L-selenomethyl modified oligopeptide compound for monascus fermentation as well as preparation method and application of L-selenomethyl modified oligopeptide compound

Abstract

The invention provides an L-selenium methyl modified oligopeptide compound for monascus fermentation as well as a preparation method and application thereof, and relates to the technical field of biological fermentation, the amino acid sequence is H2N-Arg-Gly-Asp-L-selenium methyl selenocysteine-OH, the L-selenium methyl modified oligopeptide compound is used as a fermentation accelerant, the compound is added into a monascus fermentation culture medium, the addition amount is 0.1-1.0 g / L, and the fermentation time is 1-2 hours; the growth rate of monascus can be improved, and the fermentation period is shortened; the synthesis of the monascus pigment and Monacolin K can be promoted, so that the yield of the monascus pigment is increased by 20-30%, and the yield of the Monacolin K is increased by 15-25%; the adaptability of monascus to environmental factors (such as temperature and pH value) can be enhanced, and the fluctuation in the fermentation process is reduced.

Description

Technical Field

[0001] The present invention relates to the field of biological fermentation technology, and in particular to an L-selenomethyl-modified short peptide compound for red yeast rice fermentation, and a preparation method and application thereof. Background Art

[0002] Red yeast rice fermentation is the process of utilizing the metabolic activity of Monascus koji fungi under favorable conditions to produce various beneficial ingredients, including red yeast pigments and monacolin K. However, current red yeast rice fermentation processes are plagued by challenges such as unstable fermentation efficiency, low yields of target products, and long fermentation cycles, which limit the large-scale production and application of red yeast rice-related products.

[0003] Selenium is an important trace element with multiple physiological functions in organisms. L-selenomethylselenocysteine, an organic selenium compound, exhibits high biological activity and safety. Short peptides are characterized by their small molecular weight, ease of absorption, and strong biological activity. Modifying short peptides with L-selenomethyl groups is expected to combine the advantages of both and play a positive role in red yeast rice fermentation.

[0004] At present, there are no reports on the application of L-selenomethyl-modified short peptide compounds in red yeast rice fermentation. Summary of the Invention

[0005] The purpose of the present invention is to solve the shortcomings of the prior art and to propose an L-selenomethyl-modified short peptide compound for red yeast rice fermentation and its preparation method and application.

[0006] In order to achieve the above-mentioned purpose, the present invention adopts the following technical scheme: an L-selenomethyl-modified short peptide compound for red yeast rice fermentation has an amino acid sequence of: H2N-Arg-Gly-Asp-L-selenomethylselenocysteine-OH.

[0007] Furthermore, the molecular weight is 495.5 Da.

[0008] Furthermore, the preparation method of the compound comprises the following steps:

[0009] S1: Preparation of protected amino acids: Preparation of N-protected arginine, N-protected glycine, N-protected aspartic acid and N-protected L-selenomethylselenocysteine;

[0010] S2: Synthesis of peptide chains: Using solid-phase peptide synthesis, Rink Amide MBHA resin was used as a carrier to sequentially connect N-protected arginine, N-protected glycine, N-protected aspartic acid, and N-protected L-selenomethylselenocysteine ​​to form a protected short peptide resin complex;

[0011] S3: Deprotection: Deprotect the protected short peptide resin complex using a trifluoroacetic acid system to obtain a crude product;

[0012] S4: Purification: The crude product is purified by high performance liquid chromatography to obtain the target compound.

[0013] The application of the compound in red yeast rice fermentation includes serving as a fermentation promoter, increasing the yield of target products and improving fermentation stability.

[0014] Furthermore, the amount added to the red yeast rice fermentation medium is 0.1-1.0 g / L.

[0015] Compared with the prior art, the advantages and positive effects of the present invention are:

[0016] The present invention is used as a fermentation promoter: adding the compound in a red yeast rice fermentation medium at an addition amount of 0.1-1.0 g / L can increase the growth rate of red yeast rice and shorten the fermentation cycle;

[0017] Improve target product yield: It can promote the synthesis of monascus pigment and monacolin K, increasing the yield of monascus pigment by 20-30% and the yield of monacolin K by 15-25%;

[0018] Improve fermentation stability: It can enhance the adaptability of Monascus to environmental factors (such as temperature and pH value) and reduce fluctuations during the fermentation process. DETAILED DESCRIPTION

[0019] The present invention provides an L-selenomethyl-modified short peptide compound for red yeast rice fermentation, the amino acid sequence of which is:

[0020] H2N-Arg-Gly-Asp-L-selenomethylselenocysteine-OH, molecular weight is 495.5Da.

[0021] The method for preparing the compound is characterized by comprising the following steps: S1: preparation of protected amino acids: preparation of N-protected arginine, N-protected glycine, N-protected aspartic acid and N-protected L-selenomethylselenocysteine;

[0022] S2: Synthesis of peptide chains: Using solid-phase peptide synthesis, Rink Amide MBHA resin was used as a carrier to sequentially connect N-protected arginine, N-protected glycine, N-protected aspartic acid, and N-protected L-selenomethylselenocysteine ​​to form a protected short peptide resin complex;

[0023] S3: Deprotection: Deprotect the protected short peptide resin complex using a trifluoroacetic acid system to obtain a crude product;

[0024] S4: Purification: Purify the crude product by high performance liquid chromatography to obtain the target compound;

[0025] Including acting as a fermentation promoter, increasing target product yield and improving fermentation stability;

[0026] The amount added to the red yeast rice fermentation medium is 0.1-1.0g / L;

[0027] Preparation of compounds: 1. Preparation of protected amino acids: N-protected arginine: Dissolve arginine in dimethylformamide (DMF), add fluorenylmethyloxycarbonyl (Fmoc) chloride, and react at room temperature for 2 hours to obtain Fmoc-Arg-OH.

[0028] N-protected glycine: Dissolve glycine in DMF, add Fmoc chloride, and react at room temperature for 2 hours to obtain Fmoc-Gly-OH.

[0029] N-protected aspartic acid: Dissolve aspartic acid in DMF, add Fmoc chloride and N-hydroxysuccinimide (NHS), and react at room temperature for 4 hours to obtain Fmoc-Asp(OtBu)-OH.

[0030] N-Protected L-selenomethylselenocysteine: Dissolve L-selenomethylselenocysteine ​​in DMF, add Fmoc chloride and NHS, and react at room temperature for 4 hours to produce Fmoc-Cys(SeCH3)-OH. Synthesis of the peptide chain: Swell Rink Amide MBHA resin with DMF for 30 minutes.

[0031] The Fmoc protecting group on the resin was removed using a 20% piperidine / DMF solution, and the reaction was repeated for 15 minutes.

[0032] Fmoc-Arg-OH, Fmoc-Gly-OH, Fmoc-Asp(OtBu)-OH and Fmoc-Cys(SeCH3)-OH were coupled with the resin in sequence. HCTU was used as an activating agent and N,N-diisopropylethylamine (DIPEA) was used as a base for each coupling reaction at room temperature for 2 hours.

[0033] After each coupling reaction, the resin was washed three times with DMF and three times with methanol. Deprotection: The protected short peptide resin complex was placed in a reaction flask, and a TFA solution containing 10% water and 2.5% triisopropylsilane (TIS) was added and reacted at room temperature for 2 hours.

[0034] The mixture was filtered, the filtrate was collected, and TFA was removed using a rotary evaporator to obtain a crude product.

[0035] Purification: The crude product was dissolved in water, filtered through a 0.45 μm filter membrane, and the filtrate was purified by HPLC.

[0036] HPLC conditions: C18 column (250 mm × 10 mm, 5 μm), mobile phase A was 0.1% TFA in water, mobile phase B was 0.1% TFA in acetonitrile, gradient elution (0-10 min, 5-30% B; 10-20 min, 30-50% B; 20-30 min, 50-80% B), flow rate was 5 ml / min, and detection wavelength was 220 nm.

[0037] The eluate corresponding to the target peak was collected, the solvent was removed by rotary evaporator, and the target compound was obtained by freeze-drying.

[0038] Application experiment of red yeast rice fermentation

[0039] Experimental materials: Monascus purpureus CCTCC M2015039.

[0040] Culture medium: glucose 20 g / L, peptone 10 g / L, yeast extract 5 g / L, sodium nitrate 3 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate 0.5 g / L, pH natural.

[0041] Compound: L-selenomethyl-modified short peptide compound prepared by the present invention.

[0042] Experimental method: Seed culture: Monascus was inoculated into seed culture medium and cultured at 30°C and 180 rpm for 48 hours.

[0043] Fermentation: 10% seed solution was inoculated into fermentation medium. The cells were divided into two groups: one group was supplemented with 0.5 g / L of the compound, and the other group was not supplemented with the compound. The cells were cultured at 30°C and 180 rpm for 7 days. Measurements: Samples were collected daily to measure Monascus biomass (OD600), the absorbance of the Monascus pigment (505 nm), and the Monacolin K content (HPLC method).

[0044] Experimental results: Monascus growth curve: the experimental group with added compounds entered the logarithmic growth phase on the third day of fermentation, while the control group without added compounds entered the logarithmic growth phase on the fourth day of fermentation. The biomass peak of the experimental group with added compounds was 15% higher than that of the control group.

[0045] Monascus pigment production: After fermentation, the absorbance of the Monascus pigment in the experimental group with compound addition was 1.25, while that in the control group without compound addition was 0.98, with a production increase of 27.6%.

[0046] Monacolin K content: The Monacolin K content in the experimental group with compound addition was 2.85 mg / L, while that in the control group without compound addition was 2.32 mg / L, indicating a 22.8% increase in yield.

[0047] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any other form. Any person skilled in the art may use the technical contents disclosed above to change or modify them into equivalent embodiments with equivalent changes for application in other fields. However, any simple modification, equivalent change and modification of the above embodiments made in accordance with the technical essence of the present invention without departing from the technical solution of the present invention shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. An L-selenomethyl-modified short peptide compound for red yeast rice fermentation, characterized in that: The amino acid sequence is: H2N-Arg-Gly-Asp-L-selenomethylselenocysteine-OH.

2. The compound according to claim 1, characterized in that: The molecular weight is 495.5 Da.

3. The method for preparing the compound according to claim 1, wherein The following steps are involved: S1: Preparation of protected amino acids: Preparation of N-protected arginine, N-protected glycine, N-protected aspartic acid and N-protected L-selenomethylselenocysteine; S2: Synthesis of peptide chains: Using solid-phase peptide synthesis, Rink Amide MBHA resin was used as a carrier to sequentially connect N-protected arginine, N-protected glycine, N-protected aspartic acid, and N-protected L-selenomethylselenocysteine ​​to form a protected short peptide resin complex; S3: Deprotection: Deprotect the protected short peptide resin complex using a trifluoroacetic acid system to obtain a crude product; S4: Purification: The crude product is purified by high performance liquid chromatography to obtain the target compound.

4. The use of the compound according to claim 4 in red yeast rice fermentation, characterized in that: It can be used as a fermentation promoter, increase the yield of target products and improve fermentation stability.

5. The use according to claim 1, characterized in that: The amount added to the red yeast rice fermentation medium is 0.1-1.0g / L.