2-0.3% of dipotassium phosphate, 60-65% of initial water and sterilization at 121 ℃ for 40min.
In some embodiments, the koji room is subjected to temperature and humidity control treatment, wherein the relative humidity of the koji room is controlled to be more than 50%, and the material temperature is controlled to be 28-32 ℃; fermenting and culturing for 4-8 days until the mycelium is obvious and thick and the enzyme activity is slowly increased.
Further, the preparation of the seed liquid is as follows:
(1) Slant culture
Inoculating Aspergillus niger BAK200345 to the sterilized slant solid culture medium by streaking, and culturing at 25-35 deg.C for 4-8 days; the culture medium is a Chao's culture medium: 3% of sucrose, 0.3% of sodium nitrate, 0.1% of dipotassium phosphate, 0.05% of magnesium sulfate, 0.05% of potassium chloride, 0.001% of ferrous sulfate and 1.2-1.8% of agar powder, and sterilizing for 20min at 121 ℃;
(2) Shaking culture
Scraping the bevel seeds obtained in the 1-2 ring step (1) into a secondary shake flask liquid culture medium for culture, wherein the liquid filling amount of the shake flask is 100-150 mL/bottle; culturing at 25-35 deg.C and table rotation speed of 160-200rpm for 2-6d to obtain second-stage shake flask seed solution; the formula of the culture medium is as follows: 2-6% of bran, 1-5% of peptone, 2-6% of glucose and 4-8% of maltodextrin, and sterilizing at 121 ℃ for 40min;
(3) Liquid tank culture
Inoculating the second-stage shake flask seed solution obtained in the step (2) into a third-stage liquid tank seed solution, wherein the inoculation amount is 2-10%, the temperature is 25-35 ℃, and the rotation speed is 100-180rpm, and culturing for 2-6d to obtain a third-stage seed solution; the formula of the culture medium is as follows: 2-6% of bran, 1-5% of peptone, 2-6% of glucose and 4-8% of maltodextrin, and sterilizing at 121 ℃ for 40min.
Further, the medium in step (1) may be replaced with the following medium:
sterilizing 2% of glucose, 20% of potato juice and 1.2-1.8% of agar powder at 121 ℃ for 20min; or
2-5% of bran juice, 1-3% of beef extract, 0.5-2% of peptone, 0.1-0.6% of dipotassium hydrogen phosphate, 0.2-0.4% of magnesium sulfate and 1.2-1.8% of agar powder, and sterilizing at 121 ℃ for 20min.
In some embodiments, the method further comprises a drying step and a crushing step of the fermentation product; wherein the drying temperature is 55-65 ℃.
The invention also provides an Aspergillus niger (Aspergillus niger) named as Aspergillus niger BAK200345 with the preservation number of CGMCC No.19614.
The invention also provides livestock and poultry feed which comprises the fermentation product of the Aspergillus niger BAK200345.
Compared with the prior art, the invention has the following beneficial effects:
(1) The acid pectinase-rich compound enzyme provided by the fermentation product of the invention has rich pectinase types, and pectin esterase, pectin hydrolase, pectin lyase, polygalacturonase and rhamnogalacturonase exist, so that the compound enzyme is a complete pectinase system.
(2) The acid pectinase obtained by fermentation has high enzyme activity which can reach 132960U/g to the maximum;
(3) The complex enzyme provided by the invention can reduce the feed-egg ratio, improve the laying rate of laying hens, reduce the phenomenon of sticking feces, improve the intestinal health and has a good application effect.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the technical solutions in the prior art description will be briefly introduced below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without inventive labor.
FIG. 1 shows the LC-MS spectrum results of fermentation products.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
And (3) biological preservation:
aspergillus niger BAK200345, deposited in China general microbiological culture Collection center (CGMCC; china microbiological research institute, 3, national institute of microorganisms, japan, xilu No.1, beijing, chaoyang, ltd.) at No. 4/8 of 2020, 4/8, with the preservation number: CGMCC No.19614.
And ITS identification:
the segment sequence (see sequence table) of the ITSrDNA of the strain is compared as follows: the strain used by the invention has the highest similarity with the Aspergillus gene sequence. It was therefore named aspergillus niger BAK200345.
The Aspergillus niger BAK200345 provided by the invention is a high-yield complex enzyme strain obtained by strict domestication and cultivation.
Example 1
A preparation method of a complex enzyme rich in acidic pectinase comprises the following specific steps:
1 slant culture
Inoculating Aspergillus niger BAK200345 to sterilized first-stage test tube slant solid culture medium, and culturing at 25-35 deg.C for 4-8 days. The culture medium is a Chao's culture medium: 3 percent of sucrose, 0.3 percent of sodium nitrate, 0.1 percent of dipotassium phosphate, 0.05 percent of magnesium sulfate, 0.05 percent of potassium chloride, 0.001 percent of ferrous sulfate, 1.2 to 1.8 percent of agar powder and sterilization for 20min at 121 ℃.
2 Shake flask culture
Scraping the first-stage test tube slant seeds obtained in the 1-2 ring step 1 into a second-stage shake flask liquid culture medium for culture, wherein the shake flask liquid loading is 100-150 mL/bottle. Culturing at 25-35 deg.C and shaking table rotation speed of 160-200rpm for 2-6d to obtain second-stage shake flask seed solution. The formula of the culture medium is as follows: 2-6% of bran, 1-5% of peptone, 2-6% of glucose and 4-8% of maltodextrin, and sterilizing at 121 ℃ for 40min.
3 liquid tank culture
And (3) inoculating the second-stage shake flask seed solution obtained in the step (2) into a third-stage liquid tank seed solution, wherein the inoculation amount is 2-10%, the temperature is 25-35 ℃, and the rotation speed is 100-180rpm, and culturing for 2-6d to obtain a third-stage seed solution. The formula of the culture medium is as follows: 2-6% of bran, 1-5% of peptone, 2-6% of glucose and 4-8% of maltodextrin, and sterilizing at 121 ℃ for 40min.
4 solid fermentation process
And (4) inoculating the third-level seed liquid obtained in the step (3) on a solid fermentation culture medium, uniformly stirring, filling into an aseptic fermentation breathable bag, flatly laying on a tray with the charging amount of 5-8 kg/bag, and performing fermentation culture in a koji room. During the period, the temperature and humidity control treatment is carried out, the relative humidity of the koji room is controlled to be more than 50 percent, and the material temperature is controlled to be 28-32 ℃. Fermenting and culturing for 4-8 days until the mycelium is obvious and thick and the enzyme activity is slowly increased.
The solid fermentation medium is prepared according to the following proportion: 80-90% of bran, 5-10% of corn flour, 2-5% of soybean meal, 1-2% of ammonium sulfate, 0.05-0.1% of magnesium sulfate, 0.2-0.3% of dipotassium phosphate and 60-70% of initial water, and sterilizing at 121 ℃ for 40min.
5 enzyme activity detection result
The product obtained by the solid fermentation process provided by the company is dried and crushed at the temperature of 55-65 ℃ and is sieved (40 meshes). According to the detection method of the national standard enzyme preparation, the enzyme activity is detected. According to the detection result, the fermentation product not only contains pectinase with high enzyme activity, but also contains xylanase, mannase, acid protease, cellulase, amylase, beta-glucanase and the like. Wherein the enzyme activity of the pectinase reaches 132960U/g, the xylanase is 9024U/g, the mannase is 5246U/g, the acid protease is 857U/g, the cellulase is 2620U/g, the beta-glucanase is 1264U/g, and the amylase is 2203U/g, and the result is shown in the table 1.
TABLE 1 summary of enzyme activities of major enzymes
Figure BDA0002528293860000071
Example 2
The slant medium in example 1 was replaced with the following medium;
2% of glucose, 20% of potato juice and 1.2-1.8% of agar powder, and sterilizing for 20min at 121 ℃.
Or the like, or a combination thereof,
2-5% of bran juice, 1-3% of beef extract, 0.5-2% of peptone, 0.1-0.6% of dipotassium hydrogen phosphate, 0.2-0.4% of magnesium sulfate and 1.2-1.8% of agar powder, and sterilizing at 121 ℃ for 20min.
The remaining conditions were unchanged.
The solid medium in example 1 was replaced with the following medium;
80-85% of bran, 5-10% of soybean meal, 5-10% of corn flour, 1-2% of ammonium chloride, 0.05-0.1% of magnesium sulfate, 0.5-1% of calcium carbonate, 0.2-0.3% of dipotassium phosphate, 60-65% of initial moisture and sterilization at 121 ℃ for 40min.
Or the like, or a combination thereof,
80-90% of bran, 5-10% of soybean meal, 5-10% of palm meal, 1-2% of ammonium sulfate, 0.05-0.1% of magnesium sulfate, 0.01-0.02% of manganese sulfate, 1-2% of calcium carbonate, 0.2-0.3% of dipotassium phosphate, 60-65% of initial water and sterilization at 121 ℃ for 40min.
The remaining conditions were unchanged.
The above replacement can achieve the purpose of the invention.
Example 3
A preparation method of a complex enzyme rich in acidic pectinase comprises the following specific steps:
1 slant culture
Inoculating Aspergillus niger strain CGMCC No.19614 to sterilized solid culture medium with first-stage test tube slant, and culturing at 25-35 deg.C for 6-10 days. The culture medium is a Chao's culture medium: 3 percent of sucrose, 0.3 percent of sodium nitrate, 0.1 percent of dipotassium phosphate, 0.05 percent of magnesium sulfate, 0.05 percent of potassium chloride, 0.001 percent of ferrous sulfate, 1.2 to 1.8 percent of agar powder and sterilization for 20min at 121 ℃.
2 triangular flask culture
Scraping the first-stage test tube slant spores obtained by the culture in the step 1 in a clean bench into a glass bead bottle filled with 150-200mL of sterile water (containing 100-500 muL of Tween-80), and shaking and mixing uniformly to enable the spores to be fully suspended in the sterile water. Then inoculating the seeds to a secondary triangular flask solid culture medium according to the solid seed amount of 1-3 mL/bottle for culture. Culturing at 28-35 deg.C for 2-4 days. The formula of the culture medium is as follows: 80-90% of bran, 10-15% of soybean meal, 1-5% of ammonium chloride and 55-70% of initial water, and sterilizing at 121 ℃ for 40min. The culture medium is 15-20g per bottle.
3 solid fermentation process
And (3) adding sterile water into the two-stage triangular flask solid culture medium seed obtained by fermentation culture in the step (2), and stirring to obtain a spore suspension, wherein the water addition amount is 300-500 mL/bottle. Inoculating the solid fermentation material according to the amount of 15-20 bottles per ton of solid material, and stirring and mixing uniformly. Filling into sterile fermentation air-permeable bags with the charging amount of 5-8 kg/bag, laying on a shallow tray, and performing fermentation culture in a koji room. During the period, temperature and humidity control treatment is carried out, the relative humidity of the koji room is controlled to be more than 50%, the material temperature is controlled to be 28-32 ℃, and fermentation culture is carried out for 4-8 days until hyphae are obvious and thick and the enzyme activity is slowly increased.
The solid fermentation medium is prepared according to the following proportion: 80-90% of bran, 5-10% of corn flour, 2-5% of soybean meal, 1-2% of ammonium sulfate, 0.05-0.1% of magnesium sulfate, 0.2-0.3% of dipotassium phosphate and 60-70% of initial water, and sterilizing at 121 ℃ for 40min.
Example 4
Results of enzyme protein assay
Drying and crushing a product obtained by the solid fermentation process provided by the company at 55-65 ℃, sieving the product (40 meshes), adding 8M urea solution into a sample to extract protein components in the sample, and identifying the protein components by using LC-MS. The method comprises the steps of quantifying protein, opening a three-dimensional structure of the protein through reductive alkylation, extracting peptide fragments after enzymolysis, obtaining mass spectrograms of the peptide fragments by using a mass spectrometry technology, and finally identifying the protein in a sample by using protein identification software. And (4) searching and comparing the obtained results in a protein library (an Aspergillus niger library), and further analyzing the results by comparison.
The results of the detection and analysis show that the fermentation product obtained by the solid fermentation process provided by the company contains abundant enzyme protein components (nearly 60). Is a compound enzyme system which takes pectinase as the main part and takes other enzymes as the auxiliary parts. The pectinase can be up to 18 kinds, and relates to the whole pectin hydrolysis process, and 11 kinds of xylanases, 11 kinds of cellulases and more kinds of other enzymes are involved. The LC-MS spectrogram and part of the enzyme protein components are as follows:
<xnotran> , A, A, B, D, F, I, A, B, D, E, B, X, C, α - , A, C, β - C, β -1,4- A, -1,4- β - B, -1,4- β - xlnD, 1,4- β - A, α -L- axhA, α -L- B, NAD (P) H D- xyl1, α - B, α - A, β - , A, A, α - A 1/2, β - O, β - A, I, α - , A, β - I, B, 1,3- β - eglC, β - A, B, β -1,4- D, 1,4- β -D- A, β - A, α - A, -1,3- β - EGLC, </xnotran> Endo-beta-1, 4-mannanase A, beta-galactosidase A, feruloyl esterase B, mannosyl oligosaccharide alpha-1, 2-mannosidase 1B, mannitol-1-phosphate 5-dehydrogenase, beta-galactosidase B, extracellular inulase, carboxypeptidase, subtilisin-like serine protease pepC, aspartic endopeptidase, catalase B, and the like.
Evaluation of the Effect of solid fermentation product application (Using the fermentation product of example 1)
The test is carried out in a large chicken farm in Zhejiang. 4000 laying hens with similar body weights and normal egg laying are selected for testing, wherein the laying hens are 47 weeks old. The test groups were randomly assigned to 2000 individuals each as a control group and a test group, and two tests were performed simultaneously. The test chickens freely eat and drink water, are raised in cages, the daily illumination time is 16h, the test is carried out strictly according to the feeding management, the chicken flock conditions are observed daily, and data are recorded.
TABLE 2 influence of the addition of acid pectinase preparation in the daily ration on the production performance of laying hens
Figure BDA0002528293860000101
Figure BDA0002528293860000111
As a result, compared with a control group, the solid fermentation product (test group) provided by the invention is added into the daily ration, the feed-egg ratio can be reduced, and the laying rate can be improved. Can also reduce the phenomenon of adhering feces and improve the intestinal health.
Sequence listing
<110> Hangzhou Baoankang biotechnology Limited
<120> preparation of complex enzyme rich in acidic pectinase, strain and application thereof
<141> 2020-06-05
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 576
<212> DNA
<213> Aspergillus niger BAK200345 (Aspergillus niger)
<400> 1
tgcggaagga tcattaccga gtgcgggtcc tttgggccca acctcccatc cgtgtctatt 60
ataccctgtt gcttcggcgg gcccgccgct tgtcggccgc cgggggggcg cctttgcccc 120
ccgggcccgt gcccgccgga gaccccaaca cgaacactgt ctgaaagcgt gcagtctgag 180
ttgattgaat gcaatcagtt aaaactttca acaatggatc tcttggttcc ggcatcgatg 240
aagaacgcag cgaaatgcga taactaatgt gaattgcaga attcagtgaa tcatcgagtc 300
tttgaacgca cattgcgccc cctggtattc cggggggcat gcctgtccga gcgtcattgc 360
tgccctcaag cccggcttgt gtgttgggtc gccgtccccc tctccggggg gacgggcccg 420
aaaggcagcg gcggcaccgc gtccgatcct cgagcgtatg gggctttgtc acatgctctg 480
taggattggc cggcgcctgc cgacgttttc caaccatttt ttccaggttg acctcggatc 540
aggtagggat acccgctgaa cttaagcata tcaata 576

Claims (7)

1. A method for preparing complex enzyme rich in acidic pectinase is characterized in that,
the strain is Aspergillus nigerAspergillus niger) BAK200345 with preservation number CGMCC No.19614;
the cultured fermentation product contains pectinase, xylanase, mannanase, acid protease, cellulase, amylase and beta-glucanase.
2. The method for preparing the complex enzyme rich in the acidic pectinase according to claim 1, wherein the preparation of the complex enzyme comprises a solid fermentation step;
inoculating the seed liquid on a solid fermentation culture medium, uniformly stirring, and performing fermentation culture in a koji room;
the solid fermentation medium is prepared according to the following proportion: 80-90% of bran, 5-10% of corn flour, 2-5% of soybean meal, 1-2% of ammonium sulfate, 0.05-0.1% of magnesium sulfate, 0.2-0.3% of dipotassium phosphate and 60-70% of initial water, and sterilizing at 121 ℃ for 40min.
3. The method for preparing the complex enzyme rich in the acidic pectinase according to claim 2, wherein the solid fermentation medium is replaced by the following formula:
80-85% of bran, 5-10% of soybean meal, 5-10% of corn flour, 1-2% of ammonium chloride, 0.05-0.1% of magnesium sulfate, 0.5-1% of calcium carbonate, 0.2-0.3% of dipotassium phosphate, 60-65% of initial moisture and sterilization at 121 ℃ for 40min;
or the like, or, alternatively,
80-90% of bran, 5-10% of soybean meal, 5-10% of palm meal, 1-2% of ammonium sulfate, 0.05-0.1% of magnesium sulfate, 0.01-0.02% of manganese sulfate, 1-2% of calcium carbonate, 0.2-0.3% of dipotassium phosphate, 60-65% of initial water and sterilization at 121 ℃ for 40min.
4. The method for preparing the complex enzyme rich in the acidic pectinase according to claim 2, wherein the temperature and humidity control treatment is carried out on the koji room, the relative humidity of the koji room is controlled to be more than 50%, and the material temperature is controlled to be 28-32 ℃; fermenting and culturing for 4-8 days until the mycelium is obvious and thick and the enzyme activity is slowly increased.
5. The method for preparing the complex enzyme rich in the acidic pectinase according to claim 2, which further comprises the steps of drying and crushing the fermentation product; wherein the drying temperature is 55-65 ℃.
6. A new kind of Aspergillus nigerAspergillus niger) Named as Aspergillus niger BAK200345, and the preservation number is CGMCC No.19614.
7. A livestock feed comprising the fermentation product of aspergillus niger BAK200345 as claimed in claim 1.
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CN111607574B (en) * 2020-06-08 2021-05-14 杭州保安康生物技术有限公司 Enzyme preparation mainly comprising xylanase and acid protease, and strain and application thereof
CN113897343A (en) * 2021-09-10 2022-01-07 常州大学 Method for producing pectinase by aspergillus niger solid-state fermentation

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CN106085881A (en) * 2016-07-12 2016-11-09 西北农林科技大学 A kind of Aspergillus niger strain and application thereof

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CN101591619A (en) * 2009-07-03 2009-12-02 中国农业科学院饲料研究所 Aspergillus niger strain and application thereof
CN103740601A (en) * 2013-12-23 2014-04-23 湖南鸿鹰生物科技有限公司 Aspergillus niger capable of highly yielding compound enzyme for feed and application thereof
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