阅读说明：本技术 富含酸性果胶酶的复合酶的制备及其菌株和应用 (Preparation of complex enzyme rich in acidic pectinase, strain and application thereof ) 是由 王云龙 吴勃 徐永雷 王天珍 刘广晓 于 2020-06-08 设计创作，主要内容包括：本发明提出了一种富含酸性果胶酶的复合酶的制备及其菌株和应用；培养的发酵产物中含有果胶酶、木聚糖酶、甘露聚糖酶、酸性蛋白酶、纤维素酶、淀粉酶以及β-葡聚糖酶。黑曲霉(Aspergillus niger),命名为黑曲霉BAK200345,其保藏号为CGMCC No.19614。本发明发酵产物提供的富含酸性果胶酶的复合酶中果胶酶种类丰富,果胶酯酶、果胶水解酶、果胶裂解酶、聚半乳糖醛酸酶、鼠李糖半乳糖醛酸酶均存在,是一个完整的果胶酶体系。酸性果胶酶酶活较高,最高可达132960U/g；该复合酶能够降低料蛋比,提高蛋鸡产蛋率,减少粘粪现象,改善肠道健康,应用效果好。(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, mannanase, 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 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. The enzyme activity of the acid 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.)

1. A preparation method of a compound enzyme rich in acidic pectinase is characterized in that,

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.

2. The preparation method of the complex enzyme rich in acidic pectinase according to claim 1, wherein the preparation method 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 40 min.

3. The preparation method of the complex enzyme rich in 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 40 min;

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 40 min.

4. The preparation method of the complex enzyme rich in 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 preparation method of the complex enzyme rich in 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. An Aspergillus niger (Aspergillus niger) is named as Aspergillus niger BAK200345, and its collection number is CGMCC No. 19614.

7. A livestock feed comprising the fermentation product of aspergillus niger BAK200345 of claim 1.

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 polymer compound formed by connecting galacturonic acid ester and a small amount of galacturonic acid through alpha-1, 4-glycosidic bonds; pectic acid is a straight chain of about 100 galacturonic acids connected by α -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 binds to cellulose, hemicellulose, lignin, proteins, etc. in plant tissues, and shows the inherent morphology of tissues in various regions.

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 high-molecular 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.

Animal somatic cells have no cell wall structure and can not secrete and produce pectinase, so the animal somatic cells are generally added from an external source in livestock and poultry production. Many research test results show that the addition of appropriate levels of pectinase to the diet can improve animal productivity 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 obvious 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 increase the amount of bone mineral and calcium and phosphorus in turkey.

At present, most of the application of pectinase is concentrated in food industry and the like, 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 the complex enzyme system 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 following steps,

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 40 min.

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 40 min;

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 40 min.

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 sterilized 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% 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 slant 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-; culturing at 25-35 deg.C and shaking table rotation speed of 160-; 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 40 min;

(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-; 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 40 min.

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 20 min; 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 20 min.

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 BAK 200345.

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 at most;

(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 description of the embodiments or the prior art will be briefly described below, and 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 exercise.

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 derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within 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 BAK 200345.

The Aspergillus niger BAK200345 provided by the invention is a high-yield complex enzyme strain obtained by strict domestication and cultivation.