CN114410486A - Aspergillus oryzae strain and application thereof in development of feed protein - Google Patents

Aspergillus oryzae strain and application thereof in development of feed protein Download PDF

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CN114410486A
CN114410486A CN202210316149.5A CN202210316149A CN114410486A CN 114410486 A CN114410486 A CN 114410486A CN 202210316149 A CN202210316149 A CN 202210316149A CN 114410486 A CN114410486 A CN 114410486A
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吴信
高乐
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Abstract

The invention belongs to the technical field of fermentation meal feed and application of producing single-cell protein from low-value raw materials, and relates to an aspergillus oryzae strain and application thereof. According to the invention, Aspergillus oryzae is a wild strain separated from a saprophytic microorganism, the ability of the Aspergillus oryzae to degrade a biomass material is improved in an ARTP mutagenesis mode, an Aspergillus oryzae strain A02 is obtained, Aspergillus oryzae A02 is fermented for 5 days in a liquid shake flask, the cellulase activity of the Aspergillus oryzae strain A02 can reach 12IU/ml, and the xylanase activity of the Aspergillus oryzae strain A02 can reach 210 IU/ml. The invention also provides a method for producing single-cell protein by mixing solid state fermentation of Aspergillus oryzae A02 and Aspergillus niger 60B-3DW, which greatly improves the quality and the nutritional value of meal feed. Therefore, the invention can be applied in industrialization and has better industrialization prospect.

Description

Aspergillus oryzae strain and application thereof in development of feed protein
Technical Field
The invention belongs to the technical field of fermentation meal feed and application of producing single-cell protein from low-value raw materials, and relates to an aspergillus oryzae strain and application thereof.
Background
The shortage of the raw materials of the protein feed in China coexists with the contradiction of low utilization rate and large waste of the traditional feed raw materials. On one hand, the self-sufficient rate of protein feed in China is less than 50%, the dependence degree of foreign import exceeds 80%, the situations that people and livestock fight for food and are restrained by people are increasingly severe, the livestock and poultry breeding industry is taken as the backbone industry of agriculture in China, and the transformation and upgrading requirements are very urgent; agricultural wastes are converted into unconventional protein raw materials by utilizing a biosynthesis technology, so that the traditional agricultural protein production mode is overturned. The feed protein from low-value raw materials can compete with bean pulp feed protein in aspects of protein utilization rate, nutritional function, comprehensive cost and the like, and traditional agricultural protein substitution is realized.
On the other hand, the resource utilization rate of the existing protein feed is not high, and the main reason is that the processing technology of the protein in the current industry is low, so that a large amount of protein is wasted. Meals are byproducts after oil extraction and are the most important feed protein resources. The common food contains bean pulp, cotton pulp, peanut pulp and the like, is rich in protein, has reasonable amino acid distribution, and is a common plant protein raw material in animal daily ration. However, the crude fiber content in protein resources for meal feed is high, and the problems that the utilization rate of feed protein raw materials is reduced, the animal morbidity is high and the like are caused due to the fact that anti-nutritional factors such as xylan, cellulose and beta-glucan interfere with the digestion and absorption of daily ration nutrition and the feed nutritional value is reduced. And the amino acid composition of meal feed protein is not ideal as animal-derived protein feed. The mixed fermentation of meals by using feeding strains which efficiently utilize low-value raw materials is an effective way for improving the quality of meal protein feeds and exploring the deep utilization potential of plant protein feeds.
Disclosure of Invention
According to the invention, Aspergillus oryzae is a wild strain separated from sapropel microorganisms, and the ability of Aspergillus oryzae to degrade biomass materials is improved in an ARTP mutagenesis mode, so that an Aspergillus oryzae strain A02 is obtained. When insoluble lignin/soluble lignin is used as a unique carbon source, aspergillus oryzae A02 can normally grow, which indicates that the aspergillus oryzae has strong lignin degradation capability; aspergillus oryzae A02 is fermented in a liquid shake flask for 5 days, the cellulase activity can reach 12IU/ml, and the xylanase activity can reach 210 IU/ml. Therefore, the Aspergillus oryzae fermented meal protein and the low-value biomass raw material are utilized to produce the single-cell protein, the utilization rate of the conventional protein feed is improved from two aspects of 'source opening' and 'throttling' of feed protein resources, and the development of novel feed protein is an important way for relieving the shortage of feed resources in China, so that the invention can be applied industrially and has better industrial prospect.
Accordingly, the present invention provides, in a first aspect, Aspergillus oryzae (Aspergillus oryzae) Strain a02, deposited in the china general microbiological culture collection center with accession number: CGMCC number 40043, the preservation time is as follows: at 17.1.2022, the address of the depository is: xilu No. 1, Beijing, Chaoyang, Beijing, and institute for microbiology, China academy of sciences.
And secondly, providing the application of the strain in lignin degradation, wherein a degradation substrate is insoluble lignin or soluble lignin, and the degradation substrate can be cottonseed meal, soybean meal and peanut meal.
Furthermore, after the Aspergillus oryzae A02 and the Aspergillus niger 60B-3DW are mixed and subjected to solid state fermentation for 72 hours, the crude protein content of the cottonseed meal, the soybean meal and the peanut meal is respectively increased by 11.42%, 13.64% and 11.69%. Through the mixed solid state fermentation of aspergillus oryzae A02 and aspergillus niger 60B-3DW, the essential amino acid proportion of the cottonseed meal is improved from 29.86% to 36.42%; the proportion of essential amino acid of the soybean meal is improved from 36.46% to 40.64%; the proportion of the essential amino acids of the peanut meal is improved from 26.73% to 32.78%, and the quality and the nutritional value of the meal feed are greatly improved. By using low-value raw materials (bagasse, corn straws and corncobs) as substrates, after the special aspergillus oryzae A02 and aspergillus niger 60B-3DW are mixed and fermented, the content of crude protein in single-cell protein exceeds 29 percent, and the content of amino acid exceeds 24 percent.
Among them, Aspergillus niger strain 60B-3DW, its classification name: aspergillus niger, strain Aspergillus niger 60B-3DW, was deposited in the China general microbiological culture Collection center, accession number: CGMCC No.22465 (the strain is obtained by mutagenesis of Aspergillus niger 3.316 provided by the common strain preservation center of the institute of microbiology of China academy of sciences), and the preservation time is as follows: on 2021, 07/05, the address of the depository is: xilu No. 1, Beijing, Chaoyang, Beijing, and institute for microbiology, China academy of sciences.
Accordingly, the invention provides a mixed microbial inoculum for producing single-cell protein, which comprises Aspergillus oryzae A02 and Aspergillus niger 60B-3 DW. Preferably, the strain is in the form of a spore powder or a spore suspension. More preferably, the spore concentration is 106-108one/mL.
Furthermore, the invention provides a method for producing a protein for feed, which is to obtain a single-cell protein by fermenting a lignin raw material by using mixed bacteria of aspergillus oryzae A02 and aspergillus niger 60B-3 DW. Preferably, the method also comprises the step of separating the protein for feeding. Specifically, the lignin raw material is straw raw material, specifically corn straw, corn cob, bagasse and a mixture thereof. Preferably, the lignin raw material is meal, more particularly bean meal, cotton meal, peanut meal or mixtures thereof.
In one specific embodiment, soybean meal, peanut meal and cottonseed meal are mixed with bran according to a ratio of 93:7 respectively to be used as fermentation substrates, the inoculation amounts of aspergillus oryzae A02 and aspergillus niger 60B-3DW are 5-15%, and the inoculation ratios of aspergillus oryzae A02 and aspergillus niger 60B-3DW are 1-3: 1, preferably 2: 1, the water content is 50-70%, the culture temperature is 28-32 ℃, and the fermentation is carried out for 60-90 h.
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FIG. 1 growth of Aspergillus oryzae strains on selection plates after various time periods of ARTP mutagenesis.
FIG. 2 growth of A.oryzae strain A02 on different media, wherein A: PDA panel B: soluble lignin as sole carbon source plate C: insoluble lignin is the sole carbon source plate.
Detailed Description
The invention is further illustrated by the following specific examples in order to provide a better understanding of the invention, which are not to be construed as limiting the invention.
Example 1: obtaining of Aspergillus oryzae Strain A02
1. Obtaining of original Strain
Separated from sapropel microorganisms collected in Tangshan City of Hebei province in 2021 month.
And (3) a separation process: the saprophytic microorganisms were scraped off, placed in a triangular flask containing 95 mL of sterile water and 10 beads, and shaken at 180 rpm for 30 min at 30 ℃. Taking 1 mL of bacterial suspension and carrying out 10 -1 -10 -7Serial concentration gradient dilutions were made and then 10 taken-5、10 -6、10 -7Three dilutions were plated on medium plates with lignin as the sole carbon source and cultured in an inverted format at 28 ℃ for 5 d.
And (3) purification: after the bacterial colony is formed on a culture medium plate which takes lignin as a unique carbon source, selecting a strain with the fastest growth, selecting hyphae at the edge of a single bacterial colony on a PDA culture medium plate, and continuously culturing at constant temperature of 28 ℃. Finally obtaining a pure aspergillus oryzae colony, and storing the obtained colony at 4 ℃.
The strain is identified, wherein the ITS sequencing sequence result is as follows: GACGCTCGTAAGATCTTCCGTAGGTGAACCTGCGGAAGGATCATTACCGAGTGTAGGGTTCCTAGCGAGCCCAACCTCCCACCCGTGTTTACTGTACCTTAGTTGCTTCGGCGGGCCCGCCATTCATGGCCGCCGGGGGCTCTCAGCCCCGGGCCCGCGCCCGCCGGAGACACCACGAACTCTGTCTGATCTAGTGAAGTCTGAGTTGATTGTATCGCAATCAGTTAAAACTTTCAACAATGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAACTAGTGTGAATTGCAGAATTCCGTGAATCATCGAGTCTTTGAACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTCCGAGCGTCATTGCTGCCCATCAAGCACGGCTTGTGTGTTGGGTCGTCGTCCCCTCTCCGGGGGGGACGGGCCCCAAAGGCAGCGGCGGCACCGCGTCCGATCCTCGAGCGTATGGGGCTTTGTCACCCGCTCTGTAGGCCCGGCCGGCGCTTGCCGAACGCAAATCAATCTTTTTCCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAATAAGCGGAGGAAATCTTCCTGTG are provided.
The results showed that the ITS sequence of this strain was 100% similar to the Aspergillus oryzae RP-1 strain, indicating that this strain is an Aspergillus oryzae strain.
2. Acquisition of mutant Strain A02
Performing ARTP mutagenesis and sorting on the aspergillus oryzae strain obtained in the step (a):
a. determination of mutagenesis time: 100 mul of fresh aspergillus oryzae spore suspension is adopted, and the spore concentration is 105And carrying out mutagenesis for different times. When mutagenizingSetting mutagenesis time of 0s, 60 s, 90 s, 120 s and 150 s, respectively coating plates to count the lethality rate of each mutagenesis time, and taking 70% lethality rate as ideal mutagenesis time (0s condition is used as a control);
b. evaluation by colony-well plate method after mutagenesis: the colonies after mutagenesis were picked up in a 24-well plate, cultured at 30 ℃ and 130 rpm for 1 day, and the growth rate of the colonies after mutagenesis was determined by measuring the OD 600. FIG. 1 shows the growth of Aspergillus oryzae strains on selection plates after various times of ARTP mutagenesis. The strain with the highest growth rate (No. A02) was selected and the obtained colony was stored at 4 ℃.
Example 2: growth or fermentation characteristics of Aspergillus oryzae Strain A02
Both Aspergillus oryzae A02 grew normally (as shown in FIG. 2) with PDA, and insoluble/soluble lignin as the sole carbon source, indicating that the strain has strong lignin-degrading ability.
Fermentation medium: 33g/L microcrystalline cellulose, 17 g/L corn steep liquor dry powder and KH2PO4 1.60~1.72 g/L，(NH4)2SO42.6-3.0 g/L and MgSO40.4 to 0.8 g/L. Culturing for 5 days at 24-28 ℃, pH4.8-5.2 and a rotation speed of 250-300 rpm.
Aspergillus oryzae A02 is fermented in a liquid shake flask for 5 days, the cellulase activity can reach 12.1 IU/ml, and the xylanase activity can reach 210.5 IU/ml; compared with the original Aspergillus oryzae wild strain, the Aspergillus oryzae A02 cellulase and xylanase activities are respectively improved by 17 times and 14 times.
Bacterial strains 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 fermentation meals
Seed culture medium: YPD medium: 1% glucose, 2% peptone and 1% yeast powder
The spore suspension concentrations were 10% washed from plates of Aspergillus oryzae strain A02 and Aspergillus niger 60B-3DW, respectively7Adding a seed culture medium into the culture medium per mL, culturing at 26-28 ℃ at a rotation speed of 180-200 rpm for 24 hours.
The soybean meal, the peanut meal and the cottonseed meal are respectively mixed with bran according to the ratio of 93:7 to be used as fermentation substrates, the inoculation amount of Aspergillus oryzae A02 and Aspergillus niger 60B-3DW is 10% (the inoculation ratio of Aspergillus oryzae A02 and Aspergillus niger 60B-3DW is 2: 1), the water content is 60%, the culture temperature is 30 ℃, and the fermentation is carried out for 72 hours.
Crude protein content and amino acid content of meal materials before and after fermentation are respectively determined. Crude protein GB/T6432 1994 method for determining crude protein in feed. The amino acid content is measured by an A200 amino Nova amino acid analyzer according to national standard GB/T18246-.
The results from the following table show: after the Aspergillus oryzae A02 and the Aspergillus niger 60B-3DW are mixed and subjected to solid state fermentation for 72 hours, the crude protein content of the cottonseed meal, the soybean meal and the peanut meal is respectively increased by 11.42%, 13.64% and 11.69%. The effect of the mixed solid fermentation meal of aspergillus oryzae A02 and aspergillus niger 60B-3DW is obviously better than that of the single strain fermentation meal because aspergillus oryzae strain A02 can secrete cellulase, and aspergillus niger 60B-3DW can secrete beta-glucosidase; the cellulase and the beta-glucosidase have obvious synergistic effect, and the degradation efficiency of the biomass is greatly improved after the cellulase and the beta-glucosidase are compounded; more biomass materials are degraded to generate sugar, and the sugar is utilized by thalli to generate mycoprotein, so that the crude protein content of meals is improved more after mixed solid state fermentation.
Figure 565930DEST_PATH_IMAGE001
The results in the following table show that the quality of the meal feed can be obviously improved after mixed fermentation by microorganisms. The feed strain is mixed and subjected to solid state fermentation, the proportion of the essential amino acid of the cottonseed meal is increased from 29.86 percent to 36.42 percent, and the proportion of the essential amino acid of the soybean meal is increased from 36.46 percent to 40.64 percent; the proportion of the essential amino acids of the peanut meal is improved from 26.73% to 32.78%, and the nutritional value of the meal products is greatly improved.
Figure 483070DEST_PATH_IMAGE002
Example 5: development of novel feed protein product from low-value raw materials
Respectively taking low-value raw materials (corn straws, corn cobs and bagasse), and adding water according to the material-water ratio of 1:2.5 to serve as a culture medium for solid state fermentation. Aspergillus oryzae A02 and Aspergillus niger 60B-3DW seed solutions were added to the surface of the medium for solid state fermentation of low-value materials at an inoculum size of 10% (i.e., 1: 1 ratio), and cultured at 30 ℃ for 96 hours.
The total nitrogen content, crude protein content and amino acid content were determined as shown in the table below. Therefore, the content of crude protein in single-cell protein exceeds 29% and the content of amino acid exceeds 24% for three raw materials in the mixed fermentation, so that the waste of agricultural waste resources is changed into valuable.
Material Corn stalk 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
According to the invention, from two angles of 'opening source' and 'throttling' of feed protein resources, the utilization rate of the existing feed protein resources is improved, novel feed protein resources are developed, and the current situation of shortage of feed protein resources in China is relieved. The crude protein content of the meal protein is improved by 11 percent and the proportion of essential amino acids is improved by about 5 percent after the meal protein is mixed and fermented by specific Aspergillus oryzae A02 and Aspergillus niger 60B-3DW, so that the nutritional value of the meal product is greatly improved. The low-value raw materials comprise bagasse, corn straws and corncobs as substrates, and after the low-value raw materials are mixed and fermented by specific aspergillus oryzae A02 and aspergillus niger 60B-3DW, the content of crude protein in single-cell protein exceeds 29 percent, and the content of amino acid exceeds 24 percent. Therefore, the method can be applied in industrialization, has good industrialization prospect, can change agricultural waste resources into valuable, subverts the traditional agricultural protein production mode, promotes self-sufficiency of protein raw materials in China, and realizes the important path of circular economy and agricultural sustainable development in China. Meanwhile, two problems of low resource utilization rate of agricultural wastes in China and shortage of traditional agricultural proteins are accurately solved, the comprehensive productivity and competitiveness of agriculture in China are improved, and the method has important strategic significance.
<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences
<120> Aspergillus oryzae strain and application thereof in development of feed protein
<160> 1
<210> 1
<211> 623
<212> DNA
<213> Aspergillus oryzae
<400> 1
GACGCTCGTAAGATCTTCCGTAGGTGAACCTGCGGAAGGATCATTACCGAGTGTAGGGTTCCTAGCGAGCCCAACCTCCCACCCGTGTTTACTGTACCTTAGTTGCTTCGGCGGGCCCGCCATTCATGGCCGCCGGGGGCTCTCAGCCCCGGGCCCGCGCCCGCCGGAGACACCACGAACTCTGTCTGATCTAGTGAAGTCTGAGTTGATTGTATCGCAATCAGTTAAAACTTTCAACAATGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAACTAGTGTGAATTGCAGAATTCCGTGAATCATCGAGTCTTTGAACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTCCGAGCGTCATTGCTGCCCATCAAGCACGGCTTGTGTGTTGGGTCGTCGTCCCCTCTCCGGGGGGGACGGGCCCCAAAGGCAGCGGCGGCACCGCGTCCGATCCTCGAGCGTATGGGGCTTTGTCACCCGCTCTGTAGGCCCGGCCGGCGCTTGCCGAACGCAAATCAATCTTTTTCCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAATAAGCGGAGGAAATCTTCCTGTG 623

Claims (10)

1. A strain of Aspergillus oryzae (A)Aspergillus oryzae) It is preserved in China general microbiological culture Collection center with the preservation number: CGMCC No.40043, the preservation time is as follows: 1 month and 17 days 2022.
2. Use of an Aspergillus oryzae strain according to claim 1 for lignin degradation wherein the substrate for degradation is insoluble lignin or soluble lignin.
3. The use of claim 2, wherein the substrate is cottonseed meal, soybean meal, peanut meal, or a mixture thereof.
4. A mixed bacterial agent for single-cell protein production, comprising the aspergillus oryzae strain of claim 1 and aspergillus niger 60B-3 DW.
5. The mixed bacterial preparation according to claim 4, wherein the bacterial strain is in the form of spore powder or spore suspension.
6. The mixed inoculant according to claim 5, wherein in use the spore concentration is 106-108one/mL.
7. A process for producing a feed protein from the Aspergillus oryzae strain of claim 1 and Aspergillus niger (A. niger)Aspergillus niger) 60B-3DW mixed bacteria ferment lignin raw materials to obtain protein for feeding and separate the protein for feeding, wherein Aspergillus niger 60B-3DW is preserved in China general microbiological culture Collection center with the preservation number: CGMCC No. 22465.
8. The method of claim 7, wherein the lignin raw material is straw-based raw material, meal-based raw material, or a mixture thereof.
9. The method of claim 8, wherein the straw-based raw material is corn stover, corn cobs, sugar cane bagasse, and mixtures thereof; the meal raw material is soybean meal, cotton meal, peanut meal or a mixture thereof.
10. The method of claim 7, wherein the soybean meal, the peanut meal and the cottonseed meal are mixed with bran as fermentation substrates according to a ratio of 80-95:7, respectively, the inoculation amounts of the aspergillus oryzae strain and the aspergillus niger 60B-3DW are 5-15%, and the inoculation ratios of the aspergillus oryzae A02 and the aspergillus niger 60B-3DW are 1-3: 1, the water content is 50-70%, the culture temperature is 28-32 ℃, and the fermentation is carried out for 60-90 h.
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