A solid state fermentation method for high yield of monascus yellow pigment by monascus purpureus

Abstract

The present application relates to the field of microbial fermentation, and discloses a solid-state fermentation method for high-yield production of Monascus yellow pigment by Monascus purpureus. By regulating key process parameters such as fermentation temperature, gelatinization amount, fermentation time, inoculation amount, loading amount of solid medium for fermentation, and types of carbon source matrix, the present application realizes efficient directional biosynthesis and enrichment of Monascin (MS) and Ankaflavin (AK). The method discards the traditional vague color value index, establishes a quality standard with MS and AK specific content as the core, and promotes the product from a food coloring agent to a medicinal raw material. The target components are enriched by solid-state fermentation technology, and the extraction and purification steps are omitted, thereby greatly reducing the cost. The finished product is Monascus powder, which is consistent with the traditional application scene. The final product has the high MS / AK content, low MK content, and no citrinin, and meets the high-end medicinal raw material standard.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation, specifically to a solid-state fermentation method for high-yield red yeast rice pigment using Monascus purpureus. Background Technology

[0002] Red yeast rice pigment is a widely used natural pigment, a mixture with a zineb skeleton structure. Based on its maximum absorption value, it can be divided into three main categories: yellow (330-450 nm), orange (460-480 nm), and red (490-530 nm). Due to its excellent coloring ability and certain antibacterial properties, it has wide applications in the food industry.

[0003] In the medical field, in-depth research on the active substances in red yeast rice has confirmed that MS and AK in red yeast rice yellow pigment not only have lipid-lowering effects, but also anti-inflammatory, antioxidant, anti-diabetic, immunomodulatory, Alzheimer's disease risk factor reduction, and anti-tumor effects. Furthermore, compared to another lipid-lowering metabolite of red yeast rice, Monaclin K (MK), it exhibits higher lipid-lowering activity, and no side effects (such as rhabdomyolysis) have been found. Therefore, ANKASCIN® 568-R, containing only red yeast rice yellow pigment, has received New Dietary Ingredient (NDI) certification from the US FDA, becoming the only compliant product based on red yeast rice extract currently approved for the US market. In stark contrast, high-concentration MK products are classified as unapproved drugs and are prohibited from being sold as dietary supplements.

[0004] Several preparation techniques related to red yeast rice yellow pigment have been disclosed in the prior art. For example, Chinese invention patent CN117778199B discloses a purple red yeast rice strain obtained by mutation of Monascus purpureus H1102. This mutant strain has formed a morphology conducive to pigment production, and its yellow pigment production can reach 387 U / ml through liquid fermentation. This technology mainly focuses on the liquid fermentation process. Chinese invention patent CN120173430A discloses an antibacterial and antioxidant red yeast rice yellow pigment and its preparation method and application. It involves reacting red yeast rice yellow pigment with tannic acid in solution, followed by solid-liquid separation, washing to remove unbound pigment, and then... Solid drying yields antibacterial and antioxidant red yeast rice yellow pigment; Chinese invention patent CN116875078B discloses a method for preparing high-quality red yeast rice yellow pigment by mixing and dissolving a solvent with thiourea dioxide and red yeast rice pigment raw materials, followed by alkaline reaction, desalination, concentration, and drying. This method utilizes thiourea dioxide to achieve rapid and efficient reduction of red yeast rice pigment raw materials at room temperature without the need for strong acids or bases; Chinese invention patent CN101914453B also discloses a mutant strain of Monascus purpureus MA101 and its solid-state fermentation method, aiming to simultaneously improve the color value of red yeast rice yellow pigment and red yeast rice pigment.

[0005] However, existing technologies still have many significant limitations, making it difficult to meet the pharmaceutical product demands for high-value red yeast rice yellow pigment. Firstly, metabolite regulation is non-directional. For example, technologies like CN101914453B aim for simultaneous high production of red yeast rice yellow and red pigments, with process optimization focusing on increasing the total color value. They lack the ability to target specific active components with clear pharmaceutical value, such as MS and AK. Furthermore, "color value" as a quality target cannot reflect the absolute content of specific single compounds like MS or AK. Existing technologies generally use "color value" as the core optimization target and quality indicator. This approach essentially serves the coloring function but cannot distinguish between structurally similar but functionally different components, leading to a serious disconnect between product quality standards and actual pharmaceutical efficacy. Therefore, there is an urgent need in this field for a more precise quality control method that can directly correlate with the content of specific active ingredients.

[0006] Secondly, existing mainstream liquid fermentation technologies still have a series of problems: First, downstream extraction and purification processes are complex and costly. The target pigment in the liquid fermentation product is dispersed in a large amount of liquid culture medium at a low concentration. Subsequent high-energy-consuming and high-cost separation and purification steps, such as centrifugation, solvent extraction, column chromatography, concentration, and spray drying, are required to obtain the pigment product. This process involves large equipment investment and high solvent consumption, significantly increasing production costs and potentially introducing safety and environmental risks due to the use of organic solvents. Second, the final product is of a single form and has limited applications. The final product of liquid fermentation technology is usually a refined pigment powder, which is completely different from the traditional medicinal red yeast rice and red yeast powder, making it difficult to use directly as a raw material for traditional Chinese medicine decoction pieces or "food and medicine homology" dietary supplements. First, it cannot meet consumers' demand for traditional forms of products, limiting its application in high-value-added pharmaceutical and health product fields. Second, its metabolic regulation is limited and may affect the efficacy spectrum. Although the homogenized environment of the liquid fermentation system is conducive to process control, it may not be able to fully simulate and reproduce the complex physiological state and metabolic regulation network of Monascus purpureus on natural solid substrates. This may result in a relatively simple metabolite spectrum and fail to induce the production of multiple synergistic components that may exist in solid-state fermentation to the maximum extent. Third, there are bottlenecks in product concentration and yield. The high water activity during liquid fermentation leads to high dilution of cell metabolites (pigments), and a large amount of mycelium is mixed with pigments, making subsequent separation difficult and objectively limiting the further improvement of unit volume yield.

[0007] In summary, existing liquid fermentation technologies for producing high-value red yeast rice yellow pigment suffer from a series of problems, including heavy downstream processing burdens, high product costs, product forms that are out of sync with traditional applications, and the inability to fully stimulate the metabolic potential of bacterial strains. Therefore, developing a fermentation technology that can directly produce products rich in the target active ingredient and whose end product form is closer to traditional applications has significant practical and economic value. Summary of the Invention

[0008] This invention provides a solid-state fermentation method that is simple in process, low in cost, has wide applicability to product forms, and can produce high-value red yeast rice yellow pigment in a targeted and efficient manner, providing core raw materials and technical support for the development of medicinal red yeast rice products for cardiovascular and cerebrovascular health.

[0009] On the one hand, this aspect provides a solid-state fermentation method for high-yield red yeast rice yellow pigment using *Monascus purpureus*, the steps of which are as follows:

[0010] Strain activation steps: The activated Monascus strain is propagated in a seed culture medium to obtain propagation seed solution; Solid-state fermentation steps: Gelatinize the carbon source substrate to obtain a solid fermentation medium; add the propagation seed liquid to the solid fermentation medium and incubate statically to obtain the fermentation product; Preparation steps of red yeast rice powder: The fermentation product is dried and pulverized to obtain red yeast rice powder containing red yeast yellow pigment.

[0011] Furthermore, the yellow pigment in the red yeast rice includes Monascin and Ankaflavin.

[0012] Furthermore, in the red yeast rice powder containing red yeast yellow pigment, the content of MK does not exceed 0.05g.

[0013] Furthermore, the Monascus strain is Monascus BNCC145382.

[0014] Furthermore, in the solid-state fermentation step, the carbon source matrix includes indica rice, glutinous rice, and / or japonica rice.

[0015] Furthermore, the carbon source matrix includes indica rice.

[0016] Furthermore, in the solid-state fermentation step, the static culture is carried out in a fermentation bottle; the amount of the solid fermentation medium in the fermentation bottle is 40-80 g / L.

[0017] Furthermore, the amount of the solid fermentation medium in the fermentation flask is 80 g / L.

[0018] Furthermore, in the solid-state fermentation step, the preparation process of the fermentation solid culture medium includes: soaking the carbon source matrix in water for 12 hours, draining the water to obtain the soaked carbon source matrix; adding water to the soaked carbon source matrix and cooking it to obtain the fermentation solid culture medium.

[0019] Furthermore, the cooking conditions are 121 °C, 0.1 MPa, and 15 min.

[0020] Furthermore, in the preparation process of the fermentation solid culture medium, water accounting for 20-60% of its total mass is added to the soaked carbon source matrix, and the mixture is boiled to obtain the fermentation solid culture medium.

[0021] Furthermore, in the preparation process of the fermentation solid culture medium, water accounting for 20% of its total mass is added to the soaked carbon source matrix, and the mixture is boiled to obtain the fermentation solid culture medium.

[0022] Furthermore, in the solid-state fermentation step, when the propagation seed liquid is added to the fermentation solid culture medium, the volume (mL) of the propagation seed liquid is 5%-25% of the mass (g) of the fermentation solid culture medium.

[0023] Furthermore, the volume (mL) of the propagation seed solution is 10% of the mass (g) of the fermentation solid culture medium.

[0024] Furthermore, in the solid-state fermentation step, the static culture time is 15-25 days.

[0025] Furthermore, the static incubation period was 25 days.

[0026] Furthermore, in the solid-state fermentation step, the temperature for static culture is 23-35℃.

[0027] Furthermore, the static incubation temperature is 30℃.

[0028] Furthermore, in the strain activation step, the seed culture medium comprises 60 g / L glucose, 25 g / L peptone, 2 g / L KH2PO4, 2 g / L NaNO3, and 1 g / L MgSO4·7H2O.

[0029] On the other hand, the present invention provides an application of the above-mentioned solid-state fermentation method in the preparation of red yeast rice yellow pigment.

[0030] The technical solution of this invention has the following advantages: Establishing a quality standard centered on content: This invention completely abandons the vague indicator of traditional "color value" and innovatively establishes a quality standard centered on the specific content (unit: mg / g) of Monascin and Ankaflavin. This shift elevates the product of this invention from the level of 'food colorant' to the level of 'pharmaceutical raw material'. Its content data can be directly used in drug instructions, academic research, and regulatory applications, laying the foundation for the precise use and standardization of red yeast rice yellow pigment.

[0031] Ambiguity of the target product: Existing technologies use "color value" as the core evaluation indicator, but color value is only an apparent physical indicator that measures the overall coloring ability of a sample. For complex red yeast rice pigments, a high color value cannot accurately reflect the actual content and ratio of the key medicinal components MS and AK, leading to a disconnect between product quality standards and medicinal efficacy.

[0032] Solving the problem of complex and costly downstream extraction processes: By adopting solid-state fermentation technology, the target products (red yeast rice yellow pigment MS, AK) are highly enriched in the solid matrix after fermentation. The final product can be obtained by simple drying and pulverization, which completely eliminates the complex and expensive extraction and purification steps and greatly reduces production costs.

[0033] Solving the problem of product form being out of touch with traditional applications: The final product produced by this invention is red yeast rice powder rich in Monascin (MS) and Ankaflavin (AK). Its form is consistent with traditional medicinal red yeast rice / powder, and it can be directly used as a raw material for Chinese medicine decoction pieces, dietary supplements, or functional food ingredients, perfectly matching traditional application habits and market demand for high value-added products.

[0034] Addressing the limitations and efficacy potential of metabolic regulation: By optimizing specific process parameters of solid-state fermentation (such as strain, temperature, humidity, and fermentation time), a metabolic environment conducive to the synthesis of yellow pigments rather than red pigments or Monacolin K (MK) by Monascus purpureus can be created, thereby achieving efficient and targeted production of the target active ingredients MS and AK, and obtaining a complete metabolite profile that may have a more synergistic effect.

[0035] High-quality properties of the target product: Through comprehensive control of the above key points, the final red yeast rice powder product not only has a high total MS and AK content (≥31.743 mg / g), but also has the MS / AK ratio, low MK content (≤0.05 mg / g), and absence of citrinin, etc., which are precisely controlled to meet the standards for high-end pharmaceutical raw materials. Attached Figure Description

[0036] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0037] Figure 1The results are shown in the figure. a represents the effect of different substrates on the yield and yield of yellow pigment, b represents the effect of different loading amounts on the yield and yield of yellow pigment, c represents the effect of different inoculum amounts on the yield and yield of yellow pigment, d represents the effect of different gelatinization amounts on the yield and yield of yellow pigment, e represents the effect of different temperatures on the yield and yield of yellow pigment, and f represents the effect of different fermentation times on the yield and yield of yellow pigment. Figure 2 It is a surface plot and contour lines showing the effect of the interaction between inoculation amount and loading amount on the total yellow pigment yield of red yeast rice; Figure 3 The graph and contour lines show the effect of the interaction between inoculum amount and gelatinization amount on the total yellow pigment yield of red yeast rice. Figure 4 This is a surface plot and contour lines showing the effect of the interaction between gelatinization amount and loading amount on the total yellow pigment yield of red yeast rice. Detailed Implementation

[0038] The following embodiments are provided to better understand the present invention, but the following embodiments do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the scope of protection of the present invention.

[0039] Unless otherwise specified, all experimental steps or conditions in the examples were performed according to conventional experimental procedures and conditions in the art. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0040] Example 1. A solid-state fermentation method for high-yield Monascus purpureus yellow pigment production using Monascus purpureus. 1. Seed liquid propagation process: The Monascus purpureus strain BNCC145382 (purchased from Henan Provincial Engineering Technology Research Center for Industrial Microbial Strains), preserved on slant culture medium, was activated before fermentation to obtain an activated strain; then the activated strain was inoculated into an Erlenmeyer flask containing seed liquid and propagated on a shaker.

[0041] Experimental Methods: The *Monascus purpureus* strain BNCC145382, preserved on slant agar, was inoculated onto a plate containing PDA medium using the streaking method. The plate was incubated at 28°C for 4-7 days to obtain an activated strain. 50 mL of seed culture medium was prepared and placed in a 250 mL Erlenmeyer flask. The seed culture medium consisted of 60 g / L glucose, 25 g / L peptone, 2 g / L KH₂PO₄, 2 g / L NaNO₃, and MgSO₄. . Composition: 7H2O 1 g / L; The activated strain was inoculated into the seed culture medium and cultured at 28℃ for 2 days to obtain the propagation seed solution.

[0042] 2. Solid-state fermentation process: Take 20 g of washed indica rice and place it in a 250 mL fermentation container. Soak the rice in purified water overnight (12 h), then drain. Add water equal to 50% of the total mass of the soaked rice and steam it at 121 ℃, 0.1 MPa, for 15 min to obtain a solid fermentation medium. Inoculate the propagation seed solution into the solid fermentation medium at an inoculum of 25% (v / w, mL / g) and incubate at 30 ℃ for 25 days to obtain the fermentation product.

[0043] 3. Preparation process of the end product, red yeast rice powder: After fermentation, the fermentation product is dried at low temperature (<55℃) and mechanically pulverized to obtain the red yeast rice powder end product rich in MS and AK. This process completely eliminates the complex downstream extraction steps such as centrifugation, extraction, concentration, and purification required for liquid fermentation.

[0044] Example 2. Optimization of fermentation conditions 1. Carbon source matrix: Currently, solid-state fermentation processes mostly use rice as the carbon source substrate. Differences in rice quality directly lead to significant differences in the fermentation products of red yeast rice. Different rice varieties have inherent differences in the content of amylose and amylopectin, which further determines the physicochemical properties of the cooked rice. Rice with higher amylose content has looser starch granule arrangement, facilitating oxygen permeation and mycelial invasion, making it more suitable for the growth and reproduction of *Monascus purpureus*. In this experiment, glutinous rice and japonica rice were selected as the carbon source substrate to replace indica rice in step 2, with other steps remaining the same as in Example 1. The effect on the yield of red yeast rice yellow pigment was measured to verify the carbon source substrate optimization results. The results are as follows: Figure 1 As shown in Figure a, the results indicate that the yield of red yeast rice yellow pigment is highest when the carbon source substrate is indica rice, and different carbon source substrates have different characteristics and different effects on mycelial growth and metabolic rate.

[0045] 2. Loading volume of solid fermentation medium: In solid-state fermentation, the loading amount of solid culture medium in the fermentation vessel directly affects the permeability of the carbon source matrix and the overall fermentation microenvironment. If the loading thickness of the solid culture medium is too large, it will hinder heat transfer and gas exchange, especially the introduction of oxygen (O2) and the expulsion of carbon dioxide (CO2), leading to the formation of an anaerobic core zone and heat accumulation within the fermentation system, inhibiting the aerobic growth, metabolism, and pigment synthesis processes of *Monascus purpureus*. If the loading of the solid culture medium is too shallow, it results in low fermentation scale efficiency and difficulty in maintaining stable fermentation humidity. To optimize fermentation scale and efficiency, the effects of different loading amounts of solid culture medium on cell biomass and *Monascus purpureus* yellow pigment production were investigated. 250 mL culture flasks were used to load solid culture medium, and three groups were set up according to different loading amounts of solid culture medium in the culture flasks: 20 g / flask, 30 g / flask, and 40 g / flask. Other steps were the same as in Example 1. The results are as follows: Figure 1 As shown in b, by measuring the yield of red yeast rice yellow pigment, it was found that too low a loading of the fermentation solid medium led to low fermentation scale efficiency, while too high a loading may inhibit pigment synthesis due to hindered mass and heat transfer. When the loading of the fermentation solid medium was 30 g / bottle or 40 g / bottle, the yield of red yeast rice yellow pigment was significantly improved. Considering the cost, 30 g / bottle was selected as the loading of the fermentation solid medium.

[0046] 3. Inoculation dosage: The inoculum size directly determines the initial cell concentration of the fermentation system, thus affecting the length of the lag phase and fermentation synchronicity. Too low an inoculum size prolongs the lag phase, increases the overall fermentation cycle, and raises the risk of contamination by other microorganisms; while too high an inoculum size can shorten the lag phase, it easily leads to excessive consumption of nutrients in the early stages and excessively rapid mycelial growth, resulting in insufficient oxygen supply and accumulation of metabolic waste in the early fermentation stage, which in turn inhibits the synthesis of secondary metabolites in the later stages. This study set up gradient inoculum sizes with inoculum-to-rice volume-to-mass ratios of 5%, 10%, 15%, 20%, and 25% (v / w, mL / g), with other steps identical to Example 1, to explore its regulatory effect on fermentation kinetics, cell density, and the final yield of the yellow pigment. The results are as follows: Figure 1 As shown in Figure c, by measuring the yield of red yeast rice yellow pigment, it was found that the highest yield of red yeast rice yellow pigment was achieved when the inoculum amount to rice volume-to-mass ratio was 20%. At this inoculum amount, the kinetic relationship between cell growth and product synthesis was most harmonious.

[0047] 4. Gelatinization amount: The degree of gelatinization of the carbon source substrate (rice) determines its physical structure and the ease with which it can be enzymatically hydrolyzed and utilized. Insufficient gelatinization (low water content) prevents the starch granules from fully disintegrating, resulting in a dense structure that hinders the invasion and nutrient uptake of *Monascus purpureus* mycelia. Excessive gelatinization (high water content) makes the rice grains too soft and sticky, easily caking into clumps and significantly reducing the aeration performance of the carbon source substrate (rice), thus affecting the aerobic respiration of the microorganisms. This study investigated the effects of different mass ratios of carbon source substrate (rice) to water on mycelial growth depth and pigment synthesis levels. Water was added at 20%, 30%, 40%, 50%, and 60% of the total mass of the carbon source substrate (rice) in the experiment, with other steps identical to those in Example 1, to prepare fermentation solid culture media with different degrees of gelatinization. The results are as follows: Figure 1 As shown in d, the results of the red yeast rice yellow pigment production determination show that the pigment production is highest when 40% of the total mass of water is added to the carbon source matrix (canmi), indicating that the physical structure and aeration of the carbon source matrix (canmi) reach the optimal balance at this ratio.

[0048] 5. Temperature: Fermentation temperature is a core physical parameter for regulating microbial enzyme activity and metabolic pathways. Excessively high temperatures can cause premature aging and autolysis of mycelia, and may also destroy heat-sensitive pigment components; excessively low temperatures inhibit cell growth, reduce metabolic activity, and thus prolong the fermentation cycle. Furthermore, the optimal temperatures for the growth stage and pigment synthesis stage of *Monascus purpureus* often differ. In this study, the fermentation temperatures during solid-state fermentation were set at 23℃, 25℃, 27℃, 30℃, 33℃, and 35℃, with other steps identical to those in Example 1, to investigate the effects of different culture temperatures on mycelial morphology, growth rate, and the efficiency of *Monascus purpureus* yellow pigment synthesis, and to further explore a staged temperature regulation strategy. Results are as follows: Figure 1 As shown in Figure e, by measuring the yield of red yeast rice yellow pigment, it was found that a fermentation temperature of 30℃ was most favorable for the synthesis of red yeast rice yellow pigment. Temperatures that are too high or too low significantly affect the metabolic activity of the red yeast mold cells and the stability of the products.

[0049] 6. Time: Fermentation time is a key factor determining the accumulation level of Monascus purpureus metabolites. If the fermentation cycle is too short, the Monascus purpureus mycelium growth has not yet reached the plateau phase, resulting in insufficient synthesis of secondary metabolites (such as MS and AK), ultimately leading to a low yield of the target product. Conversely, if the fermentation cycle is too long, the Monascus purpureus mycelium will undergo excessive aging. Under the combined action of nutrient depletion and its own metabolic enzymes, the synthesized monascus yellow pigment is easily degraded, and may even cause the Monascus purpureus strain to synthesize byproducts, resulting in both a waste of raw material resources and a reduction in the yield of the target product. In this study, the fermentation time in the solid-state fermentation process was set to 15 days, 17 days, 20 days, 23 days, and 25 days to monitor the changes in biomass and monascus yellow pigment yield throughout the entire fermentation cycle, thereby determining the optimal fermentation endpoint. Figure 1 As shown in f, the quantitative results of red yeast rice yellow pigment indicate that the product yield reaches its peak when the fermentation time is 25 days. Insufficient fermentation time will lead to insufficient growth of red yeast mycelium and product accumulation, while excessive fermentation time will easily cause product degradation or by-product transformation.

[0050] 7. Plackett-Burman (PB) Experiment: Screening of Key Influencing Factors: Experimental objective: To rapidly and efficiently screen key factors that significantly affect the yield of red yeast rice yellow pigment from multiple potential influencing factors (including but not limited to: carbon source matrix, loading amount of fermentation solid medium, fermentation time, fermentation temperature, gelatinization amount, and inoculum amount), so as to reduce the number of variables in subsequent optimization experiments and improve optimization efficiency.

[0051] Experimental Design: Based on the results of single-factor experiments, several factors were selected, with each factor having two levels: high (+1) and low (-1). The Plackett-Burman experimental design was used to conduct the experiment. This design can evaluate the main effects of each factor with fewer trials.

[0052] Data analysis: Multiple linear regression analysis was performed on the results of the PB experiment, and the p-values ​​and effect sizes of each factor were calculated by analysis of variance (ANOVA) and t-test. Based on the statistical analysis results, the factors with P-values ​​less than the significance level (usually set at 0.05 or 0.1) and ranking among the top three in absolute effect value were identified as the key variables with the most significant impact on xanthophyll production. These were then used as the core objects of subsequent response surface methodology optimization. The results are shown in Table 1. According to the PB experiment results, A (inoculum size): the mean value at the 15% level was 2.198 mg / g, and the mean value at the 25% level was 4.412 mg / g, with a level difference of 2.214 mg / g, significantly affecting the total xanthophyll content; B (fermentation time): the mean value at the 20-day level was 3.600 mg / g, and the mean value at the 25-day level was 3.009 mg / g, with a level difference of only 0.591 mg / g, having a small impact on the results; C (gelatinization amount): the mean value at the 40% level was 2.854 mg / g, and the mean value at the 60% level was 3.755 mg / g, with a level difference of 0.901 mg / g. The concentrations of A, C, and E (inoculum, gelatinization, and loading) significantly affected the total yellow pigment content. Temperature (A) had a mean of 2.922 mg / g at 27℃ and 3.687 mg / g at 32℃, with a difference of 0.765 mg / g, indicating a moderate impact. Loading weight (E) had a mean of 4.045 mg / g at 20 g and 2.731 mg / g at 40 g, with a difference of 1.314 mg / g, significantly affecting the total yellow pigment content. Therefore, the most suitable combination of factors for continuing the BBD experiment is: A (inoculum), C (gelatinization), and E (loading weight). The significance of the combination of factors A (inoculum), C (gelatinization), and E (loading weight) was then analyzed, and the results are shown in Table 2. All three factors (A (inoculum), C (gelatinization), and E (loading weight) significantly affected the total yellow pigment content.

[0053] Table 1. Results of the Plackett-Burman experiment on yellow pigment.

[0054] Table 2. Analysis of variance and significance test of the Plackett-Burman regression model for yellow pigment.

[0055] Note: * indicates significant.

[0056] 8. Box-Behnken Design (BBD) Response Surface Analysis: Experimental objective: Based on the three key factors screened by the PB experiment, to establish a quadratic polynomial regression model between the key factors and the yellow pigment yield, to accurately characterize the interaction between the factors, and to determine the optimal combination of process parameters that maximizes the yellow pigment yield.

[0057] Experimental Design: Using the three key factors selected from the PB experiment as independent variables, each with three levels (low (-1), medium (0), and high (+1), a three-factor, three-level Box-Behnken experimental design was adopted. This design can efficiently fit nonlinear relationships, and all experimental points are within the safe operating range.

[0058] Model Establishment and Validation: Using BBD experimental data, a quadratic polynomial regression was performed to obtain a predictive model equation with yellow pigment yield as the response value. Analysis of variance was conducted to evaluate the model's significance, lack-of-fit term, and the significance of each coefficient to ensure its effectiveness and predictive accuracy. Finally, response surface plots and contour plots were used to analyze the interaction effects between factors, and the model's numerical optimization function was used to solve for the theoretically optimal process parameter points. To verify the model's reliability, validation experiments were conducted under these optimal conditions to compare the degree of agreement between the measured values ​​and the model's predicted values.

[0059] Experimental results are as follows Figures 2-4 As shown in Table 3, this BBD experiment successfully established a quadratic regression model between total yellow pigment yield and inoculum amount, gelatinization amount, and loading amount. The model is highly significant and has an excellent fit, making it a reliable optimization model. The 3D graph presents a clear and smooth mountain peak shape, indicating that the model has a theoretical maximum point; the steep slope proves that inoculum amount and gelatinization amount are key factors affecting yield, with a significant increase of over 13 mg / g in total yellow pigment yield from the foot to the summit. The theoretically optimal process conditions were successfully found: inoculum amount 24.09%, gelatinization amount 51.18%, and loading amount 20.11g. Under these conditions, the predicted total yellow pigment yield can reach 31.69 mg / g. Furthermore, it was clarified that the key to yield influencing factors lies in the quadratic effect of each factor, meaning that each factor has an optimal level; excessively high or low levels will lead to a decrease in yield. Table 4 shows that the regression model for red yeast rice yellow pigment has P < 0.0001, indicating extreme significance; the lack-of-fit term P = 0.1569 > 0.05, indicating a reliable model. R 2 =0.9767, R adj 2 =0.9467, indicating a good fit of the regression equation. The coefficient of variation (CV) of 4.2% < 10% indicates good precision and high reliability of the experiment.

[0060] Table 3. Experimental Results of Yellow Pigment Box-Behnken Design

[0061] Table 4. Analysis of variance and significance test of the Box-Behnken Design regression model for yellow pigment.

[0062] Note: * indicates significant.

[0063] Experiment Example 1. Optimized Experimental Verification Based on the prediction of the optimal yellow pigment fermentation conditions using the BBD model and combined with actual production, the optimized conditions were: inoculum amount 25%, gelatinization amount 50%, and loading amount 20g. Four parallel verification experiments were conducted, and the results of the verification experiments are shown in Table 5.

[0064] The measured average actual value of the total yellow pigment content of red yeast rice was 33.51 mg / g. The deviation between the measured average value and the model prediction value was within an acceptable range (RE = 5.72% < 10%), demonstrating that the red yeast rice yellow pigment fermentation regression model established using response surface methodology has excellent predictive performance. The establishment of this model not only provides accurate process parameters for the efficient production of red yeast rice yellow pigment but also lays a theoretical foundation for subsequent industrial-scale production.

[0065] Table 5. Experimental Results for Predicting Optimal Conditions

[0066] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A solid-state fermentation method for high-yield red yeast rice pigment using *Monascus purpureus*, characterized in that, The solid-state fermentation method includes the following steps: Strain activation steps: The activated Monascus strain is propagated in a seed culture medium to obtain propagation seed solution; Solid-state fermentation steps: Gelatinize the carbon source substrate to obtain a solid fermentation medium; add the propagation seed liquid to the solid fermentation medium and incubate statically to obtain the fermentation product; Preparation steps of red yeast rice powder: The fermentation product is dried and pulverized to obtain red yeast rice powder containing red yeast yellow pigment; Preferably, the red yeast rice yellow pigment includes Monascin and Ankaflavin.

2. The solid-state fermentation method according to claim 1, characterized in that, In the solid-state fermentation step, the carbon source substrate includes indica rice, glutinous rice, and / or japonica rice; Preferably, the carbon source matrix includes indica rice.

3. The solid-state fermentation method according to any one of claims 1-2, characterized in that, In the solid-state fermentation step, the static culture is carried out in a fermentation bottle; the amount of the solid fermentation medium in the fermentation bottle is 40-80 g / L. Preferably, the amount of the solid fermentation medium in the fermentation flask is 80 g / L.

4. The solid-state fermentation method according to any one of claims 1-3, characterized in that, In the solid-state fermentation step, the preparation process of the fermentation solid culture medium includes: soaking the carbon source matrix in water for 12 hours, draining the water to obtain the soaked carbon source matrix; adding water to the soaked carbon source matrix and cooking it to obtain the fermentation solid culture medium. Preferably, the cooking conditions are 121 °C, 0.1 MPa, and 15 min.

5. The solid-state fermentation method according to any one of claims 1-4, characterized in that, In the preparation of the fermentation solid culture medium, water accounting for 20-60% of its total mass is added to the soaked carbon source matrix, and the mixture is boiled to obtain the fermentation solid culture medium. Preferably, in the preparation of the fermentation solid culture medium, water accounting for 20% of its total mass is added to the soaked carbon source matrix, and the mixture is boiled to obtain the fermentation solid culture medium.

6. The solid-state fermentation method according to any one of claims 1-5, characterized in that, In the solid-state fermentation step, when the propagation seed solution is added to the solid fermentation culture medium, the volume of the propagation seed solution is 5%-25% of the mass of the solid fermentation culture medium; Preferably, the volume of the propagation seed solution is 10% of the mass of the fermentation solid culture medium.

7. The solid-state fermentation method according to any one of claims 1-6, characterized in that, In the solid-state fermentation step, the static culture time is 15-25 days; Preferably, the static incubation time is 25 days.

8. The solid-state fermentation method according to any one of claims 1-7, characterized in that, In the solid-state fermentation step, the temperature for static culture is 23-35℃; Preferably, the temperature for static incubation is 30°C.

9. The solid-state fermentation method according to any one of claims 1-8, characterized in that, In the strain activation step, the seed culture medium comprises 60 g / L glucose, 25 g / L peptone, 2 g / L KH2PO4, 2 g / L NaNO3, and 1 g / L MgSO4·7H2O.

10. The application of the solid-state fermentation method according to any one of claims 1-9 in the preparation of red yeast rice yellow pigment.

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