Method for improving monascus protein yield by using cyclohexyl acetic acid

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a method for increasing the yield of monascus protein by using cyclohexyl acetic acid. Aiming at the problem that the protein production capacity of the monascus is still to be improved, the invention firstly discovers that the cyclohexyl acetic acid can improve the protein synthesis and accumulation capacity of the monascus and can improve the protein yield of the monascus by about 50%. The method provided by the invention has the advantages of simplicity and convenience in operation, low cost and the like, and provides a theoretical basis and practical guidance for optimization of monascus fermentation protein production.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a method for increasing the protein yield of Monascus purpureus using cyclohexylacetic acid. Background Technology

[0002] Monascus spp., an important food and medicinal microorganism, has wide applications in the food and pharmaceutical industries. This strain can produce functional proteins and various physiologically active substances, including red yeast pigments and Monacolin K. In recent years, with the increasing global demand for sustainable protein resources, the production of alternative proteins through microbial fermentation has become a research hotspot. Due to its high protein content and good safety profile, Monascus spp. is considered a highly promising source of food protein alternatives.

[0003] However, the protein content produced by Monascus purpureus during liquid fermentation is still not ideal, limiting its industrial application. Therefore, developing simple, inexpensive, and efficient methods to increase protein yield is of great significance for promoting the industrial application of Monascus purpureus protein.

[0004] Quorum sensing signaling molecules, as key substances for intercellular communication in microorganisms, can regulate microbial quorum behavior and metabolic processes. Recent studies have found that certain small molecule compounds may serve as quorum sensing signaling molecules in microorganisms, influencing their growth and metabolite synthesis. However, the quorum sensing signaling molecules vary considerably among different microorganisms, and their effects on microbial growth and protein synthesis differ, indicating that research in this area is still in its early stages.

[0005] Discovering small molecule compounds that can enhance the protein synthesis ability of Monascus purpureus would have a significant impact on subsequent research and applications. Summary of the Invention

[0006] The purpose of this invention is to provide a method for increasing the protein yield of Monascus purpureus using cyclohexylacetic acid.

[0007] To achieve the above-mentioned objectives, the technical solution adopted in this invention is: a method for improving the protein production capacity of Monascus purpureus or a method for producing bacterial protein, wherein cyclohexylacetic acid, hydroxyethylamine, or methoxyphenylethylamine is added during the cultivation of Monascus purpureus. The amount of cyclohexylacetic acid added is 0.5–10 μg / L.

[0008] This invention offers the following advantages: It is the first discovery that cyclohexylacetic acid can enhance the ability of Monascus purpureus to synthesize and accumulate proteins, increasing protein yield by approximately 50%. The method provided by this invention is simple to operate, low in cost, and environmentally friendly, meeting the requirements of green manufacturing and sustainable development. This invention provides a theoretical basis and practical guidance for optimizing protein production through Monascus purpureus fermentation. Attached Figure Description

[0009] Figure 1 A diagram showing the effects of different compounds on the growth of Monascus purpureus;

[0010] Figure 2 A statistical chart showing the effect of cyclohexylacetic acid on the growth of Monascus purpureus;

[0011] Figure 3 This is a graph showing the GO functional enrichment analysis after the use of cyclohexylacetic acid to affect the growth of Monascus purpureus. Detailed Implementation

[0012] This invention provides a method for improving the protein production capacity of Monascus purpureus, specifically by adding an appropriate amount of cyclohexylacetic acid during Monascus purpureus cultivation. Preferably, the amount of cyclohexylacetic acid added to the culture medium for Monascus purpureus is 0.5–10.0 μg / L, and more preferably, it is 3.0–5.0 μg / L.

[0013] 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.

[0014] The culture media and reagents used in the examples are as follows:

[0015] Seed culture medium: glucose 30g / L, peptone 10g / L, yeast extract 5g / L, pH=6.0.

[0016] Fermentation medium: glucose 30g / L, sodium nitrate 3g / L 1g / L 0.5 g / L, pH=6.0.

[0017] Example 1: Effects of different compounds on the protein production capacity of Monascus purpureus

[0018] A purple Monascus strain (CGMCC NO.42056; this strain was first disclosed in our patent CN2025115394767) that was previously screened and preserved by our research group was selected and inoculated on PDA slant medium (potato dextrose agar). The spores were cultured at 28°C for 7 days until they matured. The spores were obtained by washing with sterile water and then cultured at a concentration of 1×10⁻⁶. 4 Inoculate seed culture medium with 100 cells / mL, and culture at 30℃ and 120 rpm for 48 h with shaking to obtain seed solution ( =1.0).

[0019] Prepare stock solutions of 8 compounds (1 mg / mL, DMSO as solvent), including: cyclohexylacetic acid (CAS No.: 5292-21-7), tyrosol, isoamyl alcohol, croterol, tyramine, hydroxyethylamine, p-methylphenethylamine, and methoxyphenethylamine.

[0020] The seed culture was transferred to 500 mL Erlenmeyer flasks containing 100 mL of fermentation medium at a 5% (v / v) inoculation rate, serving as group 1. At the start of fermentation (0 h), the stock solutions of each compound were added to each group, with a final concentration of 1.0 μg / L. A blank control group was set up: no compound was added (only an equal volume of DMSO solvent was added). Three biological replicates were set up for each group. Each group was incubated at 28 °C with shaking at 120 rpm for 120 h. After fermentation, the fermentation broth was obtained, and the mycelial dry weight, protein content, and protein yield were measured and calculated.

[0021] Method for determining the dry weight of mycelium: The fermentation broth was centrifuged at 8000 rpm for 10 minutes. The mycelium was washed three times with distilled water and dried at 60℃ until constant weight was achieved. Method for determining protein content: The crude protein content of the mycelium (on a dry basis) was determined using the Kjeldahl method. Accurately weigh 0.2 g of dried mycelium and add it to a digestion tube, then add 6 g of [unspecified ingredient] sequentially. 0.4g Add 10 mL of concentrated sulfuric acid; digest at 420℃ until the solution is clear (about 2 hours), cool, and bring the volume to 100 mL; take 10 mL of the digest, add 10 mL of 40% NaOH solution, distill for 10 minutes, and receive with 2% boric acid absorption solution; titrate with 0.01 mol / L HCl standard solution to a gray-purple endpoint, and record the volume consumed (V); the conversion factor for Monascus purpureus protein is 6.25. Protein yield (g / L) = mycelial dry weight (g / L) × protein content (%).

[0022] The results are shown in Table 1 and Figure 1 As shown. Figure 1In the diagram, each column represents one condition (one compound) repeated three times. From left to right, the conditions are: cyclohexylacetic acid, tyrosol, isoamyl alcohol, tryptol, pure culture medium, tyramine, hydroxyethylamine, p-methylphenethylamine, and methoxyphenethylamine. The rightmost column is the blank control group containing only an equal amount of DMSO for Monascus purpureus. Column 5 contains empty wells containing only culture medium to observe for contamination.

[0023] Table 1. Effects of different compounds on the dry weight, protein content, and protein yield of Monascus purpureus mycelia.

[0024]

[0025] The results showed that cyclohexylacetic acid treatment increased histone content from 37.1% to 42.3% and protein yield from 1.9 g / L to 2.4 g / L, an increase of 26%, significantly outperforming all other compounds. After cultivation, cyclohexylacetic acid was not detected in the culture medium, indicating that the strain could utilize and metabolize it.

[0026] It should be noted that the patterns of the compounds in Table 1 found in this invention are universally applicable to Monascus purpureus. Experiments on three other red Monascus purpureus strains and three other purple Monascus purpureus strains showed the same trend.

[0027] Example 2: Optimization of conditions for protein production by Monascus purpureus cultured using cyclohexylacetic acid

[0028] Based on Example 1, with other conditions different, the final concentration of added cyclohexylacetic acid was adjusted to: 0.1 μg / L, 0.5 μg / L, 1.0 μg / L, 3.0 μg / L, 5.0 μg / L, 7.0 μg / L, and 10.0 μg / L. The blank control group was set up in the same way as in Example 1. Each group was also set up with three replicates. The results are shown in Table 2.

[0029] Table 2. Effects of different cyclohexylacetic acid concentrations on the dry weight, protein content, and protein yield of Monascus purpureus mycelia.

[0030]

[0031] The results showed that a cyclohexylacetic acid (CAA) concentration of 5.0 μg / L resulted in the highest protein content of 47.5% and a protein yield of 2.8 g / L, significantly outperforming other concentrations. Furthermore, although the groups in Table 2 with CAA concentrations of 0 and 1.0 μg / L had the same treatment conditions as the blank control group and CAA-treated group in Table 1, for better comparability, all groups in Table 2 underwent the same batch of experiments again, and the data for these two groups were essentially the same as the corresponding data in Table 1.

[0032] Example 3: Transcriptome analysis of the mechanism by which Monascus purpureus protein production is enhanced.

[0033] Based on Example 2, the Gene Ontology (GO) standard classification system was used to perform functional annotation of differentially expressed genes in Monascus purpureus cells from the blank control group and Monascus purpureus cells treated with 5.0 μg / L cyclohexylacetic acid. The GO database systematically elucidates the functional characteristics of genes through three major functional modules: biological process, cellular component, and molecular function. The GO functional annotation results are as follows: Figure 2 As shown.

[0034] Figure 2 The results showed that after treatment with cyclohexylacetic acid (CYA), differentially expressed genes exhibited significant enrichment across three functional dimensions: biological processes primarily involved metabolic and cellular processes; cellular components were concentrated in substructures such as membrane systems and organelles; and molecular functions were closely related to biochemical reactions such as catalytic activity and binding. Notably, the differentially expressed genes showed significant functional bias, with upregulated genes outnumbering downregulated genes in most functional categories, suggesting that CYA may affect the growth and protein accumulation of Monascus purpureus by activating specific metabolic pathways.

[0035] GO enrichment analysis was performed on the differentially expressed genes to determine the main GO functions of the genes in the gene set. The results are as follows: Figure 3 As shown in the scatter plot, the top 20 GO entries by GO enrichment level are displayed. Figure 3 The differentially expressed genes were mainly enriched in transport activities (GO:0005215), transmembrane transporter activities (GO:0022857, GO:0015116, GO:0015140), glucose phosphatase activities (GO:0050308), oxidoreductase activities (GO:0016634, GO:0016722), amino acid kinase activities (GO:0019202), and carbohydrate phosphatase activities (GO:0019203). Among these, glucose phosphatase activity (GO:0050308) and oxidoreductase activity (GO:0016634) showed the highest enrichment levels, indicating that these functions may play a key role in the growth and protein synthesis of Monascus purpureus under cyclohexylacetic acid treatment.

[0036] 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 should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for improving the protein production ability of Monascus, characterized by: Cyclohexylacetic acid, hydroxyethylamine or methoxyphenethylamine is added when Monascus is cultured.

2. The method of claim 1, wherein: The cyclohexylacetic acid is added in an amount of 0.5-10 μg / L.

3. A method of producing a bacterial protein, characterized by: The protein is produced by Monascus, and cyclohexylacetic acid, hydroxyethylamine or methoxyphenethylamine is added when Monascus is cultured.

4. The method of claim 3, wherein: The cyclohexylacetic acid is added in an amount of 0.5-10 μg / L. The protein is produced by Monascus, and cyclohexylacetic acid, hydroxyethylamine or methoxyphenethylamine is added when Monascus is cultured. The cyclohexylacetic acid is added in an amount of 0.5-10 μg / L.

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