Seawater-tolerant monascus and application thereof in high-yield protein

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a monascus capable of resisting seawater and application of the monascus in high-yield protein. According to the specific technical scheme, the monascus purpureus HXJ5 is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number of the monascus purpureus HXJ5 is CGMCC No.42056, and the preservation date of the monascus purpureus HXJ5 is July 14, 2025. The content of protein in the monascus purpureus is as high as 37%. And the monascus purpureus can tolerate salt with the concentration of 15%, and can normally grow in a high-salt environment and continuously produce the microbial protein at high yield.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a red Monascus strain that can tolerate seawater and its application in high-protein production. Background Technology

[0002] Fungal proteins, with their advantages of high quality and low cost, have gradually become a new protein source. Fungal proteins (Mycoprotein) refer to proteins obtained by culturing specific fungi. They were first discovered by British researchers in the 1960s, approved for use in food in 1985, and have been gradually promoted globally.

[0003] However, the production of fungal proteins still faces significant limitations: current research mainly focuses on Fusarium, lacking strains with greater diversity and higher protein content. On the other hand, freshwater resources are precious, and most fungi have poor tolerance to high-salt environments, making it impossible to directly utilize high-salt resources such as seawater for fermentation. If seawater could be used for direct fermentation, the production cost of fungal proteins could be further reduced.

[0004] Therefore, there is an urgent need to develop novel strains that combine high protein content and high salt tolerance in order to overcome the technical bottlenecks in the fungal protein industry. Summary of the Invention

[0005] The purpose of this invention is to provide a red Monascus strain that can tolerate seawater and its application in high-protein production.

[0006] To achieve the above-mentioned objectives, the technical solution adopted by this invention is: a strain of Monascus purpureus HXJ5, which is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 42056 and accession date of July 14, 2025; the ITS sequence of the Monascus purpureus is shown in SEQ ID NO: 1.

[0007] Accordingly, the application of the purple red Monascus in the preparation of bacterial protein.

[0008] Accordingly, microbial preparations made using the purple Monascus or containing the purple Monascus; and the application of the microbial preparations in the preparation of microbial proteins.

[0009] The present invention has the following beneficial effects: The present invention provides a new purple red Monascus strain with a protein content as high as 37%; and the purple red Monascus strain can tolerate a salt concentration of 15%, and can grow normally and continuously produce high levels of microbial protein in a high-salt environment. Attached Figure Description

[0010] Figure 1This is a diagram showing the protein content of the top 30 strains in terms of protein content.

[0011] Figure 2 A schematic diagram showing the protein content of each strain in simulated seawater culture medium;

[0012] Figure 3 A schematic diagram showing the dry weight of each bacterial strain in simulated seawater culture medium;

[0013] Figure 4 This is a schematic diagram showing the dry weight of strain 1 in culture media with different salt concentrations.

[0014] Figure 5 This is a schematic diagram showing the protein content of strain 1 in culture media with different salt concentrations.

[0015] Figure 6 This is a colony morphology diagram of strain 1 on PDA medium.

[0016] Figure 7 This is a morphological diagram of strain 1;

[0017] Figure 8 This is the phylogenetic tree of strain 1. Detailed Implementation

[0018] This invention provides a novel strain of Monascus purpureus HXJ5, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 42056 and deposit date of July 14, 2025.

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art, and the data obtained are all average values ​​obtained after at least three repetitions, and each repetition yields valid data.

[0020] Example 1: Screening for high-protein-producing strains

[0021] 1. Eighty standard strains were purchased from the China General Microbiological Culture Collection Center (CGMCC) and the American Type Culture Collection (ATCC). Combined with 270 native fungi isolated and purified from traditional yeast in our laboratory, a total of 350 fungi were used as screening targets.

[0022] 2. Initial screening of high-protein-producing strains

[0023] Primary screening medium: Potato glucose medium (PDB), with a formula of 200 g / L potato extract and 20 g / L glucose, natural pH, sterilized at 121°C for 20 minutes.

[0024] Each strain was inoculated at a rate of 5% (v / v), and the spore solution was pre-observed using microscopic techniques and diluted to 1×10⁻⁶. 5 The mycelium was inoculated at a density of 1 / mL into 100mL of PDB medium and cultured at 30℃ with shaking at 120rpm for 5 days until the end of the logarithmic growth phase. Subsequently, the fermentation broth was centrifuged at 4℃ and 8000rpm for 15 minutes, the supernatant was discarded, the mycelium was collected, the mycelium was washed 3 times with sterile water, and freeze-dried to constant weight.

[0025] The total nitrogen content of each mycelium was determined using an elemental analyzer (Elementar Vario ELⅢ). 2.0 mg of each freeze-dried mycelium was accurately weighed, placed in a tin boat, and the nitrogen content was measured using the elemental analyzer. Protein content = total nitrogen content × 6.25, where 6.25 is a universal conversion factor.

[0026] The top 30 strains in protein production were selected for secondary screening. The results are as follows: Figure 1 As shown. Figure 1 Strain number 1 in the strain has a protein content of 37%, ranking first; compared to the second-ranked Fusarium (…). Figure 1 The strain numbered 2 was 3% higher.

[0027] Example 2: Secondary screening of strains that can tolerate high salt

[0028] The culture medium formula for the simulated seawater experimental group was as follows: sucrose 30 g / L, NaNO3 3 g / L, MgSO4·7H2O 0.5 g / L, KCl 0.5 g / L, FeSO4·7H2O 0.01 g / L, K2HPO4 1 g / L, NaCl 23.0 g / L, and MgCl2 12 g / L. After preparation, the pH was adjusted to 6.5 ± 0.2, and sterilized at 121℃ for 20 minutes. NaCl and MgCl2 were used to simulate the concentration and types of salt in seawater, with a total salt concentration of 35.0 g / L.

[0029] The real seawater experimental group refers to the use of real seawater taken from the East my country Sea (26°37′N, 124°04′E), filtered to remove visible impurities, and used to replace the purified water in the culture medium of the simulated seawater experimental group to prepare the culture medium, without the addition of NaCl and MgCl2, and sterilized at 121℃ for 20 minutes; the total salt concentration of the real seawater was measured to be 34.3 g / L, and the main salts were NaCl and MgCl2.

[0030] The blank control group refers to the group that is treated the same as the simulated seawater experimental group except that NaCl and MgCl2 are not added to the culture medium.

[0031] The 30 strains obtained from the initial screening were inoculated at a rate of 5% (v / v), and the spore solution was pre-observed using microscopic techniques and diluted to 1×10⁻⁶. 5 Cells / mL were inoculated into the culture medium of the simulated seawater experimental group and cultured at 30℃ and 120 rpm for 5 days. Subsequently, the dry weight (DCW) and protein content were measured. DCW was determined by centrifuging the fermentation broth (8000 rpm, 15 min), freeze-drying the mycelium, and weighing the resulting dry weight data. Protein content was determined using the same method as the initial screening. Results are as follows: Figure 2 , 3 As shown.

[0032] Strains with no significant decrease in DCW (≥90% of the DCW level in the blank control group) and the highest protein content were selected. The results showed that strain 1 was able to tolerate simulated seawater and had the highest DCW and protein content.

[0033] 4. Tests on the high salt and seawater tolerance of strain 1

[0034] Strains were inoculated into the simulated seawater experimental group culture medium. With other conditions unchanged, the salt concentration in the culture medium was adjusted to 7%, 10%, 15% and 20% by adjusting the NaCl content. The culture was then incubated at 30℃ and 120rpm for 5 days.

[0035] The results are as follows Figure 4 and Figure 5 Strain 1 can still grow normally in an environment with a salt concentration of 15%, and the protein content can still reach 35%.

[0036] Strain 1 was inoculated into both the simulated seawater experimental group and the real seawater experimental group, with the inoculation amount and culture conditions remaining unchanged. The results showed that strain 1 grew well in both simulated and real seawater. After 5 days, the DCW in simulated seawater was 9.7 g / L, and the protein content was 37%; while in real seawater, the DCW was 9.6 g / L, and the protein content was 38%.

[0037] Example 3: Identification of strain 1 and demonstration of other properties

[0038] 1. Physiological and biochemical characteristics: Strain 1 forms pale pink, round colonies with obvious aerial hyphae on PDA solid medium; on MCD solid medium, strain 1 forms pale yellow, round colonies with obvious aerial hyphae. Experiments show that the optimal growth temperature range for strain 1 is 22–35℃, with an extreme temperature tolerance range of 4–43℃; the optimal growth pH range is 3.5–6.5, with an extreme pH tolerance range of 2.0–8.0; the optimal growth salinity range is 5–12%, with an extreme salinity tolerance range of 0–16%.

[0039] The colony morphology diagram of strain 1 is shown below. Figure 6 As shown, the morphology of the strain under the microscope is as follows: Figure 7 As shown.

[0040] 2. ITS Identification: The cultured bacterial culture was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing and identification, and its ITS sequence was sequenced. The obtained sequence is shown in SEQ ID NO:1. The obtained DNA sequence was entered into NCBI for BLAST search. Clustalx and MEGA software were used to compare and analyze the obtained nucleotide sequence with the corresponding sequences of similar strains included in NCBI, and a phylogenetic tree was constructed (e.g., Figure 8 (As shown). Strain No. 1 was found to be most closely related to Monascus purpureus (identity = 99.1%). Based on its morphological and physiological biochemical characteristics, strain No. 1 was preliminarily identified as Monascus purpureus.

[0041] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Any modifications, alterations, substitutions, or variations made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention shall fall within the protection scope defined by the claims of the present invention.

Claims

1. A strain of purple red Monascus (Monascus purpureus) HXJ5, characterized by: The purple Monascus is deposited at the China General Microbiological Culture Collection Center, accession number: CGMCC No.42056, deposit date: July 14, 2025.

2. A strain of purple red Monascus (Monascus purpureus) HXJ5, characterized by: The ITS sequence of the purple red Monascus is shown in SEQ ID NO:

1.

3. The use of the purple red Monascus according to claim 1 or 2 in the preparation of bacterial protein.

4. A bacterial preparation made using the purple Monascus as described in claim 1 or 2.

5. A bacterial preparation containing the purple red Monascus as described in claim 1 or 2.

6. The use of the bacterial preparation according to claim 4 or 5 in the preparation of bacterial protein.

7. The application according to any one of claims 3 to 6, characterized in that: In the application described, the salt concentration of the growth environment for the purple red Monascus is ≤16%.

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