阅读说明：本技术 以酸性蛋白酶为主的酶制剂的制备及其菌株和应用 (Preparation of enzyme preparation mainly containing acid protease, strain and application thereof ) 是由 王云龙 吴勃 王天珍 徐永雷 王云祥 于 2020-06-08 设计创作，主要内容包括：本发明提出了一种以酸性蛋白酶为主的酶制剂的制备及其菌株和应用,培养得到以酸性蛋白酶为主,果胶酶、木聚糖酶、淀粉酶、纤维素酶、甘露聚糖酶、葡聚糖苷酶、葡萄糖苷酶、半乳糖苷酶、阿魏酸酯酶、羧肽酶、磷酸酶多种伴生酶为辅的富含天然复杂酶类的多酶系酸性蛋白酶制剂。黑曲霉(Aspergillus niger),命名为黑曲霉BAK200389,其保藏号为CGMCC No.19613。本发明所用菌株,可一菌产多酶,具有生长稳定、产酶种类较多、安全的特性；酶活可观,日常保持在9-11万,最高可达12万左右。(The invention provides a preparation method of an enzyme preparation mainly containing acid protease, a strain and an application thereof, and the preparation method comprises the steps of culturing to obtain a multi-enzyme system acid protease preparation which is mainly containing acid protease and is supplemented with various associated enzymes such as pectinase, xylanase, amylase, cellulase, mannase, glucanase, glucosidase, galactosidase, ferulic acid esterase, carboxypeptidase and phosphatase and is rich in natural complex enzymes. Aspergillus niger (Aspergillus niger) named as Aspergillus niger BAK200389 with the preservation number of CGMCC No. 19613. The strain used by the invention can produce a plurality of enzymes by one strain, and has the characteristics of stable growth, more enzyme production types and safety; the enzyme activity is considerable, and is kept at 9-11 ten thousands daily, and the maximum can reach about 12 ten thousands.)

1. A process for preparing an enzyme preparation based on an acid protease,

the strain is Aspergillus niger BAK200389 with the preservation number of CGMCC No. 19613;

culturing to obtain a multi-enzyme system acid protease preparation which is rich in natural complex enzymes and takes acid protease as main raw materials and pectinase, xylanase, amylase, cellulase, mannase, glucanase, glucosidase, galactosidase, ferulic acid esterase, carboxypeptidase and phosphatase as auxiliary materials.

2. The preparation of an acid protease-based enzyme preparation according to claim 1, wherein the preparation of the enzyme preparation comprises: first-stage slant culture, second-stage liquid seed preparation, third-stage liquid seed preparation and first-stage solid culture of amplification culture.

3. The process for producing an acid protease-based enzyme preparation according to claim 2, wherein (1) the primary slant culture

Inoculating Aspergillus niger BAK200389 to a potato juice glucose slant culture medium, and culturing at 28-31 ℃ for 5-7 days to obtain a slant of germinated spores;

(2) secondary liquid seed preparation

Inoculating the spore and hypha mixture on the inclined plane in the step (1) to a liquid culture medium in a shake flask, and performing shake culture on a shaking table; stopping culturing after the seed bacterial liquid in the bottle is viscous, and refrigerating and storing;

(3) three stage liquid seed preparation

Opening the shake flask cultured in the step (2), taking the liquid seeds to inoculate the liquid culture medium in the third-stage shake flask, and performing shake culture on a shaking table;

(4) expanding culture

Injecting a liquid culture medium into the fermentation tank, and performing high-pressure steam sterilization for 60-80 min; stopping sterilization, and then cooling to 20-35 ℃ and keeping; taking the cultured liquid seeds in the step (3), quickly pouring the seeds into a fermentation tank through a sterilized fermentation tank opening, introducing sterile air, and stirring for culture;

(5) inoculation culture of solid culture medium

Putting the liquid culture medium cultured in the step (4) into a solid fermentation culture medium according to the weight ratio of 5-15%, uniformly mixing the solid fermentation culture medium, and standing for fermentation culture; the relative humidity is 80-90%, the material temperature is 28-35 ℃, and the fermentation period is 3-5 d;

the solid fermentation culture medium is prepared according to the following proportion: 85-95% of bran, 5-15% of soybean meal, 0.5-1.5% of ammonium sulfate, 40-55% of initial water and natural pH.

4. The preparation of an acid protease-based enzyme preparation according to claim 3, wherein the liquid medium is prepared in the following weight ratio (%): 4.0-8.0% of maltodextrin, 2.0-3.5% of glucose, 0.5-2.0% of peptone and 1.0-3.0% of fine bran, and the pH is natural;

shaking table shake culture conditions: the temperature is 28-30 ℃, the rotating speed is 180-220 r, and the culture period is 1-3 d; the sterilization requirements of the solid fermentation culture medium are as follows: at the temperature of 121 ℃, 0.1MPa, for 40-60 min; the conditions of refrigeration preservation are as follows: 4 ℃, 5-8 days;

the requirements of the fermenter culture: stirring at the temperature of 27-32 ℃ and the rotating speed of 150-200 r, and culturing for 1-3 d; the sterilization requirements of the fermentation tank are as follows: 60-80 min at 121 ℃ and 0.1 MPa;

the sterilization requirement of the tank opening of the fermentation tank is as follows: firing the alcohol fire ring for 3-5 min before inoculation operation, keeping the fire not extinguished during the inoculation period, closing the pot opening after the inoculation is finished, and evacuating and extinguishing the fire ring.

5. The preparation method of an enzyme preparation mainly comprising acid protease as claimed in claim 3, further comprising the steps of drying and pulverizing the fermentation product; wherein, drying is stopped until the water content is 5-15%.

6. An Aspergillus niger (Aspergillus niger) is named as Aspergillus niger BAK200389 with the preservation number of CGMCCNo.19613.

7. Livestock feed, characterized in that it comprises the culture product of Aspergillus niger BAK200389 of claim 1.

Technical Field

The invention relates to the field of enzyme preparations, in particular to preparation of an enzyme preparation mainly containing acid protease, and a strain and application thereof.

Background

With the increasing demand of poultry products, the importance of the breeding industry to people is increased year by year, and higher requirements on the safety, high efficiency and environmental protection of poultry and livestock breeding are provided. "birds eat as the day", the feed is regarded as one of the key links of the feeding industry, and is highly regarded. In recent years, the use of antibiotics is more and more strictly controlled, and China enters the 'banning' era in 2020, and the antibiotics in the regulation are prohibited from being detected in the feed additive. Under the two trends, the quality optimization heat tide of products such as the preparation process of the feed, additives and the like is directly promoted, wherein the products comprise enzyme preparations mainly comprising acid protease. Patent CN 110973371A discloses a feed additive of animal and poultry white enzyme, which is prepared by acid protease as one of main components according to percentage. Patent CN 109965084 a discloses a method for producing high protein feed by solid fermentation of soybean meal, wherein the solid fermentation is performed by using fermentation raw material added with acid protease.

The acidic protease is a branch of a protease, is an enzyme which can rapidly hydrolyze macromolecular protein into peptides and partial free amino acids under an acidic condition (pH value is 2-5), and the active center of a catalytic structure domain is aspartic protease. The relative molecular weight is 30-45 kDa, and the isoelectric point is 3.0-5.0. The acidic protease is mainly derived from animal viscera and microbial secretion, and comprises pepsin, chymosin and some microbial proteases. The microbial protease has the obvious characteristics of diversity and complexity, one strain can secrete one or more acid proteases, and main production strains comprise aspergillus niger, aspergillus usamii and the like. The good protein hydrolysis capability and acid resistance, so that the acid protease has wide application space, including food, medicine, light industry, leather technology and feed processing industry. The revolution of the feed field plays a positive role in promoting the application and development of the acid protease in the feed field.

In the feed industry, the addition of the acid protease can effectively improve the utilization rate of protein nutrient substances in the feed, mainly the digestive secretion of young animals is not perfect, and the addition of the acid protease can degrade macromolecular proteins into polypeptide substances, thereby being beneficial to improving the digestive absorption, reducing the stimulation of the feed to the digestive tract of the feed and reducing the nutrition disorder. The acidic protease has the growth promoting effect on livestock and poultry, so that the acidic protease becomes one of good substitutes of antibiotics. Guo Jianlai et al (2007) found that adding 0.1% acid protease to piglet diet significantly improved piglet growth performance and significantly reduced diarrhea rate.

In the research and analysis direction, the technologies of separation and purification, analytical identification, enzymology property and the like of the acid protease are increasingly mature. Xishufeng et al (2007) separated and purified Aspergillus niger acidogenic protease by ammonium sulfate salting-out and ion exchange chromatography, and analyzed the amino acid component of the enzyme. The protein produced in Aspergillus niger fermentation liquor is analyzed and compared by Wangyun (2008) through a mass spectrum fingerprint method, and the enzyme is researched in the aspect of molecular biology after being identified as Aspergillus acidic protease.

In the aspect of optimizing the productivity, the improvement of the activity and the adaptability of the acid protease is the most main promotion point in the optimization of the acid protease, and the improvement of strains and culture conditions becomes the most common optimization mode. Aqua regia et al (2002) obtain a compound enzyme high-yield strain SL2-III by a method of ultraviolet ray and nitrosoguanidine compound mutagenesis, on the basis, the solid fermentation process of a variant SL2-III is optimized by adopting single-factor search and orthogonal test by taking acid protease fermentation enzyme activity as a response value, and the enzyme activity reaches 6428U/g after optimization. The patent CN 107760612B discloses that Aspergillus niger yy07(Aspergillus niger y07) strain for producing acid protease is screened by taking Aspergillus niger as an original strain and adopting Ultraviolet (UV) and Nitrosoguanidine (NTG) combined mutagenesis technology, and the highest enzyme activity of the strain for producing the feeding acid protease by solid fermentation reaches 1646U/g. In patent CN 107586789A, a genetic engineering method for constructing an expression cassette of acid protease, introducing aspergillus niger expression host bacteria, and obtaining aspergillus niger expression strain with high yield of acid protease is disclosed. After the liquid fermentation condition of the transformed strain is optimized, the expression level of the acid protease can reach 25000-26000U/ml. CN 105199969B discloses a liquid fermentation acid protease which is prepared by liquid submerged fermentation with the feed supplement process, and the enzyme production level is 21852U/mL. CN 101948820A discloses an acid protease and a preparation method thereof, Aspergillus niger CICC2238 is used as an enzyme-producing strain, wheat bran is used as a main raw material, the optimum pH value of the acid protease produced from the wheat bran is 2.5-3.5, the optimum action temperature is 40-50 ℃, the enzyme activity of the acid protease prepared by a solid fermentation process is more than or equal to 47000U/g, the liquid enzyme yield is more than or equal to 85%, and the solid enzyme yield is more than or equal to 80%. In patent CN 110904083A, solid fermentation is used to produce acid protease, the enzyme activity of the acid protease conventionally reaches 7 ten thousand U/g, and the enzyme activity of the acid protease produced after process design optimization is 8-10 ten thousand U/g.

The huge market of enzyme preparations promotes the appearance of various enzyme preparation products. In the large category of acid protease enzyme preparations, most of the enzyme preparations are single acid protein preparations obtained by separation and purification after liquid submerged fermentation; the enzyme is a combined protease (part of products are called as compound enzyme) which is formed by mixing pure enzymes with single components such as acid protease, neutral protease, alkaline protease and the like according to the mixture ratio of percentage and the like; one or two kinds of protease are main enzyme, pectase, xylanase, etc. in the course of fermentation and metabolism, several enzyme systems of several dozen or hundreds of coenzymes are naturally produced and can be used for making synergistic action on substrate to form composite enzyme, and the product obtained by general solid fermentation is the true composite enzyme type protease. Corresponding enzyme preparations are selected for different purposes.

In the aspects of environmental protection and economic benefit, with the development of intensive livestock and poultry breeding industry, the emission of nitrogen of bred animals becomes a main substance polluting the environment. Therefore, the utilization efficiency of protein nutrient substances in the feed is improved, and the reduction of nitrogen emission is very important. After comprehensive analysis of Qiaofin and the like (2017), the results show that after the acidic protease is applied, low-protein fermentation raw materials such as bran, straws and the like are well digested and degraded, the utilization rate of nitrogen and phosphorus in livestock is improved, the discharge amount is reduced, and the environment-friendly degree is improved. The feed conversion ratio of the raised livestock is reduced, the health condition is improved, the yield is increased, and the economic benefit is steadily increased. Danish and Swiss researchers have studied the influence of adding protease to broiler ration to reduce the protein content of the ration on improving the environment, and found that the use of protease can significantly reduce nitrogen emission in livestock production.

According to the research progress of the acid protease, the current acid protease has good development trend, large market demand and gradually optimized production technology. However, the following problems still exist according to analysis:

1. in terms of application effect and optimization process, the enzyme activity of the acid protease still has a larger rising space, and the enzymolysis efficiency can be further improved.

2. The enzyme protein detection method of the enzyme preparation is mature, but the application is relatively single, and the analysis is usually carried out on a certain protein. The method is not suitable for treating solid fermentation products with more protein components and high difficulty in separation and purification, and a more suitable method needs to be found.

3. At present, no report of system research on an enzyme system of the solid fermentation acid protease is found, and unclear extensive production has adverse effects on subsequent deep research on strains, multi-enzyme interaction, widening of an optimized fermentation process idea and the like.

4. Raw materials based on fermented feed tend to agricultural residues such as bran, straws, cottonseed meal and the like more and more, and an enzyme system in an enzyme preparation needs to correspond to a complex structure of the fermented raw materials so as to achieve a good enzymolysis effect. The single enzyme or the combined enzyme and enzyme system has poor correspondence with the complex structure of the fermentation raw material, and the enzymolysis efficiency is inhibited.

5. The liquid fermentation process is mature and controllable, and is a feasible fermentation process for producing the enzyme preparation. However, the subsequent post-treatment processes such as separation, purification, freeze drying and the like consume much energy, have higher requirements on equipment and have high requirements on the cost of manufacturers. And the liquid fermentation product is less than the solid fermentation product, and the enzyme system is richer than the solid fermentation product.

Disclosure of Invention

The invention provides preparation of an enzyme preparation mainly containing acid protease, a bacterial strain and application thereof, and aims to solve the technical problems.

The technical scheme of the invention is realized as follows:

a process for preparing an enzyme preparation based on an acid protease, wherein,

the strain is Aspergillus niger BAK200389 with the preservation number of CGMCC No. 19613;

culturing to obtain a multi-enzyme system acid protease preparation which is rich in natural complex enzymes and takes acid protease as main raw materials and pectinase, xylanase, amylase, cellulase, mannase, glucanase, glucosidase, galactosidase, ferulic acid esterase, carboxypeptidase and phosphatase as auxiliary materials.

In some embodiments, the preparation of the enzyme preparation comprises: first-stage slant culture, second-stage liquid seed preparation, third-stage liquid seed preparation and first-stage solid culture of enlarged culture;

wherein (1) first-stage slant culture

Inoculating Aspergillus niger BAK200389 to a potato juice glucose slant culture medium, and culturing at 28-31 ℃ for 5-7 days to obtain a slant of germinated spores;

(2) secondary liquid seed preparation

Inoculating the spore and hypha mixture on the inclined plane in the step (1) to a liquid culture medium in a shake flask, and performing shake culture on a shaking table; stopping culturing after the seed bacterial liquid in the bottle is viscous, and refrigerating and storing;

(3) three stage liquid seed preparation

Opening the shake flask cultured in the step (2), taking the liquid seeds to inoculate the liquid culture medium in the third-stage shake flask, and performing shake culture on a shaking table;

(4) expanding culture

Injecting a liquid culture medium into the fermentation tank, and performing high-pressure steam sterilization for 60-80 min; stopping sterilization, and then cooling to 20-35 ℃ and keeping; taking the cultured liquid seeds in the step (3), quickly pouring the seeds into a fermentation tank through a sterilized fermentation tank opening, introducing sterile air, and stirring for culture;

(5) inoculation culture of solid culture medium

Putting the liquid culture medium cultured in the step (4) into a solid fermentation culture medium according to the weight ratio of 5-15%, uniformly mixing the solid fermentation culture medium, and standing for fermentation culture; the relative humidity is 80-90%, the material temperature is 28-35 ℃, and the fermentation period is 3-5 d;

the solid fermentation culture medium is prepared according to the following proportion: 85-95% of bran, 5-15% of soybean meal, 0.5-1.5% of ammonium sulfate, 40-55% of initial water and natural pH.

In some embodiments, the liquid medium is formulated in the following weight proportions (%): 4.0-8.0% of maltodextrin, 2.0-3.5% of glucose, 0.5-2.0% of peptone and 1.0-3.0% of fine bran, and the pH is natural.

In some embodiments, the shake culture conditions are: the temperature is 28-30 ℃, the rotating speed is 180-220 r, and the culture period is 1-3 d; the sterilization requirements of the solid fermentation culture medium are as follows: at the temperature of 121 ℃, 0.1MPa, for 40-60 min; the conditions of refrigeration preservation are as follows: 4 ℃ and 5-8 days.

In some embodiments, the requirements of the fermentor culture: stirring at the temperature of 27-32 ℃ and the rotating speed of 150-200 r, and culturing for 1-3 d; the sterilization requirements of the fermentation tank are as follows: 60-80 min at 121 ℃ and 0.1 MPa;

the sterilization requirement of the tank opening of the fermentation tank is as follows: firing the alcohol fire ring for 3-5 min before inoculation operation, keeping the fire not extinguished during the inoculation period, closing the pot opening after the inoculation is finished, and evacuating and extinguishing the fire ring.

In some embodiments, the method further comprises a drying step and a crushing step of the fermentation product; wherein, drying is stopped until the water content is 5-15%.

The invention also provides an Aspergillus niger (Aspergillus niger) named as Aspergillus niger BAK200389 with the preservation number of CGMCC No. 19613.

The invention also provides a livestock and poultry feed which comprises a culture product of the Aspergillus niger BAK 200389.

Compared with the prior art, the invention has the following beneficial effects:

(1) the enzymes produced by the invention have good supplement and cooperation effects, and have important synergistic effect on cracking macromolecular chemical bonds in plant cell walls, loosening compact and ordered spatial structures and generating various beneficial metabolites.

(2) The strain used by the invention can produce a plurality of enzymes by one strain, and has the characteristics of stable growth, more enzyme production types and safety.

(3) The acid protease enzyme preparation provided by the invention has more pertinence in enzyme production through specific substrate induction, and has 7 major classes of enzyme proteins and 38 enzyme proteins. Representative enzyme systems include: acid protease, pectinase, xylanase, amylase, cellulase, mannanase, glucanase, glucosidase, galactosidase, feruloyl esterase, carboxypeptidase, phosphatase, and the like.

(4) The multi-enzyme acidic protease provided by the invention has considerable enzyme activity. According to the detection of related national standards, the daily maintenance is 9-11 ten thousands, the maximum can reach about 12 ten thousands, and the activity is higher than the enzyme activity of the known feed acid protease production. The acid protease sold in the market at present is prepared by liquid fermentation, concentration and drying, the conventional enzyme activity is 50000U/g, a small part of the enzyme activity is 10 ten thousand U/g or 20 ten thousand U/g, and the enzyme activity of the solid fermentation acid protease is about 3-5 ten thousand U/g.

(5) In the invention, a combined mode of modern solid fermentation process and proteomics detection theory is adopted, and the production process under the guidance of the modern solid fermentation theory is taken as a basis: the specific substrate is induced and fermented, the fermentation is carried out through the standing of a breathing bag, and the culture parameters are strictly controlled during the fermentation; the study was performed with proteomics detection methods: all enzyme proteins and metabolites beneficial to livestock in the enzyme preparation are detected efficiently, various optimization of solid fermentation can be adjusted in a targeted manner according to detection results, and the enzyme proteins and the metabolites supplement each other.

Compared with the traditional solid-liquid fermentation process and the detection method for detecting single or a small amount of protease, the method has the advantages of simpler operation, improved hygienic indexes, more comprehensive and deep research direction, more definite functions and more direct guidance for optimizing the subsequent aspects.

(6) The multi-enzyme acidic protease provided by the invention has strong stability, and can avoid enzyme activity loss in the processes of transportation, processing and storage.

(7) The multi-enzyme acidic protease preparation provided by the invention can be used as a feed additive to be added into basic ration of most livestock, including but not limited to chicken, duck, pig, rabbit and other livestock. Has effects in stimulating immunity and improving health level.

(8) The multienzyme acidic protease preparation provided by the invention can effectively promote the improvement of the production performance of livestock, reduce the breeding cost and increase the income.

(9) The acid protease process disclosed by the invention is environment-friendly, low in energy consumption, low in cost and simple in process, and utilizes agricultural and industrial residues as production raw materials, so that the process is more environment-friendly than other fermentation modes.

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.

Aspergillus niger BAK200389, deposited in China general microbiological culture Collection center (CGMCC; China microbiological research institute, national institute of microorganisms No. 3, national institute of Western Lu 1, Beijing, Chaoyang, Ltd.) No. 4/8 of 2020, 4/8, with the deposition number: CGMCC No. 19613.

And ITS identification:

the result of alignment of the ITSrDNA segment sequences (shown in the sequence table) is as follows. As can be seen from the results, the gene sequence of the strain has the highest similarity with the Aspergillus niger, so the strain is named as Aspergillus niger CGMCC No. 19613.