Monascus purpureus with high yield of essential amino acids as well as screening method and application of monascus purpureus

Abstract

The invention belongs to the field of microorganisms, and particularly relates to monascus purpureus with high yield of essential amino acids as well as a screening method and application thereof. The invention provides a novel trans-membrane differentiation ARTP mutagenesis method aiming at filamentous fungi, aiming at the characteristics of multiple cores and thick walls of monascus, protoplast pretreatment and repair liquid components before mutagenesis are optimized, so that the fatality rate and the positive mutation rate are optimally balanced, and the problem of high instability of monascus heredity caused by conventional ARTP is solved. Based on the novel mutagenesis method, the mutant strain AZA5 is obtained through successful mutagenesis screening. The essential amino acid content of AZA5 is as high as 58%. The invention not only provides an excellent strain resource for industrial production of high-quality microbial protein, but also has important scientific value and wide industrial application potential.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, specifically relating to a strain of *Monascus purpureus* that produces high levels of essential amino acids, its screening method, and its application. Background Technology

[0002] With global population growth and rising living standards, the demand for high-quality protein is increasing. Traditional animal protein production methods face problems such as high resource consumption, severe environmental pollution, and long production cycles. Therefore, developing alternative protein sources has become an important research direction in the fields of food science and biotechnology.

[0003] Microbial proteins, as an emerging protein source, have attracted much attention in recent years due to their advantages such as short production cycles, lack of seasonal and geographical limitations, and high nutritional value. Fungal proteins, in particular, are considered one of the most promising microbial protein sources because of their balanced amino acid composition and rich content of essential amino acids.

[0004] Essential amino acids are those that humans and animals cannot synthesize themselves and must obtain from external sources. The content of essential amino acids and their proportion of total amino acids directly determine the nutritional and biological value of proteins. The higher the proportion of essential amino acids in a protein, the better it can meet the nutritional needs of humans and animals. One of the key challenges currently facing the microbial protein industry is increasing the content of essential amino acids to produce truly high-quality protein products with high nutritional value. Summary of the Invention

[0005] The purpose of this invention is to provide a high-yield strain of essential amino acids from *Monascus purpureus*, along with its screening method and applications.

[0006] To achieve the above-mentioned objectives, the technical solution adopted in this invention is: a novel *Monascus purpureus* AZA5 strain, which was deposited on July 14, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC NO.42055. The ITS sequence of *Monascus purpureus* AZA5 is shown in SEQ ID NO: 1.

[0007] Accordingly, a microbial preparation made using the aforementioned *Monascus purpureus* AZA5; or a microbial preparation containing the aforementioned *Monascus purpureus* AZA5.

[0008] Accordingly, the application of the *Monascus purpureus* AZA5 or the bacterial preparation in the preparation of microbial proteins.

[0009] Accordingly, a method for inducing mutation in Monascus purpureus includes the following steps:

[0010] (1) Synchronization pretreatment of the starting strain spores: The spore solution of the starting strain was placed in an environment of 4℃ for 18-36h to make all spores dormant;

[0011] (2) ARTP mutagenesis: The synchronized spore liquid was evenly spread on a metal slide for ARTP mutagenesis. The mutagenesis treatment time was selected to be a longer mutagenesis time within the range of 90%±2% lethality.

[0012] (3) Specific membrane repair and genetic fixation: After mutagenesis, the slides are immersed in the repair solution within 30 seconds, and then incubated at 20-25°C in the dark for 2-4 hours for repair. The repair solution contains calcium ions and sorbitol. Preferably, the repair solution formulation includes: buffer solution (e.g., MES buffer), sorbitol, calcium ions (e.g., CaCl2), and antioxidant (e.g., glutathione).

[0013] (4) Pressure screening: The strains that have undergone the first round of mutagenesis are inoculated onto the screening medium and cultured at 20-30℃ for 2-4 days. 2-4 colonies with the largest colony diameter are selected. Spores are prepared from the selected colonies and subjected to ARTP mutagenesis again according to the method in step (2). The newly mutagenized strains are then inoculated onto a new screening medium. This pressure screening process is repeated more than 5 times to obtain the desired strains. The screening medium is a medium with monosodium glutamate and aspartic acid as the sole carbon source. Preferably, the formulation of the screening medium includes: glucose, KH2PO4, Na2HPO4, MgSO4, CaCl2, ZnSO4•7H2O, FeSO4•7H2O, CoSO4•7H2O, CuSO4•5H2O, MnSO4•H2O, monosodium glutamate, and aspartic acid.

[0014] Accordingly, the red yeast rice was prepared using the method described above.

[0015] Accordingly, the application of the red yeast in the preparation of microbial proteins.

[0016] The present invention has the following beneficial effects:

[0017] To address the challenge of the multinucleate genetic instability of Monascus purpureus, this invention provides a novel transmembrane differential ARTP mutagenesis method for filamentous fungi. Taking advantage of Monascus purpureus's multinucleate and thick-walled characteristics, this invention optimizes the protoplastization pretreatment and the composition of the repair solution before mutagenesis, achieving an optimal balance between lethality and positive mutation rate, overcoming the high genetic instability of conventional ARTP in Monascus purpureus. The repair solution provided by this invention can rapidly seal membrane damage, and by adjusting the initiation timing of the SOS repair system, it promotes rapid fixation of Monascus purpureus during early nuclear division in ARTP mutagenesis, significantly reducing the phenotypic segregation rate in subsequent passages.

[0018] Based on this novel mutagenesis method, this invention successfully obtained the mutant strain AZA5 by mutagenesis screening using the preserved Monascus purpureus strain HXJ5 as the starting strain. The essential amino acid content of AZA5 was increased to 51%. On this basis, through systematic optimization of culture conditions, the essential amino acid content of AZA5 was further increased to 57.8%.

[0019] The strain AZA5 provided by this invention has an amino acid score (AAS) of over 100 for all essential amino acids, no limiting amino acids, an essential amino acid index (EAAI) of 145.8, and a biological value (BV) of 147.2, which is significantly better than traditional high-quality protein sources such as egg protein and soybean protein.

[0020] In summary, this invention successfully discovered and identified Aspergillus purpureus AZA5, which has a very high proportion of essential amino acids in the total amino acid profile. This not only provides an excellent strain resource for the industrial production of high-quality microbial proteins, but also has significant scientific value and broad potential for industrial application. Attached Figure Description

[0021] Figure 1 A schematic diagram showing the relationship between different mutagenesis times and spore lethality;

[0022] Figure 2 A schematic diagram showing the relationship between different mutagenesis rounds and the maximum colony diameter;

[0023] Figure 3 Comparison of colony morphology between strain HXJ5 (top) and strain AZA5 (bottom). Detailed Implementation

[0024] This invention provides a novel *Monascus purpureus* strain AZA5, whose ITS sequence is shown in SEQ ID NO: 1. It was deposited on July 14, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO. 42055. The *Monascus purpureus* AZA5 strain has an essential amino acid content as high as 58%.

[0025] This invention also provides a mutagenesis and screening method for improving the content and quality of essential amino acids in Monascus purpureus and obtaining Monascus purpureus with high content and high quality of essential amino acids. The method specifically includes the following steps:

[0026] 1. Synchronization pretreatment of the starting strain spores: The spore solution of the starting strain was placed in an environment of 4℃ for 18-36 hours to ensure that all spores were in a dormant period.

[0027] 2. ARTP Mutagenesis: The synchronized spore solution was evenly spread onto a metal slide. The helium flow rate was set to 10 SLM, the radio frequency power to 120W, the treatment distance to 2mm, and the treatment temperature to 25℃. The mutagenesis treatment time was selected to be a longer time within the range of 90%±2% lethality.

[0028] 3. Specific Membrane Repair and Genetic Fixation: After mutagenesis, the slides were immersed in the repair solution within 30 seconds, ensuring the slides were completely submerged. The slides were then incubated at 20–25°C in the dark for 2–4 hours. The repair solution consisted of: 10–100 mmol / L MES buffer (pH=6.0); 0.2–1.5 mol / L sorbitol; 0.1–1.4 mol / L CaCl2; and 0.03–3 mmol / L glutathione.

[0029] In CaCl2, calcium ions are the primary active component. Common calcium salts that do not significantly inhibit microbial growth, such as calcium nitrate and calcium acetate, can be used here. Besides calcium ions, magnesium ions can also play a role among other divalent cations, but their effect is not as ideal as calcium ions. Other divalent cations are either toxic to cells or cannot bind to the cell membrane, so they cannot be used here. Furthermore, glutathione can be replaced by other antioxidants that are non-toxic to microorganisms, such as vitamin C.

[0030] 4. Stress screening: Inoculate the strains that have undergone the first round of mutagenesis onto screening medium and incubate at 20–30°C for 2–4 days. Select 2–4 colonies with the largest diameter. Prepare spores from the selected colonies and perform ARTP mutagenesis again using the method in step 2. Then, inoculate the re-mutated strains onto a new screening medium. Repeat this stress screening process at least 5 times to obtain the desired strain.

[0031] The screening medium is a medium with monosodium glutamate and aspartic acid as the sole carbon sources.

[0032] 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.

[0033] Example 1: Mutagenesis to obtain strain AZA5

[0034] 1. Synchronization pretreatment of starting strain spores:

[0035] To ensure the consistency of mutagenesis, the Monascus purpureus spores needed to be synchronized. The starting strain (Monascus purpureus, HXJ5, accession number: CGMCC No. 42056, disclosed in the inventors' prior patent "A Monascus purpureus strain tolerant to seawater and its application in high protein production", patent number 202511539476.7) was inoculated onto PDA plates and cultured at 28°C for 7 days. The spores on the surface of the hyphae were eluted into the solution (sterile physiological saline containing 0.1% Tween-80) using sterile physiological saline containing 0.1% Tween-80. The mycelia were removed by filtration through four layers of sterile gauze, and the filtrate was the spore solution.

[0036] The spore suspension was subjected to a 24-hour "cold shock" treatment at 4°C to ensure all spores were in the same dormant stage (G0 phase). The suspension was then collected by centrifugation and resuspended to a concentration of 10. 6 per mL.

[0037] The purpose of this step includes:

[0038] (1) The spore suspension may contain newly awakened (S phase), dividing (M phase), and dormant (G0 phase) spores. During ARTP mutagenesis (atmospheric and room temperature plasma mutagenesis), all S phase spores die, while those in the G0 phase may not mutate. Keeping all spores in the G0 phase ensures that the energy of ARTP acts evenly on the same DNA structure, thus obtaining a stable "mutation rate" rather than a "lethal rate".

[0039] (2) Monascus purpureus is a multinucleate fungus. If the spores enter the growth and germination stage (even in the early stages), the cell nucleus may have already begun to divide (from a single nucleus to a binucleate or multinucleate nucleus). If there are already two or more nuclei in the cell at the time of mutagenesis, and ARTP only mutates one of the nuclei (mutant nucleus), while the remaining nuclei are normal (wild-type nuclei), a "heterokine" will be formed. In subsequent culture, the wild-type nucleus tends to grow faster and easily "masks" the mutant trait, causing the high-yielding strains selected to degenerate in a short period of time (most likely within a few days).

[0040] Therefore, forced cold shock synchronization can ensure that each spore is in a "monuclear" or "undivided" state when mutagenesis occurs, thus ensuring that the mutation is homozygous and improving the stability of subsequent genetics.

[0041] 2. Plotting ARTP mutagenesis and mortality curves

[0042] This step was performed using an ARTP-II mutagenesis breeding instrument. Take 10 μL of the spore solution synchronized in step 1 (diluted to a spore concentration of 1×10⁻⁶).5 Mutagenesis spores (number per mL) were uniformly coated onto a metal slide. Helium flow rate was 10 SLM, RF power was 120 W, processing distance was 2 mm, and processing temperature was 25℃. Processing times were set to 30 s, 45 s, 60 s, 75 s, 90 s, and 105 s. Lethality was determined by microscopic counting. The relationship between mutagenesis time and lethality of Monascus purpureus spores is as follows: Figure 1 As shown.

[0043] The optimal mutagenesis time should be chosen to be as long as possible, with a mortality rate of around 90%. Based on this selection criterion, a treatment time of 60 seconds resulted in a mortality rate of 91.5%, and this time was selected as the optimal mutagenesis time.

[0044] 3. Specific membrane repair and genetic fixation

[0045] After the mutagenesis in step 2 is completed in 60s, the slide is immersed in the specially prepared repair solution of this invention within 30s. The repair solution completely submerges the slide, and then it is placed at 25°C in the dark for 3 hours for repair. Shaking is strictly prohibited to prevent physical damage. The repair solution formula includes: (1) solvent: 50 mmol / L MES buffer (2-morpholinoethanesulfonic acid buffer, pH=6.0); (2) osmotic pressure regulator: 1.0 mol / L sorbitol; (3) membrane stabilizer: 0.2 mol / L CaCl2; (4) free radical scavenger: 1 mmol / L glutathione (GSH).

[0046] This step is crucial, and its specific functions include:

[0047] (1) The ARTP plasma beam can penetrate the cell wall and cell membrane, causing physical perforation. If the external solution concentration is low (hypotonic), water will rush into the cell, causing the cell to rupture (lysis). A high concentration of 1.0 mol / L of sorbitol creates a hypertonic environment, providing physical support around the cell like a "plaster splint" to prevent cell rupture. Monascus purpureus readily absorbs and metabolizes sucrose. If sucrose is used as a protectant, the cell will consume the "protective shield" while repairing, causing the osmotic pressure to drop rapidly and the protection to fail. However, Monascus purpureus metabolizes sorbitol very slowly. This allows sorbitol to remain outside the cell for a long time, maintaining a stable hypertonic environment and ensuring that the "protective shield" remains strong during the 3-hour repair period.

[0048] (2) Cell membrane phospholipids carry a negative charge. High concentrations (0.2 mol / L) of Ca 2+ (Divalent cations) can bind tightly to the phospholipid heads on the membrane, "tightening" the lipid bilayer through electrostatic interactions and rapidly sealing the tiny pores caused by ARTPs. Instantaneous fluctuations in intracellular calcium ion concentration are the signal for the cell to activate its "SOS repair mechanism." Therefore, maintaining a high-calcium environment helps regulate cell cycle checkpoints, allowing cells to temporarily halt division and focus on DNA error repair. This helps fix mutations.

[0049] (3) Glutathione can prevent the premature death of Monascus purpureus spores caused by mutagenesis. ARTP treatment not only breaks DNA but also generates a large amount of reactive oxygen species (ROS, such as hydroxyl radicals). These ROS remain in the cells after mutagenesis. This invention requires ROS to break DNA and cause mutations, but does not want ROS to kill cells. When DNA mutation has already occurred, GSH can prevent apoptosis caused by excessive oxidation. Adding GSH immediately within 30 seconds after mutagenesis can block ROS from damaging DNA.

[0050] (4) Incubation at 25°C in the dark is to preserve the mutation. Red Monascus cells contain a "photoreactivating enzyme." This enzyme rapidly repairs damaged DNA to match the wild type under light conditions. Therefore, incubation in the dark prevents the "photoreactivating enzyme" from functioning.

[0051] 3. Stress screening and mutagenesis

[0052] Selection medium (Czapek-Dox modified, unit: g / L): glucose 30, agar powder 15, KH2PO4 5, Na2HPO4 3, MgSO4 0.1, CaCl2 0.1, ZnSO4•7H2O 0.1, FeSO4•7H2O 0.1, CoSO4•7H2O 0.05, CuSO4•5H2O 0.02, MnSO4•H2O 0.01. Based on the selection medium, the sole nitrogen source was added: monosodium glutamate 5 g / L and aspartic acid 2 g / L (both non-essential amino acids). Strains that can utilize these two amino acids for rapid growth are those that synthesize essential amino acids quickly; otherwise, the strains will have difficulty growing.

[0053] The reasons for choosing glutamic acid and aspartic acid include: (1) Glutamic acid and aspartic acid are central nodes in the nitrogen metabolism of filamentous fungi and can provide amino acids for all amino acids, including human essential amino acids, through transamination. (2) Without the provision of any exogenous essential amino acids, the strain must rely entirely on de novo synthesis to build its cellular proteins. This constitutes a stringent selection pressure, which can effectively screen out mutant strains with a higher proportion of metabolic flow to the essential amino acid branch and stronger biosynthetic efficiency. (3) Aspartic acid is a direct metabolic precursor of essential amino acids such as lysine, methionine, and threonine.

[0054] Extensive testing has demonstrated that strains obtained using the pressure screening conditions of this invention exhibit a significant increase in the total amount of essential amino acids compared to the original strain.

[0055] The first-round mutated strain was inoculated onto the selection medium and cultured at 28°C for 3 days. The three colonies with the largest diameter were selected. Spores were prepared from the selected colonies and subjected to ARTP mutagenesis. The samples were then screened again in a selection medium supplemented with glutamic acid and aspartic acid. This process was repeated, often for 10 rounds, until the fastest-growing strain, AZA5, was obtained. The maximum colony diameter obtained in each of the 10 rounds of mutagenesis was as follows: Figure 2 As shown.

[0056] After 10 rounds of mutagenesis, the colony morphology of the starting strain HXJ5 and the mutant strain AZA5 after 3 days of growth on PDA medium was compared. Figure 3 As shown in the figure. The results showed that under the same culture conditions, the mutant strain grew significantly faster and had a significantly larger colony diameter than the original strain. Simultaneously, the pigment deposition on the reverse side of the mutant strain was lighter, indicating that mutagenesis effectively enhanced the strain's vegetative growth ability and may have altered its original metabolic distribution pattern.

[0057] Example 2: Identification of the mutant strain AZA5

[0058] The physiological and biochemical characteristics of strain AZA5 include: the diameter of the circular colonies formed by strain AZA5 on PDA and MCD solid media increased by approximately 15%–25% compared to the original strain, and the aerial hyphae on the surface were denser and more vigorous, indicating a significant increase in its growth rate. On PDA, the colonies maintained an orange-red color on the front, but the pigment deposition on the back changed from a deep red center in the original strain to a uniformly distributed light orange. The mutant strain exhibited a wider steady-state range: its optimal growth temperature range expanded to 22–38℃, and its growth activity at an extreme high temperature of 43℃ was significantly better than that of the original strain; the optimal growth pH range expanded to 3.0–7.0, demonstrating stronger acid tolerance.

[0059] Genomic DNA was extracted from strain AZA5, and the ITS was cloned from the genomic DNA, sequenced, and the sequencing results were compared using BLAST. The ITS sequencing results, as shown in SEQ ID NO 1, were completely identical to the originating strain HXJ5.

[0060] Based on the combined physiological and biochemical characteristics and molecular identification results, it was identified as *Monascus purpureus*. This indicates that the mutagenesis did not alter the species relationship of the originating strain.

[0061] Example 3: Amino acid content analysis of the starting strain and the mutagenized strain

[0062] 1. The starting strain HXJ5 and the mutant strain AZA5 were fermented separately. The fermentation medium was the screening medium from Example 1 with the addition of ammonium sulfate (nitrogen source), maintaining the medium at its natural pH (approximately 4.5). The carbon-to-nitrogen ratio of the fermentation medium was adjusted to 11:1 by adjusting the amount of ammonium sulfate. The spores of each strain (spore concentration of 1×10⁻⁶) were then collected. 5 The cells were inoculated into the fermentation medium at an inoculum size of 5% (v / v). After fermentation at 28℃ for 5 days, the cells were collected and the content of essential amino acids (EAA) was determined. The results are shown in Table 1.

[0063] Table 1. Comparison of essential amino acid contents between the starting strain HXJ5 and the mutant strain AZA5

[0064]

[0065] The results showed that the content of all essential amino acids in the mutant strain was significantly increased, and the proportion of essential amino acids in the total protein increased from 31% in the original strain to 51%. The amino acid scores (AAS) of the essential amino acids in the mutant strain all exceeded 100, with no limiting amino acids, and the essential amino acid index (EAAI) reached 145.8, and the biological value (BV) reached 147.2, which was significantly better than traditional high-quality protein sources such as egg protein and soybean protein.

[0066] 2. Optimization and adjustment of fermentation medium and fermentation conditions: The amount of ammonium sulfate was adjusted to achieve a carbon-to-nitrogen ratio of 7:1 in the fermentation medium, and lactic acid (acetic acid can also be used) was added to adjust the pH of the medium to 3.5. The starting strain HXJ5 and the mutant strain AZA5 were inoculated again into the new fermentation medium at an inoculum size of 5% (v / v). After fermentation at 32℃ for 5 days, the content of essential amino acids (EAA) was measured, and the results are shown in Table 2.

[0067] Table 2. Comparison of essential amino acid contents between the starting strain HXJ5 and the mutant strain AZA5

[0068]

[0069] The results showed that by systematically optimizing the culture conditions, the essential amino acid content of the mutant strain AZA5 was further increased to 57.8%.

[0070] 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 novel strain of *Monascus purpureus* AZA5, characterized by: The *Aspergillus purpureus* AZA5 was deposited at the China General Microbiological Culture Collection Center on July 14, 2025, with accession number CGMCC NO.42055.

2. The Aspergillus violaceus AZA5 according to claim 1, characterized in that: The ITS sequence of the *Aspergillus violaceus* AZA5 is shown in SEQ ID NO:

1.

3. A microbial preparation made using Monascus purpureus AZA5 as described in claim 1 or 2; or; a microbial preparation containing Monascus purpureus AZA5 as described in claim 1 or 2.

4. The use of the *Monascus purpureus* AZA5 of claim 1 or 2 or the bacterial preparation of claim 3 in the preparation of microbial proteins.

5. A method for inducing mutation of Monascus purpureus, characterized in that: The method includes the following steps: (1) Synchronization pretreatment of the starting strain spores: The spore solution of the starting strain was placed in an environment of 4℃ for 18-36h to make all spores dormant; (2) ARTP mutagenesis: The synchronized spore liquid was evenly spread on a metal slide for ARTP mutagenesis. The mutagenesis treatment time was selected to be a longer mutagenesis time within the range of 90%±2% lethality. (3) Specific membrane repair and genetic fixation: After the mutagenesis is completed, the slide is immersed in the repair solution within 30 seconds. The repair solution is used to immerse the slide and then it is placed at 20-25°C in the dark for 2-4 hours for repair. The repair solution contains calcium ions and sorbitol.

6. The method according to claim 5, characterized in that: The repair solution formulation in step (3) includes: buffer solution, sorbitol, calcium ions and antioxidants.

7. The method according to claim 5 or 6, characterized in that: The method further includes: (4) Pressure screening: Inoculate the strains that have undergone the first round of mutagenesis onto the screening medium and incubate them at 20-30℃ for 2-4 days. Select 2-4 colonies with the largest colony diameter. Prepare spores from the selected colonies and perform ARTP mutagenesis again according to the method in step (2). Then inoculate the strains that have undergone the second mutagenesis onto a new screening medium. Repeat this pressure screening process more than 5 times to obtain the desired strains. The screening medium is a medium with monosodium glutamate and aspartic acid as the sole carbon sources.

8. The method according to claim 7, characterized in that: The formulation of the screening medium includes: glucose, KH2PO4, Na2HPO4, MgSO4, CaCl2, ZnSO4•7H2O, FeSO4•7H2O, CoSO4•7H2O, CuSO4•5H2O, MnSO4•H2O, monosodium glutamate, and aspartic acid.

9. Monascus purpureus prepared by the method according to any one of claims 5 to 8.

10. The use of Monascus purpureus according to claim 9 in the preparation of microbial proteins.

Images