Fermentation medium to enhance the ability of Monacolin K synthesized by Monascus purpureus

Abstract

This invention discloses a fermentation medium for Monacolin K production by Monascus purpureus with added traditional Chinese medicine ingredients, specifically honeysuckle. Single-factor experiments were conducted to investigate the effects of honeysuckle on the growth and metabolism of Monascus purpureus. The results showed that adding 0.8 g of honeysuckle powder at 48 h of fermentation had the most significant promoting effect on Monacolin K synthesis, increasing Monacolin K production by up to 1.54 times, reaching 536.10 mg / L. Subsequent fermentation experiments indicated that honeysuckle provided abundant carbon and nitrogen sources and an acidic environment for the growth of Monascus purpureus, exhibiting a positive promoting effect on the entire metabolic process. The expression of nine genes related to Monacolin K synthesis also showed an upregulation trend. Simultaneously, the addition of honeysuckle significantly increased the content of total flavonoids and total polyphenols, and greatly enhanced the in vitro antioxidant activity of the fermentation products.

Description

Technical Field

[0001] This invention relates to the application of traditional Chinese medicine extracts in improving Monacolin K production by Monascus purpureus, and belongs to the field of biotechnology preparation. Background Technology

[0002] Monascus is a filamentous fungus that produces a variety of metabolites with practical applications. Currently, research on Monascus ruber, Monascus pilosus, and Monascus purpureus is relatively extensive. Monascus is an important microbial resource used in both food and medicine, producing various secondary metabolites such as monascus pigment, Monacolin K, γ-aminobutyric acid (GABA), and ergosterol, all of which have become research hotspots in recent years.

[0003] Studies have found that Monacolin K can competitively inhibit the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) in the cholesterol synthesis pathway, thereby controlling cholesterol synthesis levels. Monacolin K typically exists in two configurations: lactone and acid. Lactone Monacolin K is an inactive precursor that needs to be hydrolyzed by carboxyl esterases in the body to change its structure into the active hydroxyacid form before it can exert its lipid-lowering effect. Monacolin K produced by Monascus purpureus is mostly in the acid form, which can be directly utilized, has good medicinal value, and high safety, making it considered one of the ideal cholesterol-lowering agents both domestically and internationally.

[0004] The Chinese industry standard QB / T2847-2007, Functional Red Yeast Rice (Powder), requires that the Monacolin K content of functional red yeast rice must be greater than or equal to 0.4%. This demonstrates that the ability of Monacolin K synthesized by Monacolin fungi during fermentation is crucial to the quality of functional red yeast rice. However, the current Monacolin K production capacity of Monacolin fungi is relatively low, leading to higher industrial production costs and severely limiting the development of the functional red yeast rice industry. This has also become a major obstacle to the application of Monacolin fungi in food and health products. In recent years, researchers have conducted extensive and in-depth research on improving the quality of functional red yeast rice from both exogenous and endogenous perspectives. One approach involves using mutagenesis breeding methods or genetic engineering techniques to select high-yielding strains, fundamentally and directionally modifying Monacolin fungi at the genetic level to achieve efficient Monacolin K production. For example, Chen Bingmei et al. treated wild-type strains with a combination of UV-nitrite and UV-lithium chloride mutagen, ultimately obtaining three high-yielding lovastatin mutant strains, with the highest yield increase reaching 75.2%. Liang Jian et al. overexpressed four gene segments (mokC, mokD, mokE, and mokI) in the Monacolin K synthesis gene cluster, and obtained four high-yielding Monacolin K strains, with yields increased by 234.3%, 220.8%, 89.5%, and 10% respectively compared with the original strain.

[0005] On the other hand, optimizing fermentation conditions to promote Monacolin K synthesis in Monascus purpureus, such as controlling environmental factors or modifying the composition and ratio of the culture medium, is the most direct and simplest approach. The addition of carbon sources like glucose and glycerol, and nitrogen sources like ammonium chloride and peptone, can influence the secondary metabolic processes of Monascus purpureus to varying degrees, thereby promoting the synthesis of related metabolites. Additionally, some small molecules can act as regulators, accelerating the growth and metabolism of the cells. Studies have confirmed that substances such as linoleic acid and glutamic acid have shown significant promoting effects on the growth and metabolism of Monascus purpureus, and have also greatly improved the synthesis capacity of Monacolin K. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a fermentation medium that improves the production of Monacolin K by Monascus purpleis, mainly by adding the traditional Chinese medicine honeysuckle.

[0007] This invention relates to the application of traditional Chinese medicinal materials in enhancing the ability of Monacolin K synthesized by Monascus purpureus.

[0008] The present invention discovered that by adding traditional Chinese medicine ingredients to the fermentation medium of Monacolin K produced by Monacolin K from Monascus purpureus, the ability of Monacolin K to be synthesized by Monacolin K from Monascus purpureus can be greatly promoted.

[0009] Furthermore, the present invention provides a fermentation culture containing the traditional Chinese medicine honeysuckle, wherein the honeysuckle is added in the form of medicinal powder and the amount added is 0.8g.

[0010] The present invention also provides a fermentation method for improving the production of Monacolin K by purple red spores. Specifically, honeysuckle powder is prepared and added to the fermentation culture medium at an addition amount of 0.8g and an addition time of 48 hours after fermentation.

[0011] The beneficial effects of this invention are:

[0012] This invention discovered that honeysuckle, as an exogenous additive, can promote the synthesis of Monacolin K in Monascus purpureus. Single-factor experiments were used to optimize fermentation conditions, and the optimal addition conditions for co-fermentation of honeysuckle and Monascus purpureus were found to be: adding 0.8 g of honeysuckle powder at the 48th hour of fermentation, resulting in a Monacolin K yield of 330.90 mg / L. Compared with the original fermentation conditions, the Monacolin K yield increased by 41.5%. Attached Figure Description

[0013] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0014] Figure 1 Effects of honeysuckle addition form and dosage on *Monascus purpureus*. (a) Decoction-biomass; (b) Decoction-MK;

[0015] (c) Herb powder - biomass; (d) Herb powder - MK.

[0016] Figure 2 The effect of honeysuckle addition time on Monacolin K synthesis in Monacolin-1 purple.

[0017] Figure 3 Effects of honeysuckle addition on the growth of Monascus purpureus. (a) Biomass; (b) pH of fermentation broth; (c) Residual sugar concentration; (d) Protein concentration.

[0018] Figure 4 Effects of honeysuckle addition on the synthesis of secondary metabolites in Monascus purpureus. (a) Monacolin K; (b) yellow pigment; (c) orange pigment; (d) red pigment.

[0019] Figure 5 Effects of honeysuckle addition on the mycelial morphology of Monascus purpureus. (a) Control group -1500×; (b) Experimental group -1500×; (c) Control group -5000×; (d) Experimental group -5000×.

[0020] Figure 6Effects of honeysuckle addition on the expression levels of Monacolin K synthesis-related genes in Monacolin K. (a) mokA; (b) mokB; (c) mokC; (d) mokD; (e) mokE; (f) mokF; (g) mokG; (h) mokH; (i) mokI.

[0021] Figure 7 Changes in the in vitro antioxidant activity of honeysuckle-purple red frost co-fermentation products. (a) Total polyphenols; (b) Total flavonoids;

[0022] (c) Total antioxidant capacity; (d) ABTS free radical scavenging activity. Detailed Implementation

[0023] Example 1

[0024] Experimental strains and culture media

[0025] Wild-type purple Monascus strain M1, strain number CGMCC No.30568, is preserved by the Beijing Engineering Technology Research Center for Food Additives.

[0026] Plate culture medium (g / L): PDA 37, agar 5, sterilized at 121℃ for 5 min;

[0027] Liquid seed culture medium (g / L): glucose 30, soybean flour 15, peptone 10, KH2PO4 2, NaNO3 2, MgSO4·7H2O 1, glycerol 70. Inoculate and culture in shake flasks, with a liquid volume of 50mL / 250mL Erlenmeyer flask, and sterilize at 115℃ for 20min.

[0028] Liquid fermentation medium (g / L): rice flour 20, peptone 10, KH2PO4 2.5, NaNO3 5, MgSO4·7H2O 1, ZnSO4·7H2O 2, glycerol 90. Inoculate and culture in shake flasks, with a liquid volume of 50mL / 250mL Erlenmeyer flask, and sterilize at 121℃ for 15min. The liquid medium used for the control group is the same as above. The experimental group and blank group are based on the medium used for the control group, with honeysuckle added according to experimental requirements.

[0029] Instruments and equipment

[0030] Table 1 Main Instruments

[0031] Instrument Name Manufacturer High-pressure steam sterilizer Zhonghao Lab Technology Co., Ltd. SpectraMax 13 Microplate Reader Molecular Devices, USA High Performance Liquid Chromatograph 20A Shimadzu Corporation of Japan NanoDrop 2000 Nucleic Acid Quantitative Analyzer Thermo Gradient PCR Amplification Instrument Bio-Rad, Inc. (USA) CFX96 Real-time Quantitative PCR Instrument Bio-Rad, Inc. (USA) SU8020 Scanning Electron Microscope Hitachi Corporation NuAire Biosafety Cabinet Beijing Tianlin Hengtai Technology Co., Ltd. Blue pard biochemical incubator Shanghai Yiheng Scientific Instruments Co., Ltd. LYZ Thermostatic Shaker Shanghai Longyue Instrument Equipment Co., Ltd.

[0032] Experimental reagents

[0033] Honeysuckle (a type of Chinese medicinal herb) was purchased from Beijing Tongrentang; PDA culture medium, glucose, sucrose, rice flour, soybean flour, glycerol, peptone, KH2PO4, NaNO3, MgSO4·7H2O, ZnSO4·7H2O, etc. were purchased from Beijing Jinlis Technology Co., Ltd.; RNAprepPure polysaccharide and polyphenol plant total RNA extraction kit, FastQuant cDNA first strand synthesis kit, SuperRealPreMix Plus (SYBR Green) kit, etc. were purchased from Tiangen Biotech (Beijing) Co., Ltd.; Total antioxidant capacity (T-AOC) assay kit (FRAP microplate method) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; BCA protein concentration assay kit and ABTS free radical scavenging capacity assay kit were purchased from Beijing Solarbio Technology Co., Ltd.; Chromatographic grade methanol, analytical grade ethanol, glutaraldehyde, isoamyl acetate, etc. were purchased from Beijing Mairuida Technology Co., Ltd.

[0034] Experimental methods

[0035] Strain activation and culture

[0036] Purple Monascus M1 glycerol-preserved strains were taken from a -80℃ freezer, spread onto agar plates, and incubated at 30℃ for 4 days. Then, they were streaked and subcultured on the same agar plates for two generations, each incubated at 30℃ for 4 days. Liquid seed culture: The planar spores were washed with 10 mL of sterile water, filtered through a double layer of nylon cloth, and shaken well. A 5% (v / v) inoculation was added to a 50 mL Erlenmeyer flask containing seed culture medium. The flasks were incubated at 30℃ and 200 rpm for 48 hours until the color turned pale pink. Liquid fermentation culture: The seed culture was inoculated at a 10% (v / v) inoculation into a 50 mL Erlenmeyer flask containing fermentation broth medium. The flasks were incubated at 30℃ and 150 rpm for 48 hours. The temperature was then adjusted to 25℃, and fermentation continued for 15 days.

[0037] Single-factor experiments were conducted on the liquid fermentation of honeysuckle to determine the optimal addition conditions, taking into account factors such as the state, concentration, and addition time of the traditional Chinese medicine.

[0038] Preparation of decoction: Weigh 5g of honeysuckle, wash with clean water, add 500mL of distilled water and soak for 1 hour, heat and keep boiling for 30 minutes, filter out the decoction; repeat the above operation, combine the two decoctions, add distilled water to make up to 1000mL, sterilize at 121℃ for 15 minutes and set aside.

[0039] Powder preparation: Weigh a certain amount of honeysuckle, wash it with water and air dry it naturally. Grind it into powder using a grinder and pass it through an 80-mesh sieve. Sterilize it at 121℃ for 15 minutes for later use.

[0040] Honeysuckle extract at volume fractions of 0%, 2%, 4%, 8%, 12%, 16%, and 20%, and honeysuckle powder at mass fractions of 0g, 0.2g, 0.4g, 0.6g, 0.8g, and 1.0g, were added to liquid fermentation medium, and the Monacolin K content in the fermentation broth was measured. Based on the experimental results, the optimal form and amount of honeysuckle added were determined. Honeysuckle was then added at different fermentation times (0h, 24h, 48h, 72h, 96h, 120h, and 144h), and the Monacolin K content in the fermentation broth was measured to determine the optimal addition time.

[0041] Monacolin K production detection

[0042] Take 2 mL of Monascus purpureus fermentation broth into a centrifuge tube, add 6 mL of methanol solution (v / v, 75:25), and extract by sonication at room temperature for 30 min. Let it stand overnight in the dark. Take the supernatant, filter it through a 0.22 μm organic filter membrane into a liquid chromatography vial, and determine the yield using high performance liquid chromatography (HPLC).

[0043] The HPLC detection conditions were as follows: column: Inertsil ODS-3C18 (150 mm × 4.6 mm × 5 μm); mobile phase: 0.1% phosphoric acid: methanol = 1:3; flow rate: 1 mL / min; UV detector, detection wavelength: 237 nm; column oven temperature: 30 ℃; injection volume: 10 μL.

[0044] Detection of the color value of red yeast rice pigment

[0045] Take 1 mL of purple Monascus ferment broth and add 8 mL of ethanol solution (v / v, 70:30). Extract in a 60℃ water bath for 1 h. After cooling, centrifuge at 4,000 rpm for 15 min and allow to stand in the dark until analysis. Measure the absorbance of the sample solution at the maximum absorption wavelengths of the yellow, orange, and red pigments of Monascus purpureus using a UV spectrophotometer at 410, 448, and 505 nm, respectively. Perform data processing according to the quantitative formula. Quantitative formula: Monascus purpureus color value (U / mL) = Absorbance × Dilution factor

[0046] Biomass determination

[0047] Mycelial biomass was determined using the dry weight method. 3 mL of fermentation broth was transferred to sterilized, weighed filter paper and filtered. The paper was washed three times with distilled water to remove culture medium components. After drying, the paper was placed in a 60°C oven and dried to constant weight. The filter paper containing mycelium was weighed again, and the difference between the two weights was taken as the dry weight of the mycelium. Data processing was performed according to the quantitative formula.

[0048] Mycelial biomass (g / L) = Dry matter weight / Fermentation broth volume

[0049] Detection of total sugar content

[0050] The total sugar content in the fermentation broth was determined using the phenol-sulfuric acid method. After centrifugation, the supernatant of the fermentation broth was diluted 10-fold. 2 mL of the diluted sample was placed in a test tube, and 1 mL of 6% phenol solution was added, followed by 5 mL of concentrated sulfuric acid. The mixture was allowed to stand at room temperature for 10 min, then vortexed to mix, and allowed to stand at room temperature for 20 min. The OD value of the reaction solution at 490 nm was measured using a microplate reader. The sugar content in the fermentation broth was calculated based on the standard curve and the dilution factor.

[0051] pH detection

[0052] The pH values ​​of the fermentation broth in the control group and experimental group at different fermentation stages were measured using a pH meter.

[0053] Detection of total protein concentration

[0054] Take 1 mL of *Monascus purpureus* fermentation broth, add 9 mL of 1×PBS buffer, sonicate for 30 min, centrifuge at 10000 rpm for 10 min at room temperature, and collect the supernatant for later use. Subsequent experiments were performed according to the BCA protein concentration assay kit instructions, with the specific steps as follows:

[0055] 1. Dilute the standard: Take 10 μL of BSA standard and dilute it with PBS to 100 μL to make a final concentration of 0.5 mg / mL. Then dilute it to concentrations of 0.025, 0.05, 0.1, 0.15, 0.2, 0.3 and 0.4 mg / mL in sequence for later use.

[0056] 2. Before use, prepare the BCA working solution according to the quantity of standard and sample, in a ratio of BCA reagent:Cu reagent (V:V) = 50:1, and mix thoroughly.

[0057] 3. Set up blank wells (using distilled water instead of the sample), control wells (using PBS instead of BCA working solution), and assay wells. Add 20 μL of sample to each assay well of a 96-well plate, and add 200 μL of BCA working solution to each well. Incubate at 37°C for 30 min. Measure the absorbance at 562 nm using a microplate reader. Plot a standard curve with the concentration of the standard on the x-axis and the corresponding absorbance values ​​on the y-axis. Calculate the protein concentration of the sample based on the standard curve.

[0058] Scanning electron microscopy processing

[0059] Fermentation broth from day 8 was selected for treatment and observation. 2 mL of fermentation broth was transferred to a sterile centrifuge tube and centrifuged at 12,000 rpm for 5 min, discarding the supernatant. The cells were resuspended and fixed with 2.5% glutaraldehyde solution and kept for 12 h. After centrifugation and discarding the supernatant again, the cells were rinsed with 0.1 M phosphate-buffered saline (PBS, pH 7.2) to remove glutaraldehyde, repeating this process once. Subsequently, dehydration was performed sequentially using different concentration gradients of ethanol solutions (30%, 50%, 70%, 80%, 90%, 100%). After each addition, the cells were mixed, allowed to stand for 10 min, centrifuged at 12,000 rpm for 5 min, and the supernatant was discarded. Each concentration was repeated twice. The cells were then resuspended in a mixture of isoamyl acetate and ethanol (v / v, 50:50), and pure isoamyl acetate solution was added again to replace the ethanol in the cells. Finally, hexamethyl disilazane (HMDS) solvent was added until the sample was submerged. The top of the centrifuge tube was plugged with absorbent cotton to improve water absorption. The tube was then dried in a 60°C oven until the sample became powder. The morphology of the purple red Monascus mycelium was observed using a scanning electron microscope.

[0060] Transcriptional level analysis of Monacolin K synthesis-related genes

[0061] Collection and processing of bacterial cells: Fermentation broth of *Monascus purpureus* with different culture days was placed in 2 mL centrifuge tubes, washed with sterile water, and centrifuged until the supernatant was no longer red. Residual water was removed from the centrifuge tubes. Total RNA was then extracted from *Monascus purpureus* and reverse transcribed into cDNA for quantitative real-time analysis.

[0062] The experiment was conducted according to the instructions of the polysaccharide and polyphenol plant total RNA extraction kit. The specific steps are as follows:

[0063] 1. Prepare a SL mixture containing 5% mercaptoethanol in a fume hood and transfer it to a 2 mL centrifuge tube. Remove the purple Monascus cells stored at -80°C and place them in a sterile mortar. Gradually grind the cells into powder by adding liquid nitrogen in small, repeated additions. Weigh 100 mg of the powder and add it to the mixture. Immediately vortex to mix thoroughly and centrifuge at 12,000 rpm for 2 minutes.

[0064] 2. Transfer the supernatant to the CS filter column, centrifuge at 12,000 rpm for 2 min, carefully aspirate 400 μL of supernatant into a new centrifuge tube, slowly add 160 μL of anhydrous ethanol, mix well by pipetting, transfer to the CR3 adsorption column, centrifuge at 12,000 rpm for 15 s, and discard the filtrate.

[0065] 3. Add 350 μL of protein removal solution RW1 to the adsorption column CR3, centrifuge at 12,000 rpm for 15 s, and discard the filtrate.

[0066] 4. Prepare the DNase I working solution by adding 70 μL of RDD solution to 10 μL of DNase I and mixing gently. Then, add the working solution dropwise to the center of the CR3 adsorption column and let it stand for 15 min.

[0067] 5. Add 350 μL of protein removal solution RW1 to the adsorption column CR3, centrifuge at 12,000 rpm for 15 s, and discard the filtrate.

[0068] 6. Add 500 μL of rinsing buffer RW to CR3, centrifuge at 12,000 rpm for 15 seconds, and discard the filtrate. Repeat the operation once.

[0069] 7. Centrifuge an empty CR3 adsorption column for 2 min, then transfer the column to a new centrifuge tube. Add 50 μL of RNase-Free ddH2O to the center of the column for elution. Let stand for 2 min, then centrifuge at 12,000 rpm for 1 min to obtain the RNA solution. Reserve for concentration and purity testing.

[0070] The experiment was conducted according to the Fast Quant cDNA First-Strand Synthesis Kit instructions. The specific steps are as follows:

[0071] 1. Prepare the solution according to the genomic DNA removal system in Table 2, mix well by pipetting, centrifuge for 10 seconds, incubate at 42°C for 3 minutes on a PCR instrument, and place on ice for later use.

[0072] Table 2. gDNA Removal Reaction System

[0073]

[0074]

[0075] 2. Prepare the solution according to the reverse transcription system in Table 3, mix well by pipetting, centrifuge for 10 seconds, and place on ice for later use.

[0076] Table 3 Reverse Transcription System

[0077] Composition Dosage (μL) 10×King RT Buffer 2 FastKing RT Enzyme Mix 1 FQ-RT Primer Mix 2 <![CDATA[RNase-Free ddH2O]]> 5

[0078] 3. Add the solution from step 2 to the reaction system from step 1, mix well by pipetting, centrifuge for 10 seconds, incubate at 42°C for 15 minutes and 95°C for 3 minutes on a PCR instrument to obtain the cDNA solution. After testing the concentration and purity, store at -20°C for later use.

[0079] Real-time fluorescence quantitative analysis

[0080] The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was selected as an internal reference gene. The nine-segment gene sequence mokA-mokI, which is related to the synthesis of Monacolin K in Monascus purpureus and is provided on the website of the National Center for Biotechnology Information (NCBI), was used as the target gene. Primers were designed using Oligo 7.37 software. Detailed primer information is shown in Table 4.

[0081] Table 4 Primer sequences of key genes

[0082]

[0083]

[0084] Perform the experiment according to the instructions of the SuperReal PCR Premixed Reagent Enhanced Version (SYBR Green) kit. Prepare the solution according to the reaction system in Table 5, mix gently, centrifuge for 5 seconds, and place the reaction system in a real-time PCR instrument for reaction.

[0085] Table 5. Real-time quantitative PCR reaction system and conditions

[0086]

[0087] Detection of total polyphenol content

[0088] 100 μL of sample was pipetted into a centrifuge tube, 50 μL of Folin-Ciocalteu reagent was added, and the mixture was stirred and reacted at room temperature for 5 min. Then, 150 μL of 20% Na₂CO₃ solution was added, stirred, and allowed to stand at room temperature for 20 min. The absorbance of the reaction solution at 765 nm was measured using a microplate reader. A standard curve was prepared using gallic acid as a standard, and the total polyphenol content in the fermentation broth of different groups was calculated based on this curve.

[0089] Detection of total flavonoid content

[0090] 100 μL of sample was pipetted into a centrifuge tube, and 150 μL of 5% NaNO2 solution was added. After mixing, the mixture was allowed to stand in the dark for 5 min. Then, 150 μL of 10% Al(NO3)3 solution was added, mixed, and allowed to stand in the dark for 5 min. Finally, 2 mL of 4% NaOH solution was added to terminate the reaction, and the mixture was allowed to stand at room temperature for 15 min. The absorbance of the reaction solution at 510 nm was measured using a microplate reader. A standard curve was prepared using rutin as a standard, and the total flavonoid content in the fermentation broth of different groups was calculated based on this curve.

[0091] Detection of total antioxidant capacity

[0092] The experiment was conducted according to the instructions of the Total Antioxidant Capacity (T-AOC) Assay Kit (FRAP Microplate Method). The specific steps are as follows:

[0093] 1. Centrifuge the fermentation broth, and dilute the supernatant 10 times for later use.

[0094] 2. Dilute the ferrous standard solution (10mM) with distilled water to 0.05, 0.1, 0.3, 0.5, 0.7, 0.9, 1.2, and 1.5mM for later use.

[0095] 3. Prepare FRAP working solution by mixing FRAP Assay buffer, TPTZ solution and ferric chloride solution in a ratio of 10:1:1.

[0096] 4. Set up blank wells, standard wells, and assay wells according to Table 6, with at least three replicates per group. Add the solutions sequentially to the 96-well plate, mix carefully, and incubate at 37°C for 30 minutes. Measure the absorbance at 593 nm using a microplate reader and record it as A. 空白 A 标准 A 测定 .

[0097] Table 6. Reaction system for total antioxidant capacity detection.

[0098] Additives Blank well (μL) Standard well (μL) Standard well (μL) distilled water 30 — — <![CDATA[Fe 2+ Standard solution — 30 — Sample solution — — 30 FRAP working fluid 264 264 264

[0099] 5. Plot a standard curve with ferrous ion concentration (mM) on the x-axis and corresponding absorbance on the y-axis. Calculate the corresponding Fe based on the absorbance of the sample solution using the standard curve. 2+ Concentration, which represents the total antioxidant capacity of the sample.

[0100] Detection of ABTS free radical scavenging activity

[0101] The experiment was conducted according to the instructions for the ABTS free radical scavenging ability assay kit. The specific steps are as follows:

[0102] 1. Before use, prepare the working solution of Reagent 4 according to the sample volume by mixing Reagent 4 and Reagent 1 in a ratio of 1:9. Prepare the ABTS working solution according to the required amount of reagent 1, Reagent 2, and Reagent 3 working solution in a ratio of 76:5:4.

[0103] 2. Set up blank wells, control wells, assay wells, and positive control wells according to Table 7, with at least three replicates for each group. Add the solutions to the 96-well plate in sequence, mix thoroughly, and incubate at room temperature in the dark for 6 minutes. Measure the absorbance at 405 nm using a microplate reader and record it as A.空白 A 对照 A 测定 A 阳性对照 .

[0104] Table 7. Reaction systems for ABTS free radical scavenging activity assay

[0105] Additives Blank well (μL) Control well (μL) Measurement well (μL) Positive control well (μL) Sample solution — 10 10 — Vitamin C solution — — — 10 distilled water 10 — — — Reagent 4 Working Solution 20 20 — 20 ABTS working solution 170 170 — 170 Reagent 1 — — 190 —

[0106] 3. Calculate the ABTS free radical scavenging activity according to the formula.

[0107] ABTS free radical scavenging rate (%) = [[A 空白 -(A 测定 -A 对照 )]÷A 空白 ×100%

[0108] Results and Analysis

[0109] Single-factor experiments determined the optimal conditions for adding honeysuckle.

[0110] The form of exogenous substance addition is closely related to microbial growth. Therefore, the target Chinese medicinal herb honeysuckle was processed by pulverizing and grinding, and then decocting over medium heat. The honeysuckle was then added to a basal culture medium of *Monascus purpureus* at different gradient levels in both powder and decoction forms for fermentation. Biomass was used as an indicator of cell growth, and Monacolin K yield was used as an indicator of secondary metabolic levels. The results are as follows: Figure 1 As shown.

[0111] Single-factor experiments revealed that the effect of honeysuckle on Monascus purpureus varied depending on its form. The promoting effect of honeysuckle extract was not entirely positively correlated with the amount added, and even showed an inhibitory effect with increasing addition. Honeysuckle powder not only promoted cell growth but also significantly increased Monacolin K production. Under powder addition conditions, cell biomass increased with increasing addition, with the highest level of 1.0g showing the best promoting effect, reaching a maximum biomass of 39.42mg / L, which was 1.29 times higher than the control group. However, the increase in biomass also increased cell density, leading to changes in oxygen levels in the limited fermentation space, thus the regulation of secondary metabolism was not absolutely positively correlated. Adding 0.8g of honeysuckle powder had the strongest promoting effect on Monacolin K synthesis. At this addition level, the synthesis of Monacolin K was significantly promoted at each time point, with a yield of 536.10mg / L on day 15 of fermentation, nearly 1.54 times higher than the control group. Based on the changes in Monacolin K and the bacterial growth, the final selection was "addition form - powder, addition amount - 0.8g" to optimize the subsequent addition time.

[0112] The effectiveness of interventions during fermentation is related to the growth state of the microorganisms. The later the addition time, the more mature the microorganisms, and the smaller the impact on their growth and metabolism. The effect of honeysuckle addition time on Monacolin K synthesis in *Monascus purpureus* is shown in [reference needed]. Figure 2 As shown in the figure. The experimental results showed that adding honeysuckle powder at 48 h of fermentation had a better promoting effect on Monacolin K. Therefore, adding 0.8 g of powder at 48 h of fermentation was finally selected as the optimal condition for adding honeysuckle powder during fermentation.

[0113] Determination of total sugar and biomass

[0114] Fermentation was carried out under the optimal conditions for adding honeysuckle, and samples were taken at different time points during fermentation to test growth indicators. Figure 3 It can be seen that the addition of honeysuckle increased the total sugar concentration in the fermentation broth, providing a usable carbon source for Monascus purpureus. Although the residual sugar content in the experimental group's fermentation broth was relatively high, its sugar consumption increased by 38% compared to the control group in the early growth stage. After the addition of honeysuckle, the Monascus purpureus exhibited significantly improved material utilization, accelerated cell growth rate, and a 1.07-fold increase in biomass by day 5 of fermentation, with a marked increase in cell density. The experimental group also showed a better growth trend throughout the entire fermentation cycle; even at the end of fermentation, the biomass was still 85% higher than the control group.

[0115] Determination of total protein and pH

[0116] The addition of honeysuckle also provided a certain nitrogen source for the purple red yeast rice, as shown in the results. Figure 3 Honeysuckle not only contains nutrients such as protein, fat, and vitamins, but also various amino acids including aspartic acid, lysine, glutamic acid, and leucine, which can effectively promote bacterial growth and metabolism. Monascus purpureus can also effectively utilize the energy substances in honeysuckle, accelerating metabolism within the bacteria. The increased synthesis of primary metabolites such as amylase leads to a consistently higher protein concentration in the fermentation broth compared to ordinary culture media. The protein concentration in the experimental group reached 3.93 mg / mL on day 12 of fermentation, an increase of 59.1% compared to the control group and 51.1% compared to the blank group. In the later stages of bacterial fermentation, secondary metabolism dominates, with protein primarily used for bacterial growth and the synthesis of secondary metabolites, thus showing a downward trend. Furthermore, the abundant chlorogenic acid and caffeic acid in honeysuckle create a lower pH fermentation environment for Monascus purpureus, and an acidic environment has been proven to effectively promote bacterial growth and the synthesis of secondary metabolites.

[0117] Monacolin K production detection

[0118] The effect of honeysuckle addition on Monacolin K synthesis in Monacolin K of Monascus purpureus is shown in the results below. Figure 4 Overall, the addition of honeysuckle promoted the synthesis of secondary metabolites by Monascus purpureus, with the most significant effect on Monacolin K. The yield in the experimental group consistently remained higher than that in the control group, especially reaching 330.90 mg / L on day 18 of fermentation, an increase of 41.5%. Solid-state fermentation of traditional Chinese medicines such as hawthorn, Alisma plantago-aquatica, and Cassia tora not only effectively increased the yield of Monacolin K but also promoted the biotransformation of active ingredients in these herbs. This has important guiding significance for the development and application of combined lipid-lowering drugs using Monascus purpureus and traditional Chinese medicine.

[0119] Detection of the color value of red yeast rice pigment

[0120] The effect of adding honeysuckle on the synthesis of red pigment in Monascus purpleis is shown in the results below. Figure 4 The three types of red yeast rice pigments showed largely consistent trends, with the yellow pigment being significantly affected. The color value increment of the yellow pigment at the same fermentation time point consistently increased, reaching 71.04 U / mL on day 18, a 42.8% increase compared to the control group. While the red pigment also showed an increase, the fluctuation was minimal, with the best promoting effect observed in the early fermentation stage, resulting in a 24.3% increase in color value. Regarding the synthesis of red yeast rice pigments, acetyl-CoA and malonyl-CoA first generate a pentanone chromophore under the catalysis of polyketide synthase. This pentanone chromophore then reacts with medium-chain fatty acids produced through fatty acid synthesis via transesterification to generate orange pigment. The orange pigment then further generates red and yellow pigments through ammonia addition and reduction reactions, respectively. Honeysuckle, a traditional Chinese medicine with good antioxidant activity, enhances the reducing properties of the fermentation environment, presumably further influencing related reactions within the microbial cells. The increased reducing substances further promote the production of yellow pigment generated through reduction reactions. In addition, the amino acids abundant in honeysuckle can play a positive role in the synthesis of red yeast rice pigment, and can also increase the yield of pigments.

[0121] Scanning electron microscopy examination

[0122] The morphology of the bacteria was observed using scanning electron microscopy to clarify the effect of honeysuckle addition. The results are shown in [Figure Number]. Figure 5 Observation of the overall state of the mycelium at low magnification revealed that although the addition of honeysuckle increased the cell density, the compactness between cells was lower than that in the control group. The cells exhibited relatively high independence and were not affected by mutual entanglement, thus their growth and metabolism were not hindered. Simultaneously, the increased intercellular spaces enhanced cell membrane permeability, facilitating the secretion of synthetic products and further promoting the synthesis of substances such as Monacolin K. Figure 5It was also evident that the honeysuckle group had a greater accumulation of secretions on the surface of the mycelium. High-magnification observation of the mycelium morphology revealed that, compared to the control group, the spore heads and mycelium surfaces in the experimental group were relatively smooth, with less noticeable depressions and wrinkles, and little individual variation in mycelium. This may be because the addition of honeysuckle significantly promoted mycelial growth and overall density, thus affecting the morphology of the mycelium. These changes in mycelial morphology, in turn, affect various indicators of the fermentation system, ultimately impacting the yield of secondary metabolites.

[0123] Transcriptional analysis of Monacolin K synthesis-related genes

[0124] To further clarify the effect of honeysuckle on Monacolin K synthesis, the expression levels of nine key genes mokA-mokI in its synthetic gene cluster were analyzed. The results are as follows: Figure 6 As shown, the addition of honeysuckle generally promoted the expression of nine key genes related to Monacolin K synthesis, but the effects on transcriptional levels varied depending on the gene. Specifically, the transcriptional levels of mokA, mokC, and mokE genes changed significantly in the early stages of fermentation, with the greatest change observed in mokE on day 8, where its transcriptional level increased by 13.4% compared to the control group. mokA and mokC also increased by 3.54% and 0.94%, respectively. mokA and mokE mainly encode polyketide synthase, a key enzyme in the synthesis of three major secondary metabolites in Monascus purpureus: monacolin pigment, Monacolin K, and citrinin. mokB catalyzes the synthesis of the diketone portion of the Monacolin K side chain, and its expression was also upregulated in the early stages. All three play crucial roles in Monacolin K synthesis. The expression of mokC and transcription factor mokH did not change significantly, but the expression of mokG in the honeysuckle group showed a slight downregulation. It is noteworthy that the product encoded by mokI plays an efflux role, with its transcriptional level increasing in the later stages of fermentation under the influence of honeysuckle, thus enabling extracellular transport of Monacolin K and reducing feedback inhibition of the product. In summary, the addition of honeysuckle increased the basal metabolic efficiency of sugars, amino acids, and lipids within the cells during the early stages of fermentation, resulting in more acetyl-CoA substrates flowing into the secondary metabolite synthesis pathway. The expression of genes related to main chain and side chain synthesis was enhanced, and the secretion of Monacolin K became more active. The combined action of multiple genes further increased the yield of Monacolin K.

[0125] Detection of total polyphenols and total flavonoids

[0126] Microbial fermentation can effectively enhance the antioxidant activity of natural products. Figure 7It can be seen that the addition of honeysuckle, which is rich in polyphenols and flavonoids, and considering the fermentation characteristics of Monascus purpureus, significantly increased the total polyphenol and total flavonoid content of the co-fermentation products, with the total amount increasing with the extension of fermentation time. The increase in total flavonoids and total polyphenols was relatively significant in the early stage of fermentation, with the content increasing by 3.93 and 2.01 times, respectively, compared with the control group. In addition to the influence of the components contained in honeysuckle itself, the hydrolytic enzymes synthesized and secreted by Monascus purpureus during the primary metabolism may have disrupted the bonds between polyphenols and other substances, thus increasing the content of free polyphenols. In addition, the β-glucosidase and other enzymes secreted by Monascus purpureus during metabolism also have the ability to convert flavonoid glycosides into flavonoid aglycones, thereby increasing the content of flavonoid compounds in the fermentation broth.

[0127] Detection of total antioxidant capacity

[0128] After fermentation, the co-fermentation product showed a significant improvement in its ability to scavenge ABTS free radicals. Although the increase decreased with prolonged fermentation time, the overall level remained higher than that of the control group and the honeysuckle blank group at the same time point, with a maximum increase of 1.61 times. Detection of total antioxidant capacity clearly showed that the co-fermentation of honeysuckle and Monascus purpureus was bidirectionally promoting, with a better promoting effect in the early stages of fermentation. Its antioxidant capacity peaked at 3.56 mM Fe. 2+ The solution was consistent; however, it reached its peak on day 15 as the cells continued to grow. These results indicate a significant increase in antioxidant activity after fermentation, which is correlated with the increased content of polyphenols, flavonoids, and other substances.

[0129] This invention, through experiments, found that honeysuckle, as an exogenous additive, can effectively promote the growth of Monascus purpureus and the synthesis of secondary metabolites. The specific effects vary depending on the form, amount, and timing of addition. Considering both cell biomass and Monacolin K production, adding 0.8g of honeysuckle powder at 48 hours of fermentation showed the best promoting effect. Measurements of growth and metabolic indicators showed that honeysuckle provided abundant carbon and nitrogen sources and an acidic environment for the growth of Monascus purpureus. Combined with the fermentation characteristics of Monascus purpureus, both primary and secondary metabolic processes were positively affected. Specifically, the highest Monacolin K production reached 536.10 mg / L, nearly 1.54 times higher than the control group. The expression of genes related to Monacolin K synthesis also showed an upregulation trend, with significant changes in genes such as mokA, mokC, and mokE. In vitro experiments demonstrated that the total flavonoid and total polyphenol content of the honeysuckle-Monascus purpureus co-fermentation products were significantly increased, and the antioxidant capacity was greatly enhanced.

Claims

1. The use of traditional Chinese medicinal materials in improving the ability of Monascus purpureus to synthesize Monacolin K, characterized in that, The Chinese medicinal material is selected from honeysuckle, and is added in the form of medicinal powder in the liquid fermentation process, the adding time is 48 hours after the fermentation, and the adding amount is 0.8 g.

2. A fermentation method for improving Monascus purpureus production of Monacolin K, characterized by, The Chinese medicinal material is selected from honeysuckle, and is added in the form of medicinal powder in the liquid fermentation process, the adding time is 48 hours after the fermentation, and the adding amount is 0.8 g.

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