A method for preparing a product rich in monascus pigment, GABA and rare saponin by liquid fermentation of monascus on American ginseng

Abstract

The present application belongs to the field of food and medicine, and particularly relates to a method for preparing a product rich in monascus pigment, GABA and rare saponin by liquid fermentation of monascus on American ginseng. The method comprises the following steps: step 1, activating monascus spores and preparing monascus seed liquid; step 2, inoculating the supernatant of the seed liquid prepared in step 1 into American ginseng substrate to perform liquid fermentation; and step 3, after the fermentation is completed, filtering or centrifuging to separate the fermentation supernatant and monascus American ginseng mycelium. The product obtained by the method has the original functions of American ginseng and new secondary metabolites from monascus, and produces a synergistic effect.

Description

Technical Field

[0001] This invention belongs to the fields of food and medicine, and specifically relates to a method for preparing products rich in red yeast pigment, GABA and rare saponins by liquid fermentation of American ginseng with Monascus purpureus. Background Technology

[0002] American ginseng ( Panax quinquefolius Panax quinquefolius (L.), a perennial herb belonging to the genus Panax in the Araliaceae family, is a valuable resource with both medicinal and edible uses. First recorded in the *Compendium of Materia Medica Supplement*, it is cool in nature and has a sweet and slightly bitter taste. Traditional Chinese medicine believes it has the effects of replenishing qi and nourishing yin, clearing heat and promoting body fluids, and can be used clinically to improve symptoms such as qi deficiency, yin deficiency, and fatigue due to deficiency heat. Modern pharmacological studies have confirmed that the core active ingredients in American ginseng (such as ginsenosides, polysaccharides, and phenolic compounds) have rich physiological functions, including regulating immunity, resisting oxidative stress, protecting cardiovascular endothelial cells, and inhibiting abnormal proliferation of vascular smooth muscle cells. It has high application value in health products, cosmetics, and pharmaceuticals. Currently, American ginseng processing still mainly uses traditional processing methods such as steaming, decocting, and boiling. However, the products obtained by traditional processing methods have extremely low levels of rare ginsenosides, and the content of active ingredients varies significantly between different batches. Furthermore, there are problems such as violent reactions, numerous byproducts, and easy destruction of other active ingredients, which affect the final quality of the raw materials. In addition, most current research focuses only on the extraction, separation, and application of natural ginsenosides inherent in American ginseng, and has not yet achieved the enrichment of additional functional active ingredients (such as adding new functional components such as red yeast rice pigment and GABA).

[0003] Dammarane-type tetracyclic triterpenoid ginsenosides, as the main active components of American ginseng, exhibit rich structural diversity. The chemical structure and glycosylation modification of ginsenosides directly affect their bioavailability and pharmacological activity in the human body. The number, type (glucose, arabinose, xylose, etc.), linkage sites, and bonding mode of the glycosyl groups attached to the aglycone are key structural features influencing the polarity and activity of the saponin. High-polarity ginsenosides are the most abundant proto-active components in medicinal plants of the ginseng genus, but they have more glycosyl side chains, resulting in lower oral absorption efficiency and poorer bioavailability. Low-polarity ginsenosides, mostly rare low-glycosylation saponins, are more lipid-soluble, have higher in vivo transport and absorption efficiency, and exhibit more significant pharmacological activities such as anti-tumor, neuroprotective, anti-inflammatory, immunomodulatory, and metabolic regulation. In the field of neurological diseases, total saponins rich in rare saponins after fermentation with *Lactobacillus plantarum* showed a stronger ability to alleviate memory loss and motor dysfunction in an MPTP-induced Parkinson's disease mouse model. The mechanism is related to the regulation of the PI3K / AKT / mTOR and NF-κB / COX-2 / iNOS signaling pathways. Regarding anti-inflammation, a mixture of Rg3 obtained by converting the main saponins Rc, Rd, and Rb1 through heat and organic acid treatment can inhibit LPS-induced inflammatory responses in RAW264.7 macrophages and downregulate the expression of inflammatory factors such as iNOS, COX-2, and TNF-α. In metabolic diseases, studies have confirmed that the Rk1 / Rg5 complex can improve insulin resistance in 3T3-L1 cells under endoplasmic reticulum stress conditions, demonstrating potential therapeutic potential for diabetes. Currently, rare ginsenosides have become important active ingredients in the research and development of functional foods and dietary supplements. Simultaneously, as potential pharmaceutical raw materials, their development and utilization can effectively enhance the added value of deep-processed products from ginseng medicinal plants, promoting the upgrading of the ginseng industry from traditional medicinal materials to high-value-added products. With the increasing attention paid to health and functional regulation by modern populations, the transformation and synthesis of rare ginsenosides has become a core target for the high-value development of traditional Chinese medicine.

[0004] However, the active ingredients (such as ginsenosides) of natural medicinal plants (such as American ginseng) are often locked within dense cell walls composed of cellulose, hemicellulose, and lignin. Traditional extraction methods suffer from low cell wall disruption efficiency, resulting in limited yields. Currently, the development of medicinal and edible resources generally faces problems such as simplistic processing methods and unclear mechanisms of action. How to overcome the low utilization rate of active ingredients and serious resource waste through advanced processing technologies, and achieve high-value transformation and utilization of these resources, has become a key research focus and technical challenge urgently needing to be addressed in this field. Dammarane-type tetracyclic triterpenoid proto-saponins, which have relatively high natural abundance in American ginseng, have limited bioavailability due to their numerous side-chain sugars and high polarity. Furthermore, their production through traditional extraction methods is extremely complex and costly.

[0005] Microbial processing, as a highly efficient and green modern Chinese medicine processing technology, can fully explore the potential value of active ingredients in American ginseng through microbial transformation technology, and improve its modern processing technology system by combining it with modern biotechnology.

[0006] Reported microorganisms that can be used to convert ginsenosides include bacteria and fungi, such as those isolated from Korean kimchi. Lactobacillus ginsenosidimutans The EMML 3041T strain has been shown to have the ability to convert ginsenoside Rb1 into the rare ginsenoside Rg3(S). Aspergillus tubingensis and Aspergillus niger cellulase It can increase the yield of rare ginsenoside CK in American ginseng extract; isolated from soil Cladosporium xylophilum Fungi and their crude enzyme solutions can efficiently convert the main ginsenosides in Panax notoginseng flowers into rare saponins, etc. Different microorganisms vary in enzyme production characteristics, conversion efficiency, and metabolite profiles. Currently, there are no reports on using Monascus purpureus strains with specific metabolic characteristics to conduct liquid fermentation of American ginseng to simultaneously improve the bioavailability / conversion rate of American ginseng's active ingredients and enrich high-value Monascus purpureus pigments and GABA, thereby obtaining novel products with synergistic health benefits. Summary of the Invention

[0007] This invention provides a method for preparing a product rich in ginseng pigments, GABA, and rare ginsenosides from American ginseng through liquid fermentation with Monascus purpureus. This method significantly improves the utilization rate of the active ingredients in American ginseng and generates ginseng pigments and GABA during fermentation, resulting in a final product that combines the original efficacy of American ginseng with secondary metabolites derived from Monascus purpureus, and produces a synergistic antioxidant effect.

[0008] The present invention specifically adopts the following technical solution:

[0009] This invention provides a method for preparing a product rich in monaxioca pigments, GABA, and rare saponins from American ginseng using liquid fermentation with Monascus purpureus, comprising the following steps:

[0010] Step 1: Activate the Monascus purpureus strain and prepare Monascus purpureus seed solution:

[0011] Monascus strain ( Monascus purpureus After activation by inoculation into PDA medium, the mycelium cake was inoculated into malt powder liquid medium and cultured with shaking at 30 ℃ to obtain Monascus seed liquid.

[0012] In a further embodiment, the preparation method of the malt powder liquid culture medium is as follows: malt powder and water are mixed, sterilized, and then the malt powder liquid culture medium is obtained. The malt powder liquid culture medium is obtained by mixing malt powder and distilled water evenly and then sterilizing, wherein the ratio of malt powder to water is 1g:30mL.

[0013] The activation culture time is 5-7 days; the shaking culture speed is 120-180 r / min, preferably 150 r / min, and the shaking culture time is 48 h.

[0014] The classification name of Monascus redissus is... Monascus purpureus It was deposited on January 4, 2026 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 42498.

[0015] Step 2: Take the supernatant of the seed liquid prepared in Step 1 and inoculate it into the American ginseng substrate for liquid fermentation.

[0016] The method for preparing the American ginseng matrix includes: mixing American ginseng powder with water, sterilizing (121 ℃, 25 min) to obtain the American ginseng matrix; wherein the American ginseng powder is the sieve material obtained by washing, drying, pulverizing, and passing American ginseng slices through a 100-mesh sieve, and the material-liquid ratio of American ginseng powder to water is 1g:30mL.

[0017] Furthermore, the preparation method of the American ginseng matrix includes: mixing American ginseng powder, malt powder, and water, sterilizing (121 ℃, 25 min), and then preparing the American ginseng matrix; wherein the mass of American ginseng powder and malt powder are equal, and the material-to-liquid ratio in the American ginseng matrix is ​​2g:30mL. Adding malt powder to the American ginseng matrix can control the ratio of red yeast rice pigment and red yeast rice yellow pigment in the fermented product.

[0018] Furthermore, step 2 specifically involves: under aseptic conditions, inoculating the seed liquid supernatant prepared in step 1 into the American ginseng substrate at an inoculation rate of 2-5% v / v, and then placing it in a liquid fermentation environment at 30 ℃ for 6-12 days. The rotation speed during liquid fermentation is 120-180 r / min.

[0019] Step 3: After fermentation, the fermentation supernatant and red yeast rice ginseng mycelium are separated by filtration or centrifugation. The fermentation supernatant can be used directly for the development of functional foods and health products, or it can be concentrated and dried (low-temperature drying / freeze-drying) to obtain a product (powder) rich in functional components such as red yeast rice pigment, GABA, and rare saponins. The red yeast rice ginseng mycelium (including residue) is washed, dried (50℃), and pulverized to obtain a powder rich in mycelial protein, intracellular pigments, and remaining ginseng components.

[0020] The present invention also provides formulations containing products prepared by the above method.

[0021] The beneficial effects of this invention are as follows:

[0022] 1. Improved utilization of active ingredients in American ginseng: Enzymes (such as glucosidase) secreted by Monascus purpureus during liquid fermentation may biotransform ginsenosides and other components, generating rare saponins with low polarity and high bioavailability or increasing the dissolution rate. Testing showed that the product prepared by the method of this invention had a significantly higher proportion of rare ginsenoside Rg3 compared to unfermented American ginseng, and the types and contents of rare ginsenosides also showed significant changes.

[0023] 2. Effective enrichment of beneficial red yeast rice secondary metabolites: Under optimized liquid fermentation conditions, the selected specific Monascus strains can efficiently synthesize:

[0024] (1) Red yeast rice pigment: In a culture medium with American ginseng powder as the main component, the synthesis rate of red yeast rice pigment can be effectively accelerated and the total value of red yeast rice pigment inside and outside the cell can be effectively increased. With the addition of malt powder, the total color value of red yeast rice pigment inside and outside the cell can be further effectively increased. After 10 days of fermentation, the total color value of red yeast rice pigment (extracellular color value of fermentation supernatant and intracellular color value of the remaining solid mixture) can reach 182.5 U / g, giving the product a natural color and significantly enhanced antioxidant activity.

[0025] (2) Gamma-aminobutyric acid (GABA): The GABA content in the fermentation supernatant after 10 days can reach 0.78 mg / mL (PPBM group). The introduction of GABA adds potential health benefits to the product, such as helping to lower blood pressure, soothe nerves, and improve sleep.

[0026] 3. Synergistic Effect: American ginsenosides and polysaccharides themselves possess antioxidant and immunomodulatory activities. In the product of this invention, the active ingredients of American ginseng coexist with pigments derived from red yeast rice (strong antioxidants)—different types of red yeast rice pigments have different effects (this invention mainly monitors red yeast rice red pigment, red yeast rice yellow pigment, and red yeast rice orange pigment; red yeast rice pigment is a general term), GABA (neuromodulatory), and other components, which can produce a synergistic effect, making it superior to single American ginseng products or ordinary red yeast rice fermented products in terms of comprehensive antioxidant properties.

[0027] 4. Advantages of liquid fermentation: Liquid fermentation has advantages such as high mass and heat transfer efficiency, easy online monitoring and control of process parameters (temperature, pH, dissolved oxygen, stirring), relatively short fermentation cycle, and easy large-scale scaling (fermentation tank).

[0028] 5. Diverse product forms: Flexible post-processing methods can yield fermentation bacteria and fermentation supernatant powder, meeting different application needs. Attached Figure Description

[0029] Figure 1 Growth status of strain MN-001 in malt solid medium.

[0030] Figure 2 Changes in the content of 20(S)-Rg3, 20(R)-Rg3, and (Compound K)CK in the American ginseng matrix at 0 and 6 days of fermentation.

[0031] Figure 3 BPI diagrams of unfermented American ginseng and fermented American ginseng under negative ion mode; A. Unfermented American ginseng AG group, B. Fermented American ginseng FAG-6 d group under PPBM system.

[0032] Figure 4 Results of detection of ginsenoside Rb1 and ginsenoside Rd during fermentation of Monascus purpureus in PPBM substrate for 1, 2, 3 and 4 days.

[0033] Figure 5 Comparison of total color value and pigment content of red yeast rice pigments in different fermentation substrates. A. Comparison of total color value; B. Relative content of the three pigments.

[0034] Figure 6 : Dynamic trend of GABA content in PPBM and MPPCM fermentation substrates.

[0035] Figure 7 Comparison of antioxidant activity of fermentation broth.

[0036] Figure 8 Dynamic trend of citrinin content in PPBM and MPPCM fermentation substrates.

[0037] Figure 9 Dynamic changes in extracellular color value of Monascus purpureus in a fermentation system using malt powder alone as the fermentation medium. A. Trend of extracellular color value in the fermentation broth using malt powder alone as the liquid fermentation substrate. B. Statistical distribution of the color value components and total color value of Monascus purpureus red pigment, Monascus purpureus yellow pigment, and Monascus purpureus red pigment in the fermentation broth using malt powder alone as the liquid fermentation substrate.

[0038] Preservation Information

[0039] Preserved biological material: Monascus purpureus strain MN-001;

[0040] Accession number: CGMCC No. 42498;

[0041] Category Naming: Monascus purpureus ;

[0042] Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Culture Collections;

[0043] Address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing;

[0044] Preservation date: January 4, 2026. Detailed Implementation

[0045] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0046] Example 1

[0047] 1. Isolation and screening of strains

[0048] The Monascus strain used in this invention ( Monascus purpureus The strain MN 001 was isolated from red yeast rice (commercially available), a traditional Chinese medicine from Gutian County, Fujian Province. The specific isolation and screening process is as follows:

[0049] Commercially available red yeast rice was diluted using the plate dilution method. In a sterile laminar flow hood, 1 g of red yeast rice was placed in 9 mL of sterile water and shaken well. 1 mL of the diluted liquid was then added to a final volume of 10 mL with sterile water, and this process was repeated to create a serial dilution of 100 mL. -2 10 -3 10 -4 The strain was purified by multiple streak purification processes.

[0050] The obtained strains were streaked in CMC-Na selection medium and cultured at 30 °C for 7 days. The size of the clear zone of different strains in CMC-Na medium was compared and analyzed, and the strain with the largest clear zone was selected as the target strain. After Congo red staining, the diameter ratio of the clear zone of the target strain (D / d = 2.2) was measured. The target strain was inoculated into liquid esculin medium, and a black positive reaction was observed. This strain was named strain MN-001. CMC-Na selection medium: 0.05 g magnesium sulfate heptahydrate, 0.4 g potassium dihydrogen phosphate, 2 g sodium carboxymethyl cellulose, 0.4 g peptone, 0.04 g sodium chloride, 0.2 g yeast extract, 4 g agar, 200 mL distilled water.

[0051] The screened strain MN-001 was inoculated into malt solid medium and cultured. Its growth status is as follows: Figure 1 As shown. The malt powder solid culture medium used here consists of: 130.1g of malt powder, 1000mL of distilled water, and 2% agar powder, mixed evenly and then sterilized.

[0052] After identification, strain MN-001 belongs to... Monascus purpureus The red Monascus was deposited on January 4, 2026, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 42498.

[0053] Example 2

[0054] Products rich in Monascus purpureus pigment, GABA, and rare saponins are prepared from American ginseng using liquid fermentation with Monascus purpureus. These products include:

[0055] Step 1: Activate Monascus purpureus strain and prepare Monascus purpureus seed solution:

[0056] The Monascus strain from Example 1 ( M. purpureus After activation culture on PDA medium for 5 days, the mycelial cake was inoculated into malt powder liquid medium (100g malt powder, 3000 mL distilled water, mixed evenly and sterilized). The medium was then cultured at 30 ℃ and 150 r / min for 48 h with shaking to obtain seed culture. The supernatant of the seed culture was used for inoculation.

[0057] Step 2: Under aseptic conditions, the seed liquid supernatant prepared in Step 1 was inoculated at an inoculation rate of 3% (v / v) into sterilized (121 ℃, 15 min) and cooled American ginseng substrates PPBM and MPPCM, respectively. After inoculation, the substrates were placed in liquid fermentation at 30 ℃ and 150 r / min for 12 days. Each group was replicated in triplicate. On days 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 of the fermentation process, the fermentation supernatant was collected to monitor the types and contents of ginsenosides, as well as the contents of red yeast rice pigment and γ-aminobutyric acid (GABA). The fermentation supernatant at day 4 was also collected, and the antioxidant activity of the fermentation product was detected using conventional methods such as DPPH and ABTS. Additionally, the content of citrinin in the final fermentation product was determined.

[0058] An AG control group and an MN-M control group were set up. The AG control group consisted of an aqueous extract of American ginseng powder that had not undergone seed culture fermentation (obtained by mixing American ginseng powder with water, sonicating for 1 hour, and then sterilizing). The MN-M control group consisted of a malt powder liquid culture medium as the substrate for seed culture fermentation.

[0059] The method for preparing the American ginseng matrix PPBM includes: mixing American ginseng powder with water and then sonicating for 1 hour, followed by sterilization to prepare the American ginseng matrix PPBM; wherein the American ginseng powder is the sieve material obtained by washing, drying, pulverizing, and passing American ginseng slices through a 100-mesh sieve, and the material-liquid ratio of American ginseng powder to water is 1g:30mL.

[0060] The preparation method of the American ginseng matrix MPCM includes: mixing American ginseng powder, malt powder and water, and then sonicating for 1 hour to prepare American ginseng matrix MPCM; wherein the American ginseng powder is the sieve material obtained by washing, drying and crushing American ginseng slices and passing them through a 100-mesh sieve, the American ginseng powder and malt powder are of equal mass, and the material-to-liquid ratio in the American ginseng matrix MPCM is 2 g:30 mL.

[0061] The methods for detecting the types and contents of ginsenosides, as well as the contents of red yeast rice pigment and GABA, are as follows:

[0062] The types and relative contents of ginsenosides were determined using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS / MS) in negative ion mode. 0.1% formic acid aqueous solution and acetonitrile solution were used as mobile phases A and B, respectively. Mass spectrometry analysis was performed using a Waters Xevo G2-XS system with a Waters Acquity UHPLC BEHC column. 18 (100 mm × 2.1 mm × 1.7 mm). The linear gradient elution program was as follows: 0–3 min, 13% B; 3–12 min, 13%–40% B; 12–14 min, 40% B; 14–14.01 min, 40%–90% B; 14.01–16 min, 90% B; 16.01–17 min, 13% B. The flow rate was 0.3 mL / min, and the injection volume was 1 μL. Electrospray ionization (ESI) negative ion mode was used. - The mass-to-charge ratio scanning range is 100-1500 m / z, and the detection wavelength is 203 nm.

[0063] The changes in ginsenoside content were quantitatively determined by UHPLC at 203 nm. Specifically, a binary linear gradient elution program was used for separation. The mobile phase consisted of (A) 0.1% formic acid aqueous solution and (B) acetonitrile. The gradient settings were as follows: 0–13 min, 87% A; 13–22 min, 87–60% A; 20–25 min, 60% A; 25–25.01 min, 60–10% A; 25.01–27 min, 10% A; 27–27.01 min, 10–87% A; 27.01–31 min, 87% A. The flow rate was 0.3 mL / min, the injection volume was 5 μL, and the column temperature was maintained at 30°C. Separation was performed at C... 18 The procedure was performed on a reversed-phase liquid chromatography column (2.1 mm × 100 mm).

[0064] The ultraviolet absorption of red yeast rice pigment was measured at 400 nm, 450 nm and 510 nm according to national standards.

[0065] The GABA content was determined by measuring the ultraviolet absorbance at 645 nm using the deamination colorimetric principle.

[0066] Antioxidant activity was assessed using DPPH and ABTS + The conventional method for detecting in vitro antioxidant models involved diluting the samples to an appropriate factor and measuring the absorbance at wavelengths of 517 nm and 734 nm using a UV-Vis spectrophotometer. All results were measured in triplicate. The references for this method are: Xiao Xiang, Zhou Liping, Ma Xin. Effect of adding perilla leaf extract to red yeast fermentation on its antioxidant activity [J]. China Brewing, 2011, (07): 50-53. Liu A, Zhang S, Li Q, et al. Production and characterization of sorghum sourdough bread sequentially fermented with Monascus purpureus and Lactiplantibacillus plantarum [J]. Foodand Bioprocess Technology, 2024, 17 (11): 3603-3614.

[0067] The citrinin content in the fermentation end product was determined using an enzyme-linked immunosorbent assay (ELISA) kit, following the instructions. A standard curve was established: y = 0.1882x + 0.0976 (R²). 2 = 0.9989) is used for subsequent data processing.

[0068] like Figure 2 As shown, in the PPBM fermentation system, after 6 days of Monascus purpureus fermentation, the relative contents of rare ginsenosides 20-S / R-Rg3 and Compound K (CK) were significantly increased compared with those before fermentation. Specifically, compared with the unfermented control group (AG), after 6 days of Monascus purpureus fermentation of American ginseng, the contents of the three target rare ginsenosides (20(S)-Rg3, 20(R)-Rg3, and CK) all showed statistically significant increases. Among them, the relative increase of CK was the most prominent, increasing to 2 times the relative content after fermentation; the increase of 20(R)-Rg3 was 63%, and the final absolute content of 20(S)-Rg3 was the highest, with an increase of about 23%. This indicates that Monascus purpureus fermentation of American ginseng can effectively promote the conversion of the original saponins to rare ginsenosides, thereby enriching the target active ingredients.

[0069] Figure 3The images show the BPI mass spectra of unfermented American ginseng and the BPI mass spectra of fermented PPBM system. The high mass-to-charge ratio glycosides are mostly the original saponins. Figure 3 A), after fermentation and transformation, a new peak in the ratio of medium to low quality charge appears ( Figure 3 B) indicates that the substances underwent a transformation before and after fermentation. Figure 4 The results show the detection of ginsenoside components in the PPBM matrix of American ginseng during the fermentation process of Monascus purpureus strain MN-001 for 1-4 days. The results clearly reflect the dynamic conversion process of proto-ginsenoside Rb1 to secondary ginsenoside Rd: On day 1 of fermentation, ginsenoside Rb1 showed a significant characteristic peak (peak height approximately 280 mAU), while ginsenoside Rd only showed a weak signal near the baseline, indicating that proto-ginsenoside Rb1 was the main form present in American ginseng at this time, and Rd had not yet been generated in large quantities; as the fermentation time was extended to day 2, the chromatographic peak height of Rb1 decreased significantly compared to day 1 (peak height decreased to approximately 120 mAU), while the peak area of ​​Rd increased significantly, indicating that the β-glucosidase secreted by the strain had efficiently catalyzed the hydrolysis of the Rb1 sugar chain, realizing the directional conversion of Rb1 to Rd; from day 3 to 4 of fermentation, the peak signal of Rb1 continued to weaken, reaching near the baseline level by day 4, while the peak height of Rd remained at 2-3 days. After reaching its peak, it declined slightly, indicating that ginsenoside Rd, as an intermediate product, can be further hydrolyzed by glycosidases secreted by the strain in the later stage of fermentation, and transformed into rarer ginsenoside products.

[0070] like Figure 5 As shown in A, the fermentation substrate affects Monascus pigments (…). Monascus pigments The biosynthesis of Monascus purpureus pigments (MPs) is affected. During the 10-day fermentation period, the total color value of both the ginseng substrate PPBM group and the ginseng substrate MPCM group showed a continuous upward trend. Furthermore, the total color value of the MPCM group reached 182.5 U / g on day 10 of fermentation, higher than the 115.7 U / g of the PPBM group. This indicates that the malt powder nutrient substrate in the MPCM group is conducive to the accumulation of Monascus purpureus pigments. Dynamic monitoring and analysis of the relative contents of red, orange, and yellow pigments during the fermentation period yielded the following results: Figure 5 As shown in B, throughout the entire fermentation cycle, both the PPBM and MPPCM groups had yellow pigment as the main component. The relative content of yellow pigment in the PPBM group remained stable overall, while the relative proportion of red pigment in the MPPCM group gradually increased and then slowly decreased. Throughout the entire fermentation process, the relative content of red yeast rice pigment in the MPPCM group was better than that in the PPBM group.

[0071] like Figure 6As shown, during the first 5 days of fermentation, the GABA content in both the ginseng substrate PPBM group and the ginseng substrate MPPCM group did not increase significantly, and the content in both groups remained at a low and stable level. From day 6 onwards, GABA began to be rapidly biosynthesized and accumulated. The PPBM group reached a peak content of 0.78 mg / mL on day 10, while the MPPCM group reached a peak content of 0.46 mg / mL earlier on day 8. The peak GABA content of the PPBM group was approximately 1.7 times that of the MPPCM group, demonstrating a stronger GABA synthesis capacity.

[0072] like Figure 7 As shown, the antioxidant activity of the American ginseng matrix PPBM group and the American ginseng matrix MPCM group was significantly improved compared with the American ginseng powder aqueous extract without seed liquid fermentation and the MN-M control group. The MPCM group showed higher levels of DPPH and ABTS. + All groups showed strong antioxidant capacity in free radical scavenging, followed by the PPBM group, while the control groups (AG and MN-M groups) maintained relatively low antioxidant levels.

[0073] like Figure 8 As shown, the concentration of citrinin in the PPBM substrate group was 5.4 ppb on day 2 of fermentation, rising to 7.644 ppb on day 4 and then slowly increasing, reaching 9.352 ppb on day 10. In the MPPCM substrate group, the citrinin concentration remained below 10 ppb from day 1 to day 10. This indicates that the toxin content of both fermentation substrates was compliant and that the Monascus purpureus strains used were safe.

[0074] Comparative Example 1: Liquid fermentation culture of Monascus purpureus

[0075] Fermentation culture was carried out using malt powder liquid medium, PDB medium, and MRS liquid medium as fermentation substrates, including the following steps:

[0076] Step 1: Prepare seed culture of Monascus purpureus according to Step 1 of Example 2, and take the supernatant of seed culture for inoculation.

[0077] Step 2: Under aseptic conditions, the seed culture supernatant prepared in Step 1 was inoculated at an inoculation rate of 3% (v / v) into malt powder liquid medium, PDB medium, and MRS liquid medium, respectively. After inoculation, the mixture was placed in liquid fermentation at 30 ℃ and 150 r / min for 12 days. Each group was replicated in triplicate. Fermentation supernatant was collected on days 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 during the fermentation process to monitor the types and contents of ginsenosides.

[0078] Fermentation results showed that when there was no American ginseng in the fermentation substrate, ginsenosides could not be detected in the fermentation products, indicating that the American ginseng substrate is the basis for the product's efficacy.

[0079] Comparative Example 2:

[0080] Fermentation culture using malt powder liquid culture medium as the fermentation substrate includes the following steps:

[0081] Step 1: Prepare seed culture of Monascus purpureus according to Step 1 of Example 2, and take the supernatant of seed culture for inoculation.

[0082] Step 2: Under aseptic conditions, the seed liquid supernatant prepared in Step 1 was inoculated into malt powder liquid culture medium at an inoculation rate of 3% (v / v). After inoculation, the medium was placed at 30 ℃ and 150 r / min for liquid fermentation.

[0083] Fermentation results as follows Figure 9 As shown, in the system where Monascus purpureus ferments malt powder alone, significant Monascus pigment production can be observed on day 6 of fermentation. In the PPBM and MPPCM liquid fermentation systems, significant Monascus pigment production can be observed on days 3-4. This indicates that the fermentation system with American ginseng as the main substrate can promote the effective synthesis of Monascus pigment.

[0084] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a product rich in monaxioca pigment, GABA, and rare saponins from American ginseng using liquid fermentation with Monascus purpureus, characterized in that, Includes the following steps: Step 1: Activate the Monascus purpureus strain and prepare Monascus purpureus seed solution; Step 2: Take the supernatant of the seed liquid prepared in Step 1 and inoculate it into the American ginseng substrate for liquid fermentation; Step 3: After fermentation, the fermentation supernatant and red yeast rice ginseng mycelium are separated.

2. The method according to claim 1, characterized in that, The red mold is classified as Monascus purpureus, with accession number CGMCC No. 42498.

3. The method according to claim 1, characterized in that, Step 1 includes: after activating the Monascus purpureus strain by inoculating it into PDA medium, taking the mycelium cake and inoculating it into malt powder liquid medium, and shaking culture at 30 ℃ to obtain Monascus purpureus seed liquid.

4. The method according to claim 3, characterized in that, The preparation method of the malt powder liquid culture medium is as follows: malt powder and water are mixed and sterilized to obtain the malt powder liquid culture medium.

5. The method according to claim 3, characterized in that, The activation culture time is 5-7 days; the shaking culture speed is 120-180 r / min, and the shaking culture time is 48 h.

6. The method according to claim 1, characterized in that, In step 2, the method for preparing the American ginseng matrix includes: mixing American ginseng powder with water, sterilizing and then preparing the American ginseng matrix; wherein the American ginseng powder is the sieve material obtained by washing, drying, pulverizing, and passing American ginseng slices through a 100-mesh sieve, and the material-liquid ratio of American ginseng powder to water is 1g:30mL.

7. The method according to claim 1, characterized in that, In step 2, the method for preparing the American ginseng matrix includes: mixing American ginseng powder, malt powder and water, sterilizing and then preparing the American ginseng matrix; wherein the American ginseng powder is the sieve material obtained by washing, drying and crushing American ginseng slices and passing them through a 100-mesh sieve, the American ginseng powder and malt powder are of equal mass, and the material-to-liquid ratio in the American ginseng matrix is ​​2g:30mL.

8. The method according to claim 1, characterized in that, Step 2 specifically involves inoculating the supernatant of the Monascus seed liquid prepared in step 1 into the American ginseng substrate at an inoculation rate of 2-5% v / v under aseptic conditions, and then placing it in a liquid fermentation environment at 30 ℃ for 6-12 days.

9. The method according to claim 8, characterized in that, The rotation speed during liquid fermentation is 120-180 r / min.

10. A formulation comprising the product prepared by any one of claims 1-9.

Images