CN115820438B - High-yield protein strain and application thereof - Google Patents

Abstract

The invention relates to the technical field of microorganisms and foods, in particular to a strain with high protein yield and application thereof. Compared with common fusarium, the mutant strain of the fusarium provided by the invention, especially the TB02 strain with the preservation number of CGMCC No.23057, has obviously improved mycelium protein content and biomass, can use agricultural and sideline products as carbon sources or nitrogen sources, has complete amino acid types in the fermentation protein, contains essential amino acids of human bodies, and has wide application value.

Description

High-yield protein strain and application thereof

Technical Field

The invention relates to the technical field of foods, in particular to fusarium venenatum with high yield of hypha protein and application thereof in the production of the hypha protein.

Background

At present, the mycelium protein product contains abundant proteins, generally 40% -80%, has complete amino acid types and proper proportion, contains various amino acids required by animals, especially has higher lysine content, contains rich tryptophan, threonine and isoleucine, contains rich carbohydrate, functional sugar, nucleic acid, vitamin, inorganic salt and the like, contains various active substances such as enzyme, hormone, free nucleotide and the like, and can promote the absorption and utilization of nutrient substances by the organisms. The mycelium protein can be produced by fully utilizing industrial wastewater, waste gas, agricultural byproducts and other wide raw materials as a culture medium and then purifying and drying the raw materials, the production process is simple, the production efficiency is high, the protein production efficiency per unit area is 8000 times higher than that of planted soybeans, 80000 times higher than that of cattle, and the green continuous production can be realized. Chinese patent CN108077595A discloses a method for preparing protein powder by using mycelium protein, which comprises the steps of mixing protein powder and corn steep liquor according to a specific proportion to prepare the protein powder. Chinese patent CN103627695A discloses a method for improving the protein content of tuckahoe mycelium and the biomass of liquid fermentation, which mainly aims at the problems of low protein content and slow biomass. However, the hyphal proteins produced directly by microbial fermentation have yet to be developed and studied further.

Disclosure of Invention

Aiming at the actual demand, the technical problem to be solved by the invention is how to obtain the strain capable of producing protein in high yield and/or how to obtain the strain with fast biomass and/or how to obtain the strain with wide substrate utilization and/or how to obtain the strain suitable for mycelium protein production.

The invention provides a fusarium venenatumFusarium venenatum) A mutant strain of TB01 comprising one or more non-synonymous mutations selected from the group consisting of a K12N mutation of Zn (II) 2cys6 zinc clusterin, a T428I mutation of aspartic protease, an S99I mutation of an ATF 4-activated transcription factor, a Q40L mutation of a cell differentiation protein; the preservation number of the fusarium venenatum TB01 is CGMCC NO.20740, and the fusarium venenatum TB01 is classified and namedFusarium venenatumThe microbial strain is preserved in China general microbiological culture Collection center (CGMCC) for 10 months and 12 days in 2020, and the preservation unit address is North Chenxi Lu No. 1 and No. 3 in the Chaoyang district of Beijing city.

Specifically, the mutant strain contains a homozygous mutation for the mutation.

Preferably, the mutant strain is fusarium venenatum TB02 with the collection number of CGMCC No.23057 and the classification of the mutant strain is named asFusarium venenatumThe microbial strain is preserved in China general microbiological culture collection center (CGMCC) for 7.27 days in 2021, and the preservation unit address is North Chen West Lu No. 1 and No. 3 in the Korean region of Beijing city.

The invention also provides application of the mutant strain in preparing hypha protein.

Preferably, it ferments the mutant strain by means of fermentation to produce hyphal proteins.

More preferably, the agricultural byproduct material is fermented. More specifically, the agricultural byproduct material is soybean molasses and/or sugarcane molasses.

In one embodiment, the fermentation is performed using a glucose fermentation medium, which is: every 1000mL contains glucose 60. 60 g, ammonium dihydrogen phosphate 2.1 g, ammonium sulfate 6.0 g, potassium sulfate 2.1 g, magnesium sulfate 0.87 g, ferric chloride 3.64 mg, zinc sulfate 19.30 mg, manganese sulfate 15.46 mg, copper sulfate 1.85mg, and the pH is adjusted to 5.5;

in another embodiment, the fermentation medium is a cane molasses fermentation medium comprising, per 1000mL, cane molasses 60. 60 g, monoammonium phosphate 2.1 g, ammonium sulfate 6.0 g, potassium sulfate 2.1 g, magnesium sulfate 0.87 g, ferric chloride 3.64 mg, zinc sulfate 19.30 mg, manganese sulfate 15.46 mg, copper sulfate 1.85mg, adjusted pH to 5.5;

in another embodiment, the fermentation medium is a soy molasses fermentation medium containing, per 1000mL, soy molasses 60 g, monoammonium phosphate 2.1 g, ammonium sulfate 6.0 g, potassium sulfate 2.1 g, magnesium sulfate 0.87 g, ferric chloride 3.64 mg, zinc sulfate 19.30 mg, manganese sulfate 15.46 mg, copper sulfate 1.85mg, and adjusting the pH to 5.5.

Compared with common fusarium, the mutant strain of the fusarium provided by the invention, especially the TB02 strain with the preservation number of CGMCC No.23057, has obviously improved mycelium protein content and biomass, can use agricultural and sideline products as carbon sources or nitrogen sources, has complete amino acid types in the fermentation protein, contains essential amino acids of human bodies, and has wide application value.

Drawings

FIG. 1 shows the lethal curves of F.Veneti TB02.

FIG. 2 shows the protein content of F.Veneti TB02.

Biological material preservation information:

fusarium venenatum TB01 with preservation number of CGMCC NO.20740 and classified nameFusarium venenatumThe microbial strain is preserved in China general microbiological culture Collection center (CGMCC) for 10 months and 12 days in 2020, and the preservation unit address is North Chenxi Lu No. 1 and No. 3 in the Chaoyang district of Beijing city.

Fusarium Veneti TB02 with preservation number of CGMCC NO.23057, and classified and named asFusarium venenatumThe microbial strain is preserved in China general microbiological culture collection center (CGMCC) for 7.27 days in 2021, and the preservation unit address is North Chen West Lu No. 1 and No. 3 in the Korean region of Beijing city.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

The preparation method of the culture medium in the embodiment of the invention comprises the following steps:

the pH of the spore-forming solid culture medium is 5.5, and the composition is as follows: cellulose 20. 20 g/L, monoammonium phosphate 2.1 g/L, ammonium sulfate 6.0 g/L, potassium sulfate 2.1 g/L, magnesium sulfate 0.87 g/L, and water the balance. The preparation method comprises the following steps of (1000 mL): glucose 20 g, monoammonium phosphate 2.1 g, ammonium sulfate 6.0 g, potassium sulfate 2.1 g, magnesium sulfate 0.87 g, water to 1000mL, adjusting pH to 5.5, and sterilizing at 121deg.C for 15min.

The pH value of the inorganic salt liquid culture medium is 5.5, and the composition is as follows: glucose 20. 20 g/L, ammonium dihydrogen phosphate 2.1 g/L, ammonium sulfate 6.0 g/L, potassium sulfate 2.1 g/L, magnesium sulfate 0.87 g/L, ferric chloride 3.64 mg/L, zinc sulfate 19.30 mg/L, manganese sulfate 15.46 mg/L, copper sulfate 1.85mg/L, and water as the remainder. The preparation method comprises the following steps of (1000 mL): glucose 20. 20 g, ammonium dihydrogen phosphate 2.1 g, ammonium sulfate 6.0 g, potassium sulfate 2.1 g, magnesium sulfate 0.87 g, ferric chloride 3.64 mg, zinc sulfate 19.30 mg, manganese sulfate 15.46 mg, copper sulfate 1.85mg, water to 1000mL, adjusting pH to 5.5, and sterilizing at 121deg.C for 15min.

The pH of the inorganic salt solid culture medium is 5.5, and the composition is as follows: glucose 20. 20 g/L, ammonium dihydrogen phosphate 2.1 g/L, ammonium sulfate 6.0 g/L, potassium sulfate 2.1 g/L, magnesium sulfate 0.87. 0.87 g/L, ferric chloride 3.64 mg/L, zinc sulfate 19.30 mg/L, manganese sulfate 15.46 mg/L, copper sulfate 1.85mg/L, agar 20 g/L and water as the remainder. The preparation method comprises the following steps of (1000 mL): glucose 20. 20 g, monoammonium phosphate 2.1 g, ammonium sulfate 6.0 g, potassium sulfate 2.1 g, magnesium sulfate 0.87 g, ferric chloride 3.64 mg, zinc sulfate 19.30 mg, manganese sulfate 15.46 mg, copper sulfate 1.85mg, agar 20 g, water to 1000mL, pH adjusted to 5.5, and sterilization at 121℃for 15min.

The pH of the glucose fermentation medium was 5.5, the composition was: glucose 60. 60 g/L, ammonium dihydrogen phosphate 2.1 g/L, ammonium sulfate 6.0 g/L, potassium sulfate 2.1 g/L, magnesium sulfate 0.87 g/L, ferric chloride 3.64 mg/L, zinc sulfate 19.30 mg/L, manganese sulfate 15.46 mg/L, copper sulfate 1.85mg/L, and water as the rest. The preparation method comprises the following steps of (1000 mL): glucose 20. 20 g, ammonium dihydrogen phosphate 2.1 g, ammonium sulfate 6.0 g, potassium sulfate 2.1 g, magnesium sulfate 0.87 g, ferric chloride 3.64 mg, zinc sulfate 19.30 mg, manganese sulfate 15.46 mg, copper sulfate 1.85mg, water to 1000mL, adjusting pH to 5.5, and sterilizing at 121deg.C for 15min.

The pH of the soy molasses fermentation medium is 5.5, and the composition is: 60. 60 g/L of soybean molasses, 2.1 g/L of ammonium dihydrogen phosphate, 6.0 g/L of ammonium sulfate, 2.1 g/L of potassium sulfate, 0.87 g/L of magnesium sulfate, 3.64 mg/L of ferric chloride, 19.30 mg/L of zinc sulfate, 15.46 mg/L of manganese sulfate, 1.85mg/L of copper sulfate and the balance of water. The preparation method comprises the following steps of (1000 mL): glucose 20. 20 g, ammonium dihydrogen phosphate 2.1 g, ammonium sulfate 6.0 g, potassium sulfate 2.1 g, magnesium sulfate 0.87 g, ferric chloride 3.64 mg, zinc sulfate 19.30 mg, manganese sulfate 15.46 mg, copper sulfate 1.85mg, water to 1000mL, adjusting pH to 5.5, and sterilizing at 121deg.C for 15min.

The pH value of the cane molasses fermentation medium is 5.5, and the composition is as follows: cane molasses 60. 60 g/L, monoammonium phosphate 2.1 g/L, ammonium sulfate 6.0 g/L, potassium sulfate 2.1 g/L, magnesium sulfate 0.87 g/L, ferric chloride 3.64 mg/L, zinc sulfate 19.30 mg/L, manganese sulfate 15.46 mg/L, copper sulfate 1.85mg/L, and the balance being water. The preparation method comprises the following steps of (1000 mL): glucose 20. 20 g, ammonium dihydrogen phosphate 2.1 g, ammonium sulfate 6.0 g, potassium sulfate 2.1 g, magnesium sulfate 0.87 g, ferric chloride 3.64 mg, zinc sulfate 19.30 mg, manganese sulfate 15.46 mg, copper sulfate 1.85mg, water to 1000mL, adjusting pH to 5.5, and sterilizing at 121deg.C for 15min.

Example 1 obtaining Fusarium Venetian TB02

In this example, fusarium venenatum TB01 (deposited in this laboratory, see chinese patent CN112226373 a) was used as the starting strain, and fusarium venenatum TB02 was prepared by means of ARTP mutagenesis:

preparation of bacterial Strain spore suspension

And (3) streaking and inoculating the fusarium venenatum TB01 preserved by an inclined plane into a spore production culture medium plate, culturing at the constant temperature of 28 ℃ for 7-9 d, slightly scraping spores on the surface of the fusarium venenatum plate by using an inoculating loop, flushing three times by adding normal saline, sucking spore suspension, transferring the spore suspension into a conical flask, adding few glass beads, oscillating for 10 min to fully disperse the spores, and filtering absorbent cotton to obtain fusarium venenatum spore suspension. Calculating spore concentration of Fusarium Veneti by blood cell counting method, and adjusting the final concentration of spore suspension to 1x10 ⁷ CFU/ml. Preserving at 4 ℃ for standby.

ARTP mutagenesis screening

Lethal curve: 10 mu L of prepared fusarium venenatum suspension is sucked and uniformly coated on the surface of a sterile metal slide, the metal slide is placed in an ARTP mutagenesis blind instrument after being air-dried, and mutagenesis time is set to 10 s, 20 s, 40 s, 60 s, 80 s, 100s, 120 s, 140 s, 160 s, 180 s and 200 s respectively, and the mutagenesis power is 120W, the ventilation quantity of helium (He) is 10 SLM and the radiation distance is 2 mm. After mutagenesis, the metal slide is transferred into a centrifuge tube of 5 mL filled with sterile water, fully oscillated to form a new fusarium venenatum suspension, and the suspension under different treatment time is diluted and coated into an inorganic salt solid culture medium plate for culturing for 48 hours at 30 ℃. The mortality of the strains was calculated by plate colony counting using untreated samples (C s) as controls, and a strain mortality curve (FIG. 1) was drawn, from which it was found that at 60 spores had a mortality rate of 90% or more, thus 60 s was selected as the mutagenesis time. The calculation formula is as follows:

the mortality formula is: mortality = (U-T)/U100%, U is the number of non-mutagenized colonies (C s), T is the number of colonies after mutagenesis.

And (3) primary mutagenesis screening: 60 s was selected as the mutagenesis time based on the lethal curve of Fusarium Veneticum spores. Absorbing 10 mu L of prepared fusarium venenatum suspension, uniformly coating the suspension on the surface of a sterile metal slide, air-drying, placing the metal slide into an ARTP mutagenesis blind instrument, setting the mutagenesis time to 60 s, and carrying out mutagenesis power of 120W, helium (He) ventilation of 10 SLM and radiation distance of 2 mm. After mutagenesis, the metal slide was transferred to a 5 mL centrifuge tube containing sterile water, and the tube was thoroughly shake-coated on a plate of inorganic salt solid medium and incubated at 30℃for 48 hours. Selecting 350 monoclonals into inorganic salt liquid culture medium 30 ml, culturing at 30deg.C for 48 hr, filtering with Buchner funnel, washing with sterile water 150 ml, oven drying at 105deg.C for 3 h to constant weight, and selecting 35 strains (respectively numbered as TB01-1 to TB01-35, wherein TB01-1 is TB 02) with biomass exceeding 3 g/L.

And (3) re-screening: the 35 strains obtained by primary screening are respectively inoculated into a triangular flask of 30 ml inorganic salt liquid for culturing in 100 mL, 2 mL strains are taken after culturing in 30 ℃ for 48 hours, and are inoculated into a triangular flask of 500 mL with 100 mL inorganic salt liquid for culturing in 30 ℃ for 48 hours. After fermentation, the mixture was filtered through a Buchner funnel, washed with sterile water 500 ml and dried at 105℃for 3 h to constant weight. Weighing 0.1000. 0.1000 g dry mycelium into 20 mL sterile water, shaking uniformly, completely mixing the mycelium uniformly by utilizing ultrasonic waves of an ultrasonic instrument to form a uniform solution, measuring the total nitrogen content by utilizing a total organic carbon/total nitrogen analyzer (N/C2100S), and calculating the protein content of the mycelium.

As shown in FIG. 2, it was found from FIG. 2 that the biomass of strain TB01-1 was the largest, reaching 5.3.+ -. 0.1. 0.1 g/L and the protein content was the largest, 55.2.+ -. 0.2%, to obtain a mutant strain designated as F.Venetii TB02.

Example 2 Fusarium Venetian TB02 protein amino acid analysis

Fusarium venenatum TB01 and TB02 were inoculated from plates and incubated with 30 ml inorganic salt in flasks of 100 mL, respectively, at 30℃for 48h. 5 mL is inoculated with 100 mL inorganic salt liquid and cultured in a 500 mL triangular flask at 30 ℃ for 48h. Filtering with Buchner funnel after fermentation, collecting thallus, and oven drying at 105deg.C for 3 h to constant weight.

Weighing 0.1g of dry mycelium into 20 ml sterile water, shaking uniformly, completely mixing the mycelium uniformly by utilizing ultrasonic waves of an ultrasonic instrument to form a uniform solution, measuring the total nitrogen content by utilizing a total organic carbon/total nitrogen analyzer (N/C2100S), and calculating the protein content of the mycelium. The content of TB01 protein is 46.3+/-0.3%, and the content of TB02 protein is 55.1+/-0.4%.

The hyphal proteins from TB01 and TB02 were analyzed by LC-MS and the results are shown in Table 1. Compared with TB01, TB02 increases the ratio of essential amino acids in mycelium protein, increases the range from 26.18% -147.57%, and increases the nutritive value of mycelium protein.

TABLE 1 hyphal protein amino acid analysis

Example 3 fermentation of Fusarium Venetian TB02 glucose

Fusarium venenatum TB01 and TB02 were inoculated from plates and incubated with 30 ml inorganic salt in flasks of 100 mL, respectively, at 30℃for 48h. 5 mL is inoculated with 200 mL inorganic salt liquid and cultured in a 500 mL triangular flask at 30 ℃ for 24 hours. 100 mL seed liquid is inoculated into a 5L fermentation tank, wherein the fermentation tank contains 2.8L glucose fermentation medium, the pH value is regulated to 5.5 by ammonia water, the rotating speed is 300 rpm, the oxygen ventilation amount is 1ppm, and the fermentation time is 72 h. After the fermentation, the fermentation broth was filtered through a buchner funnel, washed with 15L distilled water, and dried at 105℃for 3 h to constant weight. TB01 gave 39.6.+ -. 0.2 g, i.e.13.2.+ -. 0.2 g/L, and TB02 gave 51.3.+ -. 0.3 g dry weight, i.e.17.1.+ -. 1 g/L.

Example 4 fermentation of Fusarium Venetian TB02 Soybean molasses

Fusarium venenatum TB01 and TB02 were inoculated from plates and incubated with 30 ml inorganic salt in flasks of 100 mL, respectively, at 30℃for 48h. 5 mL is inoculated with 200 mL inorganic salt liquid and cultured in a 500 mL triangular flask at 30 ℃ for 24 hours. 100 mL seed liquid is inoculated into a 5L fermentation tank, wherein the fermentation tank contains a soy molasses fermentation medium of 2.8L, the pH value is regulated to 5.5 by ammonia water, the rotating speed is 300 rpm, the oxygen ventilation amount is 1ppm, and the fermentation time is 72 h. After the fermentation, the fermentation broth was filtered through a buchner funnel, washed with 30L distilled water, and dried at 105℃for 3 h to constant weight. TB01 gave 50.46.+ -. 0.2 g, i.e.17.4.+ -. 0.2 g/L, and TB02 gave 64.09.+ -. 0.3 g dry weight, i.e.22.1.+ -. 0.3 g/L.

Example 5 fermentation of Fusarium Venetian TB02 sugar cane molasses

Fusarium venenatum TB01 and TB02 were inoculated from plates and incubated with 30 ml inorganic salt in flasks of 100 mL, respectively, at 30℃for 48h. 5 mL is inoculated with 200 mL inorganic salt liquid and cultured in a 500 mL triangular flask at 30 ℃ for 24 hours. 100 mL seed liquid is inoculated into a 5L fermentation tank, wherein the fermentation tank contains 2.8L cane molasses fermentation medium, the pH value is regulated to 5.5 by ammonia water, the rotating speed is 300 rpm, the oxygen ventilation amount is 1ppm, and the fermentation time is 72 h. After the fermentation, the fermentation broth was filtered through a buchner funnel, washed with 15L distilled water, and dried at 105℃for 3 h to constant weight. TB01 gave 59.68.+ -. 0.2 g, i.e.20.58.+ -. 0.2 g/L, and TB02 gave 77.43.+ -. 0.3 g dry weight, i.e.26.7.+ -. 1 g/L.

Example 6 genetic variation of Fusarium Veneticum TB02

By performing whole genome resequencing on fusarium venenatum TB02, non-synonymous mutations were accumulated in fusarium venenatum TB023 as compared to the starting strain TB01 by comparative genomic analysis, and the results are shown in table 2.

Genomic DNA and sequencing libraries of the strains Fusarium venenatum TB01 and TB02 were extracted and constructed by Northhe origin (Beijing, china) and sequenced by a 150bp double-ended sequencing method using a Illumina HiseqXten-PE150 high-throughput sequencing platform. To be used forFusarium venenatumA3/5 is the reference genome (RefSeq assembly accession: GCF_ 900007375.1), 44.3 million Clean reads are obtained, sequencing depth 147x and sequencing coverage of 97.8%.

TABLE 2 non-synonymous mutant genes accumulated in Fusarium Veneti TB02

Note that: WT: wild type; MT: mutation; homo: homozygosity; het: and (5) heterozygosis. The smaller the SIFT Score, the greater the impact of the mutation on protein structure and function.

Single nucleotide polymorphism (single nucleotide polymorphism, SNP) mutations and insertion/deletion (InDels) mutations in strains were evaluated according to the genomic analysis procedure reported by DePristo et al (related literature: dePristo MA, banks E, poplin R, et al A framework for variation discovery and genotyping using next-generation DNA sequencing data Nat Genet.2011;43 (5): 491-498.). Then, prediction analysis of the effect of mutations on protein function was performed on non-synonymous mutations occurring in the coding region of the protein using SIFT (related literature: wagih O, galarnini M, busby BP, memon D, typas A, beltrao P. A resource of variant effect predictionsof single nucleotide variants in model organisms. MolSyst biol. 2018.14 (12): e 8430.).

Using the above method, non-synonymous mutations were screened by genomic sequencing analysis, PCR amplification of mutant DNA fragments was performed using genomic DNAs of F.Venetian TB01 and F.Venetian TB02 as templates, and the results were shown in Table 2. In comparison with F.Veneti TB01, 4 homozygous mutations were found in F.Veneti TB02, FVRRES_03473 ^K12N 、FVRRES_11063 ^T428I 、FVRRES_12511 ^S99I And FVRRES_13504 ^Q40L 。

Claims (8)

1. Fusarium Veneticum with high protein yieldFusarium venenatum) A mutant strain of TB01, which is fusarium venenatum TB02 and has a collection number of CGMCC No. 23057.

2. Use of a mutant strain according to claim 1 for the preparation of hyphal proteins.

3. Use according to claim 2, characterized in that it ferments the mutant strain by means of fermentation to produce hyphal proteins.

4. Use according to claim 3, characterized in that the fermentation is carried out using agricultural by-product raw materials.

5. The use as claimed in claim 4, wherein the agricultural by-product raw material is soybean molasses and/or sugarcane molasses.

6. Use according to claim 3, characterized in that the fermentation is carried out with a glucose fermentation medium, which is: every 1000mL contains glucose 60. 60 g, ammonium dihydrogen phosphate 2.1 g, ammonium sulfate 6.0 g, potassium sulfate 2.1 g, magnesium sulfate 0.87 g, ferric chloride 3.64 mg, zinc sulfate 19.30 mg, manganese sulfate 15.46 mg, copper sulfate 1.85mg, and the pH is adjusted to 5.5.

7. Use according to claim 3, characterized in that the fermentation is carried out using a sugar cane molasses fermentation medium containing, per 1000mL, sugar cane molasses 60 g, monoammonium phosphate 2.1 g, ammonium sulphate 6.0 g, potassium sulphate 2.1 g, magnesium sulphate 0.87 g, ferric chloride 3.64 mg, zinc sulphate 19.30 mg, manganese sulphate 15.46 mg, copper sulphate 1.85mg, the pH being adjusted to 5.5.

8. Use according to claim 3, characterized in that the fermentation is carried out using a soy molasses fermentation medium containing, per 1000mL, soy molasses 60 g, monoammonium phosphate 2.1 g, ammonium sulphate 6.0 g, potassium sulphate 2.1 g, magnesium sulphate 0.87 g, ferric chloride 3.64 mg, zinc sulphate 19.30 mg, manganese sulphate 15.46 mg, copper sulphate 1.85mg, the pH being adjusted to 5.5.

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