CN1651569A - A kind of aspergillus niger strain and its application - Google Patents

Abstract

The invention relates to an Aspergillus niger strain (Aspergillus niger) MAFIC-005, and its preservation number is CGMCC No.1067. The strain is obtained by screening and isolating a soil sample of a corn field in Wuxi as a starting strain, and is obtained through multiple mutagenesis selections. The aspergillus niger strain provided by the invention can produce xylanase, beta-glucanase, cellulase and pectinase at the same time in solid-state fermentation to produce compound enzymes for feed. The solid-state fermentation composite enzyme can be widely used in various livestock and poultry feeds, especially in ruminant feeds. It not only improves the economic benefits, but also protects the ecological environment and promotes the sustainable and healthy development of my country's animal husbandry.

Description

A kind of aspergillus niger strain and its application

technical field

The invention relates to a microorganism and its application, in particular to an Aspergillus niger strain (Aspergillus niger) and its application for solid-state fermentation to produce a compound enzyme for feed.

technical background

In recent decades, due to the rapid increase of population and the continuous improvement of living standards, the demand for meat, eggs and milk has also increased year by year. Feed is the food of livestock and poultry. How to improve the utilization rate of feed, how to protect the environment and ensure the sustainable, healthy and stable development of agriculture and animal husbandry have become important problems that human beings need to solve urgently. The feed compound enzyme produced by microbial fermentation is a kind of non-toxic, harmless, and pollution-free green feed additive, in which non-starch polysaccharase (mainly including xylanase, glucanase, cellulase, and mannanase) are important components of feed enzymes. Adding an appropriate amount of non-starch polysaccharases to feeds with high crude fiber content can significantly improve feed utilization and breeding benefits, and at the same time reduce environmental pollution.

The key to the production of compound enzymes for feed by microbial fermentation is: in addition to selecting the appropriate culture conditions to make the strains exert the best production performance, it is also necessary to screen out the strains that can obtain high-yield enzymes.

Contents of the invention

The object of the present invention is to provide a kind of Aspergillus niger strain capable of high-yield compound enzyme for feed.

Another object of the present invention is to provide the use of the Aspergillus niger strain in the production of compound enzymes for feed by solid-state fermentation.

The purpose of the present invention is achieved through the following technical solutions:

The Aspergillus niger strain (Aspergillus niger) MAFIC-005 provided by the present invention was deposited in "General Microorganism Center of China Microbiological Culture Collection Management Committee" on December 8, 2003, and its preservation number is CGMCCNo.1067.

Aspergillus niger strain (CGMCC No.1067) MAFIC-005 provided by the invention has the following microbiological characteristics:

1. Morphological characteristics:

The colony grows rapidly on Chase's medium, with a diameter of 45-60 mm at 25°C for 6 days; the texture is velvety or slightly flocculent; the conidia are numerous, brown-black, without exudate; the reverse side of the colony is slightly yellow.

2. Characteristics of culture:

Conidia heads spherical to radial, 150-450 microns in diameter; conidiophores occur in the stroma, spore stems (1000-3000) microns × (12-20) microns, yellow or yellowish-brown, with smooth walls; apical capsule Spherical or nearly spherical, 40-70 microns in diameter, fertile on the entire surface; sporulation structure double-layered, stem base (10-20) microns × (4.5-7.0) microns, phialide (6-9) microns × (2.5- 3.0) microns, conidia spherical or nearly spherical, diameter 3.0-4.5 microns, rough wall.

The method for using the Aspergillus niger bacterial strain of the present invention to be used for solid-state fermentation to produce feed compound enzyme comprises the steps:

1) Preparation of culture medium: prepare culture medium according to the following weight ratios (calculated as dry matter): 85.0% of wheat bran; 2.0% of sugar beet pulp; 2.0% of apple peel powder; 4.0% of ammonium sulfate; %; potassium dihydrogen phosphate 1.0%;

2) Preparation of koji: Put 50 g of the koji of the above medium with a water content of 50% in a conical flask (500ml capacity) and sterilize at 121°C for 40 to 50 minutes, and inoculate 2ml of koji containing 2 to 8×10 ⁸ after cooling. The spore liquid of the Aspergillus niger bacterial strain of the present invention of each/ml, the bottle mouth is sealed with 16 layers of sterile medical gauze, and cultivated at a constant temperature of 28～32°C for 4 days.

3) solid-state fermentation: the koji of the above-mentioned medium with a water content of 50-65% is sterilized at 110-121°C for 40-150 minutes; after cooling, 0.5-2.0% of the koji (calculated by weight) is inoculated, Cultivate for 3-4 days at 24-32 DEG C and a relative humidity above 85%, and then air-dry at 45-50 DEG C for 24-48 hours to obtain a fermentation product—a compound enzyme for feed.

The Aspergillus niger strain provided by the invention can simultaneously high-yield xylanase, beta-glucanase, cellulase and pectinase in the production of compound enzymes for feed by solid-state fermentation. The solid-state fermentation composite enzyme can be widely used in various livestock and poultry feeds, especially in ruminant feeds. It not only improves the economic benefits, but also protects the ecological environment and promotes the sustainable and healthy development of my country's animal husbandry.

Detailed ways

Embodiment 1, the acquisition of Aspergillus niger strain (Aspergillus niger) MAFIC-005

The mutagenesis selection process of the aspergillus niger bacterial strain (Aspergillus niger) MAFIC-005 provided by the present invention is:

1. Screening of starting strains

Prepare solid potato agar medium, sterilize it at 121°C for 40 minutes, pour it into a sterile plate with a diameter of 9 cm, and set it aside after solidification; the solid potato agar medium is prepared according to the following ratio : Peeled potato 100g, pectin 10g, carmellose sodium 10g, ammonium sulfate 5g, distilled water 1000ml, penicillin 10ppm, pH 6.7～7.0.

Take 1.0 g of soil sample from a corn field in Wuxi, dilute it with sterile water, and prepare bacterial suspensions with concentrations of 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 , 10 ^-6 , and 10 ^-7 . Spread evenly on the solid potato agar medium respectively, and spread 0.5ml evenly in each plate. After constant temperature cultivation at 28-30°C for 6 days, a strain of Aspergillus niger was isolated on a 10 ^-5 dilution solid medium plate as the starting strain for subsequent mutagenesis screening.

2. Mutation breeding

Mutation screening and solid-state fermentation of the starting strains were carried out to analyze and measure their production performance, mainly to analyze the production of xylanase and cellulase.

Mutagenesis selection process:

Starting strain→UV mutagenesis→γ-ray mutagenesis→NTG mutagenesis

After each mutagenesis, carry out plate separation and screening, select 20-30 single colonies, do a solid-state fermentation test in a Erlenmeyer flask, and measure the enzyme production capacity. The production of xylanase and glucanase is used as the main detection index, and 2-30 colonies are selected. Three strains with high enzyme activity production were used as high-yield strains for repeated experiments, and a strain with the highest yield was selected as the starting strain for the next stage of mutagenesis.

The power of the ultraviolet lamp is 15W, the irradiation distance is 20cm, and the irradiation time is 10 minutes. The spore bacterial suspension is prepared with sterile physiological saline, the spore concentration of the bacterial suspension is 10 ⁶ -10 ⁷ spores/ml, and the thickness of the liquid layer is 0.3 - 0.5 cm. After irradiation, culture at 28-30°C for 8 hours in the enrichment solution (2 grams of pectin, 2 grams of sodium carboxymethyl cellulose, 2 grams of ammonium sulfate, 1 gram of yeast powder, 1000 ml of distilled water, pH 6.8-7.0) , and then diluted and spread on potato solid agar medium, and cultivated at a constant temperature of 28-30°C for 6 days. Select a single colony, conduct solid-state fermentation in a triangular flask, measure production performance, and select a high-yielding strain as the starting strain for the next step of mutagenesis.

The gamma rays come from Co ⁶⁰ , the spore concentration of the bacterial suspension to be treated is 10 ⁵ -10 ⁶ spores/ml, and the irradiation dose is 600-800 Curies. After irradiation, it is cultured statically at 28-30°C for 8 hours in the enrichment solution, then diluted and spread on the potato solid agar medium, and cultivated at a constant temperature of 28-30°C for 6 days. Select a single colony, conduct solid-state fermentation in a triangular flask, measure production performance, and select a high-yielding strain as the starting strain for the next step of mutagenesis.

The concentration of the NTG mutagen is 300-500ppm, the spore concentration of the bacterial suspension to be treated is ^105-106 /ml, the action time is ^20-30 minutes, and the action temperature is 35-40°C. The mutated spore liquid is cultured statically at 28-30°C for 8 hours in the enrichment liquid, then diluted and spread on the potato solid agar medium, and cultured at a constant temperature of 28-30°C for 6 days. Select a single colony, conduct solid-state fermentation in a triangular flask, measure production performance, and select a high-yielding strain as the starting strain for the next step of mutagenesis.

The entire mutagenesis cycle was carried out for 4 rounds, and finally a non-starch polysaccharase high-producing strain with relatively stable genetic properties was obtained. It was identified as Aspergillus niger (Aspergillus niger) MAFIC-005 by the Institute of Microbiology, Chinese Academy of Sciences, and was deposited in the "General Microbiology Center of the Chinese Microbiological Culture Collection Management Committee" on December 8, 2003, and its preservation number is CGMCC No.1067.

The obtained Aspergillus niger strain (CGMCC No.1067) MAFIC-005 has the following microbiological properties:

1. Morphological characteristics:

The colony grows rapidly on Chase's medium, with a diameter of 45-60 mm at 25°C for 6 days; the texture is velvety or slightly flocculent; the conidia are numerous, brown-black, without exudate; the reverse side of the colony is slightly yellow.

2. Characteristics of culture:

Conidia heads spherical to radial, 150-450 microns in diameter; conidiophores occur in the stroma, spore stems (1000-3000) microns × (12-20) microns, yellow or yellowish-brown, with smooth walls; apical capsule Spherical or nearly spherical, 40-70 microns in diameter, fertile on the entire surface; sporulation structure double-layered, stem base (10-20) microns × (4.5-7.0) microns, phialide (6-9) microns × (2.5- 3.0) microns, conidia spherical or nearly spherical, diameter 3.0-4.5 microns, rough wall.

Embodiment 2, cultivate kind of song in Erlenmeyer flask

Medium ratio (dry matter calculation): 85.0% of wheat bran; 2.0% of beet pulp; 2.0% of apple peel powder; 4.0% of ammonium sulfate; 4.0% of cotton meal powder;

The water content of the koji is 50.0%.

The koji is divided into 500ml triangular flasks, and the charging amount is 50 grams/bottle. Sterilize and sterilize at 121 ℃ for 40 minutes, inoculate the spore liquid (2×10 ⁸ /ml, every bottle inoculates 2ml) of slant strain after cooling. The bottle mouth was sealed with 16 layers of sterile medical gauze, and cultured at a constant temperature of 32°C for 4 days to obtain the seed koji.

Embodiment 3, in Erlenmeyer flask, solid-state fermentation produces feed compound enzyme

The ratio of the medium is the same as in Example 2, and the water content of the koji is 50%.

The koji is divided into 500ml triangular flasks, and the charging amount is 50 grams/bottle. Sterilize at 115°C for 80 minutes, inoculate 1.0 g of the koji cultured in Example 2 after cooling, cultivate for 4 days at 32°C and a relative humidity above 85%, and then air-dry at 48°C for 24 hours; The fermented product—composite enzyme for feed was obtained, the water content of the dry koji was below 8.0%, crushed through a 60-mesh sieve, and the enzyme activity of the sample was measured. See Table 1 for the results.

Embodiment 4, solid-state fermentation production feed compound enzyme in tray

The ratio of the medium is the same as in Example 2, and the water content of the koji is 60%.

The koji was sterilized at 121° C. for 50 minutes, cooled and inoculated with the koji cultivated in Example 2, and every 1000 gram of koji (calculated as dry matter) was inoculated with the koji cultivated in 5 g of Example 2. Mix evenly, pack in trays of 20×20cm ² , the thickness of the koji is 3.0-3.5cm. Then cultivate at a constant temperature of 28° C. for 72 hours, and the relative humidity of the air in the incubator is controlled above 85%. At the end of the cultivation, it was air-dried at 45°C for 48 hours to obtain a fermented product—a compound enzyme for feed. The water content of the dry koji was below 9.0%, and it was crushed through a 60-mesh sieve to measure the enzyme activity of the sample. See Table 1 for the results.

Embodiment 5, pilot-scale production of compound enzyme for feed in solid-state fermentation in tray

The ratio of the culture medium is the same as in Example 2, the feeding amount is 300kg/batch, and the initial water content of the koji is 65%.

The koji was sterilized at 110° C. for 150 minutes, and inoculated with the koji cultivated in 3000 g of Example 2 after cooling. Mix evenly, pack in trays of 40×60cm ² , the thickness of the koji is 3.5-4.0cm. The trays are scattered on the brackets with a vertical spacing of 20-25cm. During the cultivation process, the relative humidity of the air in the bent room is controlled at more than 85%. After 84 hours of constant temperature cultivation at 24°C, it is transferred to a drying room, and air-dried at 50°C for 48 hours to obtain a fermentation product—a compound enzyme for feed. The water content of the koji is below 9.0%, crushed through a 60-mesh sieve, and the enzyme activity is determined. The results are shown in Table 1.

Enzyme Activity Definition and Measurement Results

Various enzyme activities are defined as:

Xylanase activity: at 40°C and pH 5.5, the amount of enzyme needed to degrade and release 1 μmol of reducing sugar from a xylan (SigmaX0627) solution with a concentration of 5 mg/ml per minute is an enzyme activity unit u .

β-glucanase activity: at 40°C and pH 5.5, the amount of enzyme required to degrade and release 1 μmol of reducing sugar from a β-glucan (Sigma G6513) solution with a concentration of 4 mg/ml per minute is A unit of enzymatic activity u.

Cellulase activity: at 40°C and pH 5.5, the amount of enzyme required to degrade and release 1 μmol of reducing sugar from a solution of sodium carboxymethylcellulose (Sigma C5678) with a concentration of 4 mg/ml per minute is one enzyme Live unit u.

Pectinase activity: at 40°C and pH5.5, the amount of enzyme required to degrade and release 1 μmol of reducing sugar from a polygalactose (SigmaP9135) solution with a concentration of 4 mg/ml per minute is an enzyme activity unit u.

Table 1: Determination results of product enzyme activity

Enzyme activity (u/g) Erlenmeyer flask culture Incubator tray culture Pilot tray fermentation

Xylanase 6270 6402 5423

β-glucanase 3750 3812 3245

Cellulase 61.2 64.3 46.5

Pectinase 142 165 125

As can be seen from Table 1, the compound enzyme for feed that adopts the Aspergillus niger strain provided by the invention to carry out solid-state fermentation to obtain contains multiple non-polysaccharide amylase activities, and under pilot production conditions, the content of xylanase in every gram of dry koji The activity can reach 5423u, the activity of β-glucanase can reach 3245u, the activity of cellulase can reach 46.5u, and the activity of pectinase can reach 125u. The production cost of the compound enzyme for feeding by the method is low, less than 8 yuan per kilogram, which improves economic benefits, protects the ecological environment, and promotes the sustainable and healthy development of animal husbandry in my country.

Claims (3)

1. A kind of Aspergillus niger MAFIC-005, which was deposited on December 8, 2003 in the "General Microorganism Center of China Committee for the Collection of Microorganisms", and its preservation number is CGMCC No.1067. 2. The Aspergillus niger bacterial strain as claimed in claim 1, which has the following microbiological properties: 1) Morphological characteristics: The colony grows rapidly on Chase medium, with a diameter of 45-60 mm at 25°C for 6 days; the texture is velvety or slightly flocculent; the structure of conidia is large, brown-black, without exudate; the reverse side of the colony is slightly yellow; 2) Characteristics of culture: Conidia heads spherical to radial, 150-450 microns in diameter; conidiophores occur in the stroma, spore stems (1000-3000) microns × (12-20) microns, yellow or yellowish-brown, with smooth walls; apical capsule Spherical or nearly spherical, 40-70 microns in diameter, fertile on the entire surface; sporulation structure double-layered, stem base (10-20) microns × (4.5-7.0) microns, phialide (6-9) microns × (2.5- 3.0) microns, conidia spherical or nearly spherical, diameter 3.0-4.5 microns, rough wall. 3. An application of the Aspergillus niger strain according to claim 1 in the production of compound enzymes for feed by solid-state fermentation.