CN114410486B - Aspergillus oryzae strain and application thereof in development of feed protein - Google Patents

Abstract

The invention belongs to the technical field of application of fermented meal feeds and single-cell protein sources for producing low-value raw materials, and relates to an Aspergillus oryzae strain and its application. In the present invention, Aspergillus oryzae is a wild strain isolated from rot wood microorganisms, and the ability of degrading biomass materials is improved through ARTP mutagenesis to obtain an Aspergillus oryzae strain A02, which is fermented in a liquid shake flask for 5 days, and its The cellulase activity can reach 12IU/ml, and the xylanase activity can reach 210IU/ml. The invention also provides a method for producing single-cell protein by mixed solid-state fermentation of Aspergillus oryzae A02 and Aspergillus niger 60B-3DW, which greatly improves the quality and nutritional value of meal feed. Therefore, the present invention can be applied industrially and has a good industrialization prospect.

Description

A strain of Aspergillus oryzae and its application in the development of feed protein

technical field

The invention belongs to the technical field of application of fermented meal feed and single-cell protein sources for producing low-value raw materials, and relates to an Aspergillus oryzae strain and its application.

Background technique

Insufficient supply of protein feed raw materials in my country coexists with the low utilization rate and large waste of traditional feed raw materials. On the one hand, my country's protein feed self-sufficiency rate is less than 50%, the dependence on foreign imports exceeds 80%, and the situation of "human and animal competition for food" and "subject to people" is becoming more and more serious. Very urgent; use biosynthesis technology to convert agricultural waste into unconventional protein raw materials, subverting the traditional agricultural protein production model. Feed protein derived from low-value raw materials can compete with soybean meal feed protein in many aspects such as protein utilization, nutritional function, and comprehensive cost, and realize the substitution of traditional agricultural protein.

On the other hand, the utilization rate of existing protein feed resources is not high, mainly due to the low protein processing technology in the current industry, resulting in a large amount of protein waste. Meal is a by-product after oil extraction and is the most important feed protein resource. The common ones are soybean meal, cotton meal, peanut meal, etc., which are rich in protein and have a reasonable distribution of amino acids, and are commonly used vegetable protein raw materials in animal diets. However, the crude fiber content of meal feed protein resources is high, and there are anti-nutritional factors such as xylan, cellulose, β-glucan, etc., which interfere with the digestion and absorption of dietary nutrients, reduce the nutritional value of feed, and lead to the utilization of feed protein raw materials. declining rates and high animal morbidity. And the amino acid composition of meal feed protein is not as ideal as that of animal-derived protein feed. It is an effective way to improve the quality of meal protein feed and tap the deep utilization potential of plant-based protein feed by using feed strains that efficiently utilize low-value raw materials and fermented meal.

SUMMARY OF THE INVENTION

The present invention adopts Aspergillus oryzae, a wild strain isolated from rot wood microorganisms, and improves its ability to degrade biomass materials through ARTP mutagenesis to obtain an Aspergillus oryzae strain A02. When insoluble lignin/soluble lignin was used as the only carbon source, A02 of Aspergillus oryzae could grow normally, indicating that this strain of Aspergillus oryzae had strong lignin degradation ability; Aspergillus oryzae A02 was fermented in a liquid shake flask for 5 days, and its cellulase enzyme The activity can reach 12IU/ml, and the xylanase activity can reach 210IU/ml. Therefore, using the Aspergillus oryzae fermented meal protein and low-value biomass raw materials of the present invention to produce single-cell protein, from the two aspects of "open source" and "reduction" of feed protein resources, improve the utilization rate of existing protein feed, and develop new feed Protein is an important way to alleviate the shortage of feed resources in my country, so the invention can be applied industrially and has a good industrialization prospect.

Therefore, the present invention first provides Aspergillus oryzae strain A02, which is preserved in the General Microbiology Center of the China Microorganism Culture Collection and Administration Commission, the preservation number is: CGMCC No. 40043, and the preservation time is: January 17, 2022, The address of the preservation unit is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

Secondly, the application of the strain in lignin degradation is provided, and the degraded substrate is insoluble lignin or soluble lignin, specifically cotton meal, soybean meal, and peanut meal.

Further, after 72 hours of mixed solid-state fermentation of Aspergillus oryzae A02 and Aspergillus niger 60B-3DW in the present invention, the crude protein content of cotton meal, soybean meal and peanut meal was increased by 11.42%, 13.64% and 11.69% respectively. Through mixed solid-state fermentation of Aspergillus oryzae A02 and Aspergillus niger 60B-3DW, the proportion of essential amino acids in cotton meal increased from 29.86% to 36.42%; the proportion of essential amino acids in soybean meal increased from 36.46% to 40.64%; the proportion of essential amino acids in peanut meal increased from 26.73% to 32.78 %, greatly improving the quality and nutritional value of meal feed. Using low-value raw materials (bagasse, corn stalk, corn cob) as substrates, the present invention undergoes specific mixed fermentation of Aspergillus oryzae A02 and Aspergillus niger 60B-3DW, the crude protein content in the single-cell protein exceeds 29%, and the amino acid content Both exceed 24%.

Among them, Aspergillus niger strain 60B-3DW, its taxonomic name: Aspergillus niger, strain Aspergillus niger 60B-3DW is preserved in the General Microbiology Center of China Microorganism Culture Collection Management Committee, the preservation number is: CGMCC No.22465 (the The strain was obtained by mutation of Aspergillus niger 3.316 provided by the General Culture Collection Center of the Institute of Microbiology, Chinese Academy of Sciences. , Institute of Microbiology, Chinese Academy of Sciences.

Accordingly, the present invention provides a mixed bacterial agent for single-cell protein production, which comprises Aspergillus oryzae A02 and Aspergillus niger 60B-3DW. Preferably, the strain is in the form of a spore powder or a spore suspension. More preferably, the spore concentration at the time of use is 10 ⁶ -10 ⁸ /mL.

Furthermore, the present invention provides a method for producing feed protein, which is to obtain single-cell protein by fermenting lignin raw materials with mixed bacteria of Aspergillus oryzae A02 and Aspergillus niger 60B-3DW. Preferably, the step of isolating the feed protein is also included. Specifically, the lignin raw material is a straw-like raw material, specifically corn stover, corn cob, bagasse and mixtures thereof. Preferably, the lignin raw material is meal, more specifically soybean meal, cotton meal, peanut meal or mixtures thereof.

In a specific embodiment, soybean meal, peanut meal and cotton meal are mixed with bran at a ratio of 93:7 as the substrate for fermentation, the inoculum amount of Aspergillus oryzae A02 and Aspergillus niger 60B-3DW is 5-15%, and the rice The inoculation ratio of Aspergillus A02 and Aspergillus niger 60B-3DW is 1-3:1, preferably 2:1, the water content is 50%-70%, the culture temperature is 28-32°C, and the fermentation is 60-90h.

Description of drawings

Fig. 1 The growth of Aspergillus oryzae strains on screening plates after ARTP mutagenesis at different times.

Figure 2 The growth of Aspergillus oryzae strain A02 on different media, among which, A: PDA plate B: Soluble lignin as the sole carbon source plate C: Insoluble lignin as the sole carbon source plate.

Detailed ways

The present invention will be further described below through specific examples, so as to better understand the present invention, but it does not constitute a limitation of the present invention.

Example 1: Acquisition of Aspergillus oryzae strain A02

1. Obtaining the original strain

In May 2021, it was isolated from rot wood microorganisms collected in Tangshan City, Hebei Province.

Separation process: scrape the decayed wood microorganisms, put them into a triangular flask containing 95 mL of sterile water and 10 glass beads, and shake at 30 °C and 180 rpm for 30 min. Take 1 mL of bacterial suspension for 10 ^-1 -10 ^-7 serial concentration gradient dilution, then take 10 ^-5 , 10 ^-6 , 10 ^-7 three dilutions and spread it to the medium plate with lignin as the sole carbon source was incubated upside down at 28°C for 5 days.

Purification: After the colony was formed on a medium plate with lignin as the sole carbon source, the fastest growing strain was selected, the mycelium at the edge of the single colony was picked on the PDA medium plate, and the culture was continued at a constant temperature of 28°C. Finally, a pure Aspergillus oryzae colony was obtained, and the obtained colony was stored at 4°C.

对该菌株进行鉴定，其中ITS测序序列结果如下： GACGCTCGTAAGATCTTCCGTAGGTGAACCTGCGGAAGGATCATTACCGAGTGTAGGGTTCCTAGCGAGCCCAACCTCCCACCCGTGTTTACTGTACCTTAGTTGCTTCGGCGGGCCCGCCATTCATGGCCGCCGGGGGCTCTCAGCCCCGGGCCCGCGCCCGCCGGAGACACCACGAACTCTGTCTGATCTAGTGAAGTCTGAGTTGATTGTATCGCAATCAGTTAAAACTTTCAACAATGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAACTAGTGTGAATTGCAGAATTCCGTGAATCATCGAGTCTTTGAACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTCCGAGCGTCATTGCTGCCCATCAAGCACGGCTTGTGTGTTGGGTCGTCGTCCCCTCTCCGGGGGGGACGGGCCCCAAAGGCAGCGGCGGCACCGCGTCCGATCCTCGAGCGTATGGGGCTTTGTCACCCGCTCTGTAGGCCCGGCCGGCGCTTGCCGAACGCAAATCAATCTTTTTCCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAATAAGCGGAGGAAATCTTCCTGTG。

The results showed that the ITS sequence of this strain was 100% similar to that of Aspergillus oryzae RP-1 strain, indicating that the strain was an Aspergillus oryzae strain.

2. Obtainment of mutant strain A02

Carry out ARTP mutagenesis and sorting to the above-obtained Aspergillus oryzae strain:

a. Determination of mutagenesis time: use 100µl of fresh Aspergillus oryzae spore suspension with a spore concentration of 10 ⁵ , and mutagenize at different times. When the mutagenesis times of 0s, 60s, 90s, 120s, and 150s were set for the mutagenesis, the lethality of each mutagenesis time was calculated by plating the plates separately, and the ideal mutagenesis time was 70% lethality (the case of 0s was used as a control). );

b. Evaluation of colony orifice plate method after mutagenesis: After mutagenesis, colonies were picked into 24-well plates, cultured at 30°C and 130 rpm for 1 day, and their OD600 was measured to determine the growth rate of colonies after mutagenesis. Figure 1 shows the growth of Aspergillus oryzae strains on screening plates after ARTP mutagenesis at different times. Pick the strain with the fastest growth rate (number A02), and store the obtained colony at 4°C.

Example 2: Growth or fermentation characteristics of Aspergillus oryzae strain A02

When PDA and insoluble lignin/soluble lignin were used as the sole carbon source, Aspergillus oryzae A02 could grow normally (as shown in Figure 2), indicating that the strain has strong lignin degradation ability.

Fermentation medium: microcrystalline cellulose 33g/L, corn steep liquor powder 17 g/L, KH ₂ PO ₄ 1.60~1.72 g/L, (NH ₄ ) ₂ SO ₄ 2.6~3.0 g/L and MgSO ₄ 0.4~0.8 g/L. 24℃~28℃, pH 4.8~5.2, rotating speed 250~300 rpm, cultured for 5 days.

Aspergillus oryzae A02 was fermented in liquid shake flask for 5 days, its cellulase activity could reach 12.1 IU/ml, and its xylanase activity could reach 210.5IU/ml; and xylanase activities were increased by 17 times and 14 times, respectively.

strain Cellulase activity (IU/ml) Xylanase activity (IU/ml) Aspergillus oryzae wild strain 0.67 13.9 Aspergillus oryzae A02 12.1 210.5

Example 3: Mixed fermented meal

Seed medium: YPD medium: 1% glucose, 2% peptone, 1% yeast powder

The spore suspension concentration of 10 ⁷ /mL was washed from the plates of Aspergillus oryzae strain A02 and Aspergillus niger 60B-3DW, respectively, and the seed medium was added.

Soybean meal, peanut meal, and cotton meal were mixed with bran at a ratio of 93:7 as the substrate for fermentation, and the inoculation amount of Aspergillus oryzae A02 and A. 2:1), the water content is 60%, the culture temperature is 30°C, and the fermentation is 72h.

The crude protein content and amino acid content of the meal materials before and after fermentation were determined respectively. Crude protein GB/T 6432-1994 "Determination of crude protein in feed". Amino acid content was determined using A200 amino Nova amino acid analyzer with reference to the National Standard of the People's Republic of China GB/T 18246-2000 "Determination of Amino Acids in Feed".

The results from the table below show that the crude protein content of cotton meal, soybean meal and peanut meal increased by 11.42%, 13.64% and 11.69% respectively after 72h mixed solid-state fermentation of Aspergillus oryzae A02 and Aspergillus niger 60B-3DW. Aspergillus oryzae A02 and Aspergillus niger 60B-3DW mixed solid-state fermented meal is significantly better than single-strain fermented meal, because Aspergillus oryzae strain A02 can secrete cellulase, Aspergillus niger 60B-3DW can secrete β-glucosidase; fiber Enzyme and β-glucosidase have obvious synergistic effects, and the degradation efficiency of biomass is greatly improved after compounding; more biomass materials are degraded to produce sugar, and sugar is used by bacteria to generate bacterial protein, so after mixed solid-state fermentation, The crude protein content of meal increased more.

The results from the table below show that the quality of meal feed can be significantly improved after microbial mixed fermentation. Through the mixed solid-state fermentation of feed strains, the proportion of essential amino acids in cotton meal was increased from 29.86% to 36.42%; the proportion of essential amino acids in soybean meal was increased from 36.46% to 40.64%; the proportion of essential amino acids in peanut meal was increased from 26.73% to 32.78%, which greatly improved the meal. nutritional value of products.

Example 4: Development of new feed protein products from low-value raw materials

The low-value raw materials (corn stalk, corn cob, bagasse) were taken respectively, and water was added according to the ratio of material to water at 1:2.5 as the medium for solid-state fermentation. Both Aspergillus oryzae A02 and Aspergillus niger 60B-3DW seed liquids were added to the medium surface of the low-value raw material solid-state fermentation medium according to 10% inoculum amount (ie, 1:1 ratio), and cultured at 30°C for 96 hours.

The total nitrogen content, crude protein content and amino acid content were determined as shown in the table below. It can be seen that for the three raw materials of mixed fermentation, the crude protein content of single-cell protein exceeds 29%, and the amino acid content exceeds 24%, realizing the transformation of agricultural waste resources into treasures.

Material Corn stalks bagasse Corn cob Total nitrogen content (%) 4.71 4.92 4.81 Crude protein content (%) 29.44 30.75 30.06 Amino acid content (%) 24.11 24.53 24.76

The present invention improves the utilization rate of existing feed protein resources, develops new feed protein resources, and alleviates the current situation of shortage of feed protein resources in my country from the perspectives of "open source" and "reduction" of feed protein resources. Meal protein is the most commonly used feed protein resource in my country. After specific mixed fermentation of Aspergillus oryzae A02 and Aspergillus niger 60B-3DW, the crude protein content of meal protein increased by 11%, and the proportion of essential amino acids was also increased by about 5%. Improve the nutritional value of meal products. Low-value raw materials include bagasse, corn stalk, and corn cob as substrates. After the specific mixed fermentation of Aspergillus oryzae A02 and Aspergillus niger 60B-3DW, the crude protein content in the single-cell protein exceeds 29%, and the amino acid content exceeds 29%. twenty four%. Therefore, the present invention can be applied industrially, has good industrialization prospects, can realize the transformation of agricultural waste resources into treasure, subvert the traditional agricultural protein production mode, promote the self-sufficiency of protein raw materials in my country, and realize the recycling economy and agricultural sustainable development in China. an important way of sustainable development. At the same time, it is of strategic significance to accurately solve the two major problems of low utilization rate of agricultural waste in my country and the shortage of traditional agricultural protein, and to improve the comprehensive productivity and competitiveness of my country's agriculture.

<110> Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences

<120> A strain of Aspergillus oryzae and its application in the development of feed protein

<160> 1

<210> 1

<211> 623

<212> DNA

<213> Aspergillus oryzae

<400> 1

GACGCTCGTAAGATCTTCCGTAGGTGAACCTGCGGAAGGATCATTACCGAGTGTAGGGTTCCTAGCGAGCCCAACCTCCCACCCGTGTTTACTGTACCTTAGTTGCTTCGGCGGGCCCGCCATTCATGGCCGCCGGGGGCTCTCAGCCCCGGGCCCGCGCCCGCCGGAGACACCACGAACTCTGTCTGATCTAGTGAAGTCTGAGTTGATTGTATCGCAATCAGTTAAAACTTTCAACAATGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAACTAGTGTGAATTGCAGAATTCCGTGAATCATCGAGTCTTTGAACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTCCGAGCGTCATTGCTGCCCATCAAGCACGGCTTGTGTGTTGGGTCGTCGTCCCCTCTCCGGGGGGGACGGGCCCCAAAGGCAGCGGCGGCACCGCGTCCGATCCTCGAGCGTATGGGGCTTTGTCACCCGCTCTGTAGGCCCGGCCGGCGCTTGCCGAACGCAAATCAATCTTTTTCCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAATAAGCGGAGGAAATCTTCCTGTG 623

Claims (9)

1. A strain of Aspergillus oryzae , which is preserved in the General Microbiology Center of the China Microorganism Culture Collection and Administration Commission, the preservation number is: CGMCC No.40043, and the preservation time is: January 17, 2022. 2. The application of the Aspergillus oryzae strain as claimed in claim 1 in lignin degradation, wherein the degraded substrate is insoluble lignin or soluble lignin. 3. The use of claim 2, wherein the substrate is cotton meal, soybean meal, peanut meal or a mixture thereof. 4. A mixed inoculum for single-cell protein production, comprising the aspergillus oryzae strain and aspergillus niger ( Aspergillus niger ) 60B-3DW as claimed in claim 1, wherein the aspergillus niger 60B-3DW is preserved in Chinese microbial strains The General Microbiology Center of the Depository Management Committee, the deposit number is: CGMCC No.22465. 5. The mixed inoculum of claim 4, wherein the bacterial strain exists in the form of spore powder or spore suspension. 6. a method for producing forage protein, it is with aspergillus oryzae bacterial strain as claimed in claim 1 and aspergillus niger ( Aspergillus niger ) 60B-3DW mixed bacteria fermentation lignin raw material to obtain forage protein and separate forage protein Step, wherein Aspergillus niger 60B-3DW is preserved in the General Microbiology Center of China Microorganism Culture Collection Management Committee, and the preservation number is: CGMCC No.22465. 7. The method of claim 6, wherein the lignin raw material is a straw-based raw material, a meal-based raw material, or a mixture thereof. 8. The method of claim 7, wherein the straw-based raw materials are corn stover, corncob, bagasse and mixtures thereof; and the meal-based raw materials are soybean meal, cotton meal, peanut meal or a mixture thereof. 9. method as claimed in claim 6 is characterized in that, is mixed with bran according to 80-95:7 with soybean meal, peanut meal, cotton meal respectively as the substrate of fermentation, and described Aspergillus oryzae strain and black The inoculation amount of Aspergillus 60B-3DW is 5-15%, the inoculation ratio of the Aspergillus oryzae strain and Aspergillus niger 60B-3DW is 1-3:1, the water content is 50%-70%, the culture temperature is 28-32 ° C, and the fermentation 60-90h.

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