CN112226373B

CN112226373B - Strain for producing protein and application thereof

Info

Publication number: CN112226373B
Application number: CN202011415776.1A
Authority: CN
Inventors: 马延和; 李德茂; 齐显尼; 曾艳; 王钦宏; 孙媛霞
Original assignee: Tianjin Institute of Industrial Biotechnology of CAS
Current assignee: Tianjin Institute of Industrial Biotechnology of CAS
Priority date: 2020-12-07
Filing date: 2020-12-07
Publication date: 2021-04-20
Anticipated expiration: 2040-12-07
Also published as: CN112226373A

Abstract

The invention relates to the technical field of microorganisms and food, in particular to a strain capable of producing protein and application thereof. The substrate which can be utilized by the strain is very rich, and the substrate raw materials which are wide in source and low in price, such as industrial wastewater, agricultural and sideline products and the like, can be fully utilized as a carbon source or a nitrogen source. The protein content in the mycelium obtained by fermentation is very high, the variety of amino acids is complete, and the mycelium has wide application value.

Description

Strain for producing protein and application thereof

Technical Field

The invention relates to the technical field of food, in particular to fusarium capable of producing hyphal protein and application thereof in producing hyphal protein.

Background

At the end of the last 60 s, the food companies Ranks Hovis McDougall (RHM) in the uk first initiated research and development work on fungal proteins and, through extensive global search, discovered fusarium as a sufficient and effective protein source in the soil near the wheat fields in the Marlow region in the uk. The hypha protein product contains rich protein, generally 40-80%, complete amino acid types and proper proportion, contains various amino acids required by animals, particularly has higher lysine content and also contains the limiting amino acids required by the growth of livestock and poultry, and can furthest ensure the growth requirements of the livestock and poultry; the product is rich in carbohydrate, functional sugar, nucleic acid, vitamins, inorganic salt, etc., and also contains active substances such as various enzymes, hormones, free nucleotides, etc., and can promote the absorption and utilization of nutrient substances by the body.

Compared with other protein sources, the mycelium protein has comprehensive nutrition, easily obtained production raw materials, short period, high yield per unit area, no influence of environment and climate, continuous production, environmental protection and the like, and has wide market prospect. The production of the hypha protein can fully utilize extensive raw materials such as industrial wastewater, agricultural and sideline products and the like as a culture medium, and then the hypha protein is prepared by purifying and drying, the production process is simple, the production efficiency is high, the production efficiency of the hypha protein per unit area is 8000 times higher than that of planted soybeans, and is 80000 times higher than that of cow proteins, and green continuous production can be realized. Chinese patent document CN108077595A discloses a protein powder obtained by subjecting mycelium protein liquid produced in various plants to some treatments. Chinese patent document CN102860432A discloses a processing technology of mushroom mycelium protein fish feed, which comprises mixing crop straws, fresh pig manure and fresh chicken manure, stacking and fermenting to prepare fermented compost; inoculating mushroom mycelium strains into the fermented compost, and culturing to obtain a mushroom mycelium protein raw material; drying protein material of mushroom mycelium, and pulverizing.

However, hyphal proteins directly produced by microbial fermentation with high efficiency need to be further developed and researched.

Disclosure of Invention

Aiming at the practical requirements, the inventor obtains a fusarium capable of efficiently producing hyphal protein through research and screening, and obtains a hyphal protein product with very high protein content by utilizing different nitrogen sources.

Therefore, the invention firstly provides fusarium TB01, wherein the preservation number of the fusarium TB01 is CGMCC number 20740, and the fusarium TB01 is named as fusariumFusarium venenatumAnd is preserved in China general microbiological culture Collection center (the preservation address is No. 3 of No.1 Xilu-Beijing) of the China general microbiological culture Collection center (Beijing) in the morning and Yangxi district, 10 months and 12 days in 2020. The fusarium TB01 is obtained by screening from soil and is identified as fusariumFusarium venenatum。

The invention further provides application of the fusarium in production of hyphal protein.

Further, the invention provides a method for producing hyphal protein by using fusarium, which is characterized in that the hyphal protein is produced by fermenting the fusarium, wherein a nitrogen source of a fermentation medium of the fusarium is an inorganic nitrogen source or an organic nitrogen source.

In some embodiments, the inorganic nitrogen source is urea, or (NH4)₂SO₄Or the organic nitrogen source is peptone, yeast powder or bean cake powder. More preferably, a collagen peptide, a whey protein peptide, or a whey protein-optimized nitrogen source is added to the fermentation medium of fusarium. More preferably, the collagen peptide, whey protein peptide, or whey protein is added to the medium in an amount of 0.5g/L to 2g/L, and more preferably in an amount of 1 g/L.

In other embodiments, the organic nitrogen source is collagen produced from skins or bones of pigs, cattle, sheep, and aquatic leftovers by enzymolysis. Wherein, the aquatic leftovers are preferably fish skin, fish scales or fish bones.

In other embodiments, the organic nitrogen source is a whey by-product formed from the production of cheese or casein.

The fusarium provided by the invention has abundant available substrates, can fully utilize cheap substrate raw materials with wide sources such as industrial wastewater, agricultural and sideline products (such as aquatic product processing byproducts and cheese processing byproducts) and the like as a carbon source or a nitrogen source, and can greatly reduce the industrial application cost; the contents of hypha proteins in obtained mycelia are more than 40% and even more than 60% by fermentation under different nitrogen source conditions, and meanwhile, the hypha proteins are used as carriers for enriching the protein content, the amino acid types are complete, no toxin is detected according to the requirements of food toxin detection standards, and the food application foundation is good. The fusarium products are rich in variety, can be fermented to obtain various nutritional ingredients including protein, lipid, dietary fiber and the like, and have wide application value.

Drawings

FIG. 1 is a colony morphology of Fusarium TB01 strain;

FIG. 2 is a morphological diagram of the mycelium of Fusarium TB01 strain;

FIG. 3 is a diagram based on ITS sequence structureFusarium builtFusarium venenatumTB01 phylogenetically develops trees.

Detailed Description

The technical solution of the present invention is clearly and completely described by the following embodiments. The described embodiments are only some embodiments of the invention, 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.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. The materials, reagents and the like used in the examples are commercially available unless otherwise specified.

Example 1 FusariumFusarium venenatum Acquisition of TB01

Rhizosphere soil of the wheat land is collected in a new coastal area of Tianjin city in 07 months in 2020.

And (3) a separation process: the rhizosphere soil sample is mixed evenly, 5g is weighed and put into a triangular flask containing 95 mL of sterile water and 10 glass beads, and the mixture is shaken at 180 rpm and 30 ℃ for 30 min. Taking 1 mL of soil suspension for 10^-1-10 ^-7Serial concentration gradient dilutions were made and then 10 taken^-5、10^-6、10 ^-7Three dilutions were plated onto plates of synthetic low nutrient SNA medium and cultured in an inverted format at 28 ℃ for 2 d.

And (3) purification process: purifying by gradually transplanting mycelium ends. After the colonies are formed on the plate, hyphae at the edge of a single colony are picked and placed on a PDA culture medium plate, constant-temperature culture is continued at 28 ℃ until a pure colony is obtained, and the obtained colony is stored at 4 ℃.

And (3) re-screening: inoculating the obtained colony to a 98-hole enzyme label plate containing 200 mul of inorganic salt culture medium, and using a Microscreen instrument to detect the growth speed at 28 ℃ and select 20 strains with the highest growth speed; and centrifuging the obtained culture solution of 20 strains of bacteria, determining the protein content of the culture by using a total organic carbon/total nitrogen analyzer (N/C2100S), screening out the strain with the highest protein content (No. TB 01), and storing the obtained bacterial colony at 4 ℃ for morphological characteristics and molecular identification.

Wherein the formula of the synthetic low-nutrient agar SNA culture medium is as follows: KH (Perkin Elmer)₂PO₄ 1 g/L，KNO₃ 1g/L，MgSO₄0.5g/L, KCl 0.5g/L, sucrose 0.2 g/L, agar 20 g/L, autoclaved (121 ℃ C.) for 20 min. Adding a proper amount of antibiotics and chloramphenicol (inhibiting gram-positive bacteria) into the SNA culture medium, wherein the mother solution is 50 mg/mL, and the working concentration is 50 mg/L; streptomycin (inhibiting gram-negative bacteria), the mother liquor is 350 mg/mL, and the working concentration is 350 mg/L; and uniformly mixing the prepared two antibiotics according to the ratio of 1:1 for later use, and adding the SNA culture medium according to the ratio of 1:1 when the culture medium is cooled to 50 ℃.

Wherein, the formula of the PDA culture medium is as follows: weighing 200 g of potato, cleaning, peeling, cutting, adding 1000 ml of water, boiling for half an hour, filtering with gauze, adding 20 g of glucose and 20 g of agar, dissolving completely, filtering with gauze, packaging, and sterilizing with high pressure steam (121 deg.C) for 20 min.

Inorganic salt culture medium: na (Na)₃C₆H₅O₇·2H₂O 2.6 g/L，KNO₃2.52 g/L，(NH4)₂SO₄2.88 g/L，KH₂PO₄ 1.6 g/L，MgSO₄0.2 g/L, 2% glucose was added, and the mixture was sterilized by autoclaving (121 ℃ C.) for 20 min.

Example 2 morphological characteristics of the Strain TB01

Strain TB01 was inoculated into PSA medium at 24-25 ℃ for 4 d. The strain is identified according to the growth speed, hypha shape, colony color, the number and shape of small conidia and large conidia, spore-producing cells, the shape and existence of chlamydospores, sporophore types and the like.

Culture properties: 4 d, the diameter of the colony is 3.8 cm, hypha on the PSA culture medium is flocculent at the initial stage, then the hypha is changed into flocculent to powder, the color is white to pink, the surface of the substrate is white, and the substrate does not change color;

morphological characteristics: the large conidia are uniform in size, thick and fat, uniform in middle, wedge-shaped in top cells, without heel of basal cells, separated by 3-4, and mostly separated by 4-6. The measurement is 28.32-42.50 μm × 4.63-4.54 μm; the number of microconidia is small or small; no chlamydospore; the single bottle of peduncle produces spores, and the sexual stage is not seen.

Wherein, potato sucrose agar medium (PSA medium): weighing 200 g of potato, cleaning, peeling, cutting, adding 1000 ml of water, boiling for half an hour, filtering with gauze, adding 20 g of sucrose and 20 g of agar, dissolving completely, filtering with gauze, packaging, and sterilizing with high pressure steam (121 deg.C) for 20 min.

Example 3 ITS sequence analysis of Strain TB01

Inoculating the strain TB01 into a PDA culture medium, culturing for 24 h at 30 ℃ and 180 rpm by a shaking table, collecting thalli, extracting total DNA, and then carrying out PCR amplification on ITS gene sequences by using the strain TB01 as a template and adopting the following general primers of the ITS gene sequences of eukaryotes according to a conventional method: FP1: 5'-AGTAAAAGTCGTAACAAGGT-3' and FP2: 5'-TTCACTCGCCGTTACTAGGG-3'.

After the amplification product is separated by 1% agarose gel electrophoresis, the amplification product is recovered by a gel recovery kit and submitted to sequencing by Beijing Ongzhike Biotechnology Co. Sequencing results show that the ITS gene sequence of the strain is shown in SEQ ID No. 1. It was aligned to sequences in the GenBank database and subjected to multiple sequence homology analysis using MEGA 7.0 software, and a phylogenetic tree was constructed as shown in fig. 3.

According to morphological characteristics and TIS sequence analysis, the strain TB01 is fusariumFusarium venenatumIs named as fusariumFusarium venenatumTB 01. The strain is preserved in China general microbiological culture Collection center (CGMCC) at 10 months and 12 days in 2020, and the preservation number is CGMCC number 20740.

Example 4 production of hyphal proteins by fermentation with different Nitrogen sources

Inoculating fusarium TB01 into a PDA plate seed culture medium, and culturing for 48 h at 30 ℃. The activated strain was inoculated with 3 loops of inoculum rings into a 100 mL shake flask containing 20 mL seed medium and incubated at 30 ℃ and 250 rpm for 48 h. According to the inoculation amount of 5 percent, 5 ml of seed liquid is inoculated into a 250 ml shake flask containing 95 ml of different nitrogen source fermentation culture media, and the seed liquid is cultured for 48 hours at 30 ℃ and 250 rpm. Centrifugally collecting the zymocyte protein, washing for three times by using sterile water, and drying for 24 hours at 60 ℃. Weighing 0.1g of dry mycelium into 20 ml of sterile water, shaking up, then using an ultrasonic instrument to completely and uniformly mix the mycelium to form a uniform solution, using a total organic carbon/total nitrogen analyzer (N/C2100S) to measure the total nitrogen content and calculating the protein content of the mycelium.

The influence of different nitrogen sources on the protein content is studied, and when ammonium sulfate is used as the nitrogen source, the protein content is increased along with the increase of the concentration of the ammonium sulfate; when urea is used as a nitrogen source, the protein content can also reach more than 45 percent; the highest protein content of peptone in the organic nitrogen source can reach 55.8%.

TABLE 1 Effect of different nitrogen sources on protein content

As can be seen from the above table, although different nitrogen sources are used, the contents of hyphal proteins in the obtained mycelia are all more than 40%, different nitrogen sources have different influences on the protein content, and when ammonium sulfate is used as the nitrogen source, the protein content increases with the increase of the concentration of the ammonium sulfate; when urea is used as a nitrogen source, the protein content can also reach more than 45 percent; the highest protein content of peptone in the organic nitrogen source can reach 55.8%.

Among them, the seed culture medium used in this example: 2.6 g/L Na₃C₆H₅O₇·2H₂O，2.52 g/L KNO₃，2.88 g/L (NH₄)H₂PO₄，1.6 g/L KH₂PO₄，0.2 g/L MgSO₄·7H₂O，0.1 g/L CaCl₂·2H₂O, adding 2% of glucose; the different nitrogen source fermentation culture media are as follows: 2.6 g/L Na₃C₆H₅O₇·2H₂O，2.52 g/L KNO₃，1.6 g/L KH₂PO₄，0.2 g/L MgSO₄·7H₂O，0.1 g/L CaCl₂·2H₂O, nitrogen source type and content are shown in Table 1, and glucose is 5%.

Example 5 fermentation production of hyphal proteins by addition of collagen peptide

Inoculating fusarium TB01 into a PDA plate seed culture medium, and culturing for 48 h at 30 ℃. The activated strain was inoculated with 3 loops of inoculum rings into a 100 mL shake flask containing 20 mL seed medium and incubated at 30 ℃ and 250 rpm for 48 h. According to the inoculation amount of 5 percent, 5 ml of seed liquid is inoculated into a 250 ml shake flask containing 95 ml of collagen peptide nitrogen source fermentation medium, and the culture is carried out for 48 h at 30 ℃ and 250 rpm. Centrifugally collecting the zymocyte protein, washing for three times by using sterile water, and drying for 24 hours at 60 ℃. Weighing 0.1g of dry mycelium into 20 ml of sterile water, shaking up, then using an ultrasonic instrument to completely and uniformly mix the mycelium to form a uniform solution, using a total organic carbon/total nitrogen analyzer (N/C2100S) to measure the total nitrogen content and calculating the protein content of the mycelium. The fermentation medium without collagen peptide was set as a control (i.e., no collagen peptide was added relative to the collagen peptide nitrogen source fermentation medium) in the experiment, which was repeated three times, and the results were recorded and calculated. The experimental result shows that the content of protein in mycelium with collagen peptide as nitrogen source is 46.82 + -0.62%, the content of mycelium protein obtained by fermentation without adding collagen peptide is only 44.87 + -0.53%, and the ratio of protein content increase is 4.34%. This experiment also showed that the protein content of the hyphal proteins was very high (up to 44.87%) even without the addition of collagen peptide as a nitrogen source. But compared with fermentation without adding collagen peptide, the content of the mycelium protein is improved a certain amount by adding the collagen peptide.

Wherein the seed culture medium: 2.6 g/L Na₃C₆H₅O₇·2H₂O，2.52 g/L KNO₃，2.88 g/L (NH₄)H₂PO₄，1.6 g/L KH₂PO₄，0.2 g/L MgSO₄·7H₂O，0.1 g/L CaCl₂·2H₂O, adding 2% of glucose; the collagen peptide nitrogen source fermentation medium comprises: 10 g/L yeast powder, 2.6 g/L Na₃C₆H₅O₇·2H₂O，2.52 g/L KNO₃，2.88 g/L (NH₄)H₂PO₄，1.6 g/L KH₂PO₄，0.2 g/L MgSO₄·7H₂O，0.1 g/L CaCl₂·2H₂O, 1g/L of collagen peptide, and 5% of added glucose.

Example 6 production of hyphal protein by fermentation with addition of whey protein peptide

Inoculating fusarium TB01 into PDA plate seed culture medium, and culturing at 30 deg.C for 48 h. The activated strain was inoculated with 3 loops of inoculum rings into a 100 mL shake flask containing 20 mL seed medium and incubated at 30 ℃ and 250 rpm for 48 h. According to the inoculation amount of 5 percent, 5 ml of seed liquid is inoculated into a 250 ml shake flask containing 95 ml of whey protein peptide nitrogen source fermentation medium, and the culture is carried out for 48 hours at 30 ℃ and 250 rpm. Centrifugally collecting the zymocyte protein, washing for three times by using sterile water, and drying for 24 hours at 60 ℃. Weighing 0.1g of dry mycelium into 20 ml of sterile water, shaking up, then using an ultrasonic instrument to completely and uniformly mix the mycelium to form a uniform solution, using a total organic carbon/total nitrogen analyzer (N/C2100S) to measure the total nitrogen content and calculating the protein content of the mycelium.

The fermentation medium without whey protein peptide was set as a control in the experiment (i.e. no whey protein peptide was added relative to the whey protein peptide nitrogen source fermentation medium), and the experiment was repeated three times, and the results were recorded and calculated. The experimental result shows that the hypha protein is obtained by adding the lactalbumin peptide for fermentation, and the content of the protein reaches 48.04 +/-0.81 percent through detection. The content of hypha protein obtained by fermentation of the fermentation medium without adding the whey protein peptide is 44.87 +/-0.53%. Therefore, the content of the hypha protein obtained by adding the whey protein peptide for fermentation is improved by 7.06 percent.

Wherein the seed culture medium: 2.6 g/L Na₃C₆H₅O₇·2H₂O，2.52 g/L KNO₃，2.88 g/L (NH₄)H₂PO₄，1.6 g/L KH₂PO₄，0.2 g/L MgSO₄·7H₂O，0.1 g/L CaCl₂·2H₂O, adding 2% of glucose; the whey protein peptide nitrogen source fermentation medium comprises: 10 g/L yeast powder, 2.6 g/L Na₃C₆H₅O₇·2H₂O，2.52 g/L KNO₃，2.88 g/L (NH₄)H₂PO₄，1.6 g/L KH₂PO₄，0.2 g/L MgSO₄·7H₂O，0.1 g/L CaCl₂·2H₂O, 1g/L of whey protein peptide, and 5% of added glucose.

Example 7 production of hyphal protein by fermentation with addition of whey protein

Fusarium TB01 was inoculated into PDA plate medium and cultured for 48 h at 30 ℃. The activated strain was inoculated with 3 loops of inoculum rings into a 100 mL shake flask containing 20 mL seed medium and incubated at 30 ℃ and 250 rpm for 48 h. According to the inoculation amount of 5 percent, 5 ml of seed liquid is inoculated into a 250 ml shake flask containing 95 ml of whey protein nitrogen source fermentation medium, and the culture is carried out for 48 h at 30 ℃ and 250 rpm. Centrifugally collecting the zymocyte protein, washing for three times by using sterile water, and drying for 24 hours at 60 ℃. Weighing 0.1g of dry mycelium into 20 ml of sterile water, shaking up, then using an ultrasonic instrument to completely and uniformly mix the mycelium to form a uniform solution, using a total organic carbon/total nitrogen analyzer (N/C2100S) to measure the total nitrogen content and calculating the protein content of the mycelium.

The experiment was repeated three times with the results recorded and calculated by setting the fermentation medium without whey protein addition as a control (i.e. without whey protein addition relative to the whey protein nitrogen source fermentation medium). The experimental result shows that the content of the mycelium protein produced by adding the whey protein through fermentation can reach 61.64 +/-0.79 percent through detection. The content of hypha protein obtained by fermentation of the fermentation medium without adding the whey protein peptide is 44.87 +/-0.53%. Therefore, the content of the mycelium protein produced by fermentation by adding the whey protein is increased by 37.37 percent.

The obtained hyphal proteins were analyzed by LC-MS, and the results are shown in Table 5. The whey protein is added as an organic nitrogen source, so that the proportion of essential amino acid in the mycelium protein is increased, the range is increased from 10% to 77%, and the nutritional value of the mycelium protein is increased.

TABLE 2 amino acid analysis of hyphal proteins

Note: essential amino acids: valine, isoleucine, methionine, tryptophan, threonine, lysine, phenylalanine, leucine.

As can be seen from the table above, the fermentation medium added with whey protein can obviously improve the proportion of essential amino acid in the mycelium protein, the improvement range is from 10% to 77%, and the nutritive value of the mycelium protein is increased. Seed medium used in this example: 2.6 g/L Na₃C₆H₅O₇·2H₂O，2.52 g/L KNO₃，2.88 g/L (NH₄)H₂PO₄，1.6 g/L KH₂PO₄，0.2 g/L MgSO₄·7H₂O，0.1 g/L CaCl₂·2H₂O, adding 2% of glucose; the whey protein nitrogen source fermentation medium comprises: 10 g/L yeast powder, 2.6 g/L Na₃C₆H₅O₇·2H₂O，2.52 g/L KNO₃，2.88 g/L (NH₄)H₂PO₄，1.6 g/L KH₂PO₄，0.2 g/L MgSO₄·7H₂O，0.1 g/L CaCl₂·2H₂O, 1g/L of whey protein, and 5% of added glucose.

Sequence listing

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Claims

1. A strain of Fusarium which produces protein isFusarium venenatumThe microbial inoculum is preserved in China general microbiological culture Collection center on 12 months 10 and 2020, and the preservation number is CGMCC 20740.

2. Use of fusarium of claim 1 for the production of hyphal proteins.

3. A method for producing hyphal protein by using the fusarium of claim 1, wherein the fusarium is fermented to produce the hyphal protein, and a nitrogen source of a fermentation medium of the fusarium is an inorganic nitrogen source or an organic nitrogen source.

4. A method for producing hyphal protein by using fusarium of claim 3, wherein the inorganic nitrogen source is urea or (NH4)₂SO₄。

5. A method for producing hyphal protein by using fusarium of claim 3, wherein the organic nitrogen source is peptone, yeast powder or soybean cake powder.

6. A method for producing hyphal protein by using fusarium of any one of claims 3 to 5, wherein collagen peptide, whey protein peptide or whey protein is added in a fermentation medium of the fusarium.

7. A method for producing hyphal protein by using fusarium of claim 6, wherein the collagen peptide, the whey protein peptide or the whey protein is added in an amount of 0.5g/L-2g/L in a culture medium.

8. A method for producing hyphal protein by using fusarium of claim 3, wherein the organic nitrogen source is collagen prepared by enzymolysis of skin or bone of pig, cow and sheep and aquatic leftovers.

9. A method for producing hyphal protein by using fusarium of claim 8, wherein the aquatic leftovers are fish skin, fish scales or fish bones.

10. A method for producing hyphal proteins using Fusarium of claim 3 wherein the organic nitrogen source is a whey by-product formed from the production of cheese or casein.