A method for producing carotenoids by in situ fermentation of rice by chrysosporium

Abstract

The application discloses a method for producing carotenoids by in-situ fermentation of rice by Chrysosporium merdarium, and belongs to the technical field of biological fermentation engineering and functional food processing technology. In the method, 10-50 ppm of beta-ionone is added to specifically activate the mevalonate (MVA) pathway on the 2th-4th day of liquid seed culture of the Chrysosporium merdarium; the seed liquid is inoculated into rice substrate with a water content of 50-60%, and is first cultured at 20-25 DEG C for 2-4 days to promote mycelium colonization, and then is cultured at 28-35 DEG C for 2-4 days to induce pigment synthesis; finally, the finished product is obtained through low-temperature drying. Through precise timing control and environmental stress, the application forces the metabolic flow of the Chrysosporium merdarium to deflect from vegetative growth to secondary metabolism. The application has the advantages of simple process, no need of complex extraction equipment, and the obtained product can be directly used as a functional staple food or further processed, and has high industrialization popularization value and market application prospect.

Description

Technical Field

[0001] This invention relates to a method for producing carotenoids from rice through in-situ fermentation of rice by Auricularia aurea, belonging to the fields of bio-fermentation engineering and functional food processing technology. Background Technology

[0002] Rice is the staple food for more than half of the world's population, but its nutritional structure is significantly limited, consisting mainly of carbohydrates and lacking in protein and micronutrients. To address this deficiency, biofortification of grains using microbial fermentation technology has become a research hotspot in the food industry, such as Monascus purpureus fermentation of rice to produce Monacoline K, or Ganoderma lucidum fermentation of grains to accumulate polysaccharides. However, existing grain fermentation technologies still struggle to simultaneously achieve both "staple food form" and "in-situ enrichment of highly active ingredients," especially for Auricularia auricula-judae (Ganoderma lucidum). Tremella aurantialba The compound fermentation system consisting of rice and other ingredients faces the following pressing technical challenges: (1) Solid-state fermentation of Auricularia auricula-judae is difficult to regulate metabolism, and the synthesis efficiency of carotenoids (mainly β-carotene) is extremely low. Auricularia auricula-judae is rich in carotenoids and has excellent antioxidant and hypoglycemic potential. However, β-carotene is a tetraterpenoid compound, and its biosynthesis is extremely sensitive to the physiological state of the fungus and environmental factors. In the existing technology, Auricularia auricula-judae is mostly extracted by liquid fermentation, which is complicated and costly. Moreover, the heat-sensitive active ingredients are easily degraded during the extraction and drying process. If we try to ferment directly on solid grains (such as rice), due to limited mass transfer, uneven dissolved oxygen, and difficulty in metabolic regulation, it is difficult to activate the mevalonate (MVA) pathway in Auricularia auricula-judae, resulting in extremely low in-situ synthesis efficiency of β-carotene. The product content is usually insufficient to meet the needs of functional foods.

[0003] (2) The compatibility between *Auricularia auricula-judae* and rice substrates is poor, making it difficult to simultaneously achieve mycelial colonization and product accumulation. *Auricularia auricula-judae* mycelia are slender, and their growth competitiveness is far weaker than that of wood-rotting fungi such as *Ganoderma lucidum*, and they have high requirements for the growth environment. As a high-starch substrate, rice has a dense crystalline structure that restricts the deep penetration of mycelia. At the same time, during solid-state fermentation, if the substrate moisture content is too high, the rice is prone to gelatinization and adhesion after sterilization, resulting in a viscous system, insufficient oxygen supply, and inhibition of mycelial respiration. If the moisture content is too low, mycelial growth is hindered and metabolism is slow. In addition, existing processes mostly use constant temperature fermentation, which cannot simultaneously meet the differentiated needs of the two stages of "rapid mycelial colonization" and "efficient synthesis of secondary metabolites", resulting in long fermentation cycles, poor batch stability, and the final product is prone to contamination or morphological disintegration.

[0004] (3) The product form is disconnected from the attributes of staple food, and there is a lack of whole fermented staple food that can be directly consumed. At present, most of the fungal fermentation products on the market are fermentation liquid, extracts or compound compressed candies, which consumers cannot directly use as a substitute for daily staple food. How to achieve deep enrichment and stable retention of active ingredients (β-carotene, polysaccharides, flavonoids, etc.) of Auricularia auricula-judae while maintaining the integrity of rice grains and not damaging the cooking characteristics of staple food is still a technological gap in this field. There are no reports of mature processes in the existing technology that can solve the above problems at the same time and achieve the directional enrichment of active ingredients of Auricularia auricula-judae in rice matrix with intact grain morphology.

[0005] In summary, there is an urgent need to develop a simple, low-cost solid-state fermentation method that can achieve the targeted enrichment of active ingredients in rice matrix from *Auricularia auricula-judae* (a type of fungus) to solve the problems of low content of active ingredients, poor stability, and inability to possess the attributes of staple food in existing fermented grain foods. Summary of the Invention

[0006] Technical issues This invention aims to solve the technical problems in existing grain fermentation technology, such as the difficulty of deep colonization of Auricularia auricula-judae mycelium in rice substrate, low efficiency of carotenoid synthesis and easy degradation, and the inability of the final product to combine the form of staple food with high-activity nutrition.

[0007] Technical solution This invention provides a method for in-situ fermentation of rice by *Auricularia auricula-judae* to produce carotenoids. By adding β-ionone at a specific time during the liquid seed culture stage, combined with two-stage variable-temperature solid-state fermentation, the method solves the problems of difficult colonization of *Auricularia auricula-judae* in rice substrate and low efficiency of carotenoid synthesis. The rice fermented by *Auricularia auricula-judae* is orange-yellow, with intact grains and no sticking together. The method includes the following steps: (1) Directed domestication of seed liquid: Take activated Auricularia auricula mycelium, inoculate it into primary corn bran liquid culture medium, add β-ionone on the 2nd-4th day, the amount added is 10-50 ppm, and obtain Auricularia auricula seed liquid; (2) Construction of solid fermentation substrate: 20 g to 80 g of rice is placed in a fermentation container, water is added to adjust the moisture content to 10 to 60%, sterilization is performed, and fermentation substrate is obtained; the fermentation container can be an eggplant bottle, a stainless steel lunch box, a triangular bottle, or a polypropylene bacterial bag, tray or fermentation tank suitable for industrial production. (3) Two-stage variable temperature fermentation: Inoculate with Auricularia auricula seed liquid, the inoculation amount is 20~50% (mL / 100g) of the volume mass ratio of seed liquid to the fermentation substrate, first culture at 20~25℃ for 2~4 days to promote the rapid colonization of mycelium inside the rice; then raise the temperature to 28~35℃ and culture for 2~4 days. (4) Low-damage post-treatment: Dry at 45℃ to retain heat-sensitive active substances and obtain rice fermented by Auricularia aurea. The rice fermented by Auricularia aurea is orange-yellow, with intact grains and no sticking.

[0008] In some embodiments of the present invention, the *Auricularia auricula-judae* (… Tremella aurantialba Purchased from the China Center for Type Microorganisms (CCTCC HF 20081008). *Auricularia auricula-judae*, rich in polyphenols, flavonoids, saponins, polysaccharides, carotenoids, proteins, and other active ingredients, possesses high medicinal and pharmacological value and effects, including immunomodulation, anti-inflammation, anti-hyperglycemia, and lipid-lowering properties.

[0009] In some embodiments of the present invention, the primary corn bran liquid culture medium in step (1) is: according to g·L -1 The formula is as follows: glucose 20-40g, magnesium sulfate heptahydrate 2-3g, potassium dihydrogen phosphate 3-4.5g, corn flour 1-2g, wheat bran 1-2g; for example, by g·L -1 The ingredients are: glucose 20g, magnesium sulfate heptahydrate 2g, potassium dihydrogen phosphate 3g, corn flour 1g, and wheat bran 1g.

[0010] In some embodiments of the present invention, the culture temperature in step (1) is 25~30℃, the time is 5~7 days, the rotation speed is 150~180 rpm, and β-ionone is added on the 2nd-4th day at an amount of 10-50 ppm to obtain the golden ear seed liquid.

[0011] In some embodiments of the present invention, the rice used in step (2) is a finished product made from paddy rice after cleaning, hulling, milling and other processes. The preferred amount is 20-80 g, and the rice is soaked in advance to control the moisture content to be 10-60%, preferably 50%-60%. If the moisture content of the substrate exceeds 60%, the rice and water will become too viscous after sterilization, resulting in insufficient oxygen supply, which is not conducive to microbial fermentation. If the moisture content of the substrate is less than 10%, fermentation can still proceed smoothly, but the mycelial production efficiency will be low.

[0012] Preferably, the volume percentage of the *Auricularia auricula-judae* broth used for inoculation into the fermentation substrate is 20-60%.

[0013] In some embodiments of the present invention, the method for preparing the *Auricularia auricula-judae* seed solution in step (1) includes the following steps: ① Strain activation: Inoculate the strain into glucose potato agar medium for activation at a temperature of 28-30℃ for 5-8 days; ② Liquid culture: Take 1 cm of the activated mycelium 2 After chopping the corn bran with an inoculation needle, it was inoculated into 80 mL of primary corn bran liquid culture medium for culture. Primary corn bran liquid culture medium (g·L⁻¹) -1): Glucose 20g, magnesium sulfate heptahydrate 2g, potassium dihydrogen phosphate 3g, corn flour 1g, wheat bran 1g. Culture temperature 25-30℃, time 5-7 days, rotation speed 150-180 rpm. β-ionone is added on days 2-4 at a concentration of 10-50 ppm to obtain the *Auricularia auricula-judae* seed culture for inoculation.

[0014] In some embodiments of the present invention, stirring is performed during the fermentation process in step (3), preferably with a stirring time interval of 1 to 3 days.

[0015] In some embodiments of the present invention, the low-temperature drying time in step (4) is 24 to 48 hours.

[0016] [Beneficial Effects] The rice fermented with Auricularia auricula-judae mycelium and its preparation method provided by this invention break through the limitations of traditional grain fermentation, which only focuses on flavor improvement or single-component fortification. It has made significant progress in three aspects: metabolic regulation mechanism, solid-state fermentation process coupling, and functional design of staple food, as detailed below: (1) A novel metabolic regulation strategy coupled with temporal precursor induction and temperature stress was developed to overcome the challenge of in-situ synthesis of β-carotene. In existing technologies, *Auricularia auricula-judae* mainly extracts polysaccharides through liquid fermentation, while the synthesis efficiency and stability of β-carotene in solid substrates are low. This invention, through in-depth research on the metabolic mechanism of *Auricularia auricula-judae*, found that the synthesis of β-carotene in *Auricularia auricula-judae* is subject to strict spatiotemporal specific regulation. Precise timing of precursor addition: 10-50 ppm of β-ionone is added on the 2nd to 4th day of seed liquid preparation. On the one hand, it can specifically activate the mevalonate pathway (MVA pathway) in *Auricularia auricula-judae* cells, providing precursors for pigment synthesis in subsequent solid-state fermentation and avoiding the inhibition of mycelial growth by high concentrations of precursors; on the other hand, β-ionone can be evenly distributed to ensure that each *Auricularia auricula-judae* mycelium receives the induction signal. When transferred to rice, the state of the entire batch of strains is consistent, ensuring the uniformity of subsequent fermentation products. Compared with the absence of β-ionone, the present invention increases the carotenoid content in the final product from 2.13±0.05 mg / g to over 3.03±0.08 mg / g, an increase of more than 42%.

[0017] (2) Traditional solid-state fermentation often falls into the dilemma of "high humidity leads to rot, low humidity leads to stagnation". This invention controls the moisture content of rice to 50-60% (w / w). This water activity range can ensure that the rice grains maintain their integrity while providing sufficient porosity for mycelial propagation, thus solving the problems of metabolic stagnation under low water activity and root rot due to lack of oxygen under high water activity. SEM showed that when the moisture content of rice is 50-60%, the mycelium of Auricularia auricula-judae can penetrate deep into the rice to form a dense network structure, which significantly improves the bioconversion efficiency of starch, protein and other matrices.

[0018] When the moisture content drops to 5%, the carotenoid content plummets to 0.22±0.01 mg / g; when the fermentation process is not stirred, the product shows obvious caking, and the carotenoid content is only 1.94±0.04 mg / g.

[0019] (3) The temperature transition during fermentation simulated natural environmental stress. The low temperature range of 20-25 ℃ ensured rapid colonization of mycelium in the rice substrate, while the high temperature range of 28-35 ℃ artificially created environmental stress, forcing the golden ear fungus to switch from "vegetative growth" to "secondary metabolism (pigment synthesis)," thereby significantly increasing the synthesis rate of β-carotene and polysaccharides. Compared with constant temperature fermentation throughout the process, the variable temperature strategy significantly improved the synergistic accumulation of pigments and polysaccharides.

[0020] (4) Unlike fortifying a single nutrient, this invention simultaneously achieves high-density enrichment of multiple components in the same process: carotenoids ≥ 3.03±0.08 mg / g, polysaccharides ≥ 264.7±13.24 mg / g, total amino acids ≥ 278.5±8.97 mg / g, starch content ≥ 457.7±16.93 mg / g, protein ≥ 122.14±0.76 mg / g, saponin content ≥ 15.78±0.26 mg / g, flavonoid content ≥ 2.55±0.11 mg / g, and total phenol content ≥ 3.98±0.17 mg / g. Meanwhile, the rice fermented with golden ear fungus exhibits a golden-yellow color, uniform luster, distinct grains, stable texture, and excellent sensory characteristics.

[0021] The above-mentioned active ingredients are all synthesized in situ by Auricularia auricula-judae mycelium and stably distributed in rice matrix without the need for exogenous addition or compounding. This fundamentally avoids the defects of uneven distribution and poor stability of active ingredients in traditional compound foods, and has the advantage of in situ synthesis.

[0022] (5) This invention not only achieves the accumulation of highly active ingredients during the fermentation cycle, but also the carotenoid retention rate of the fermented rice obtained from the golden ear fungus is still greater than 90% and the polysaccharide retention rate is greater than 95% after being stored for 6 months under normal temperature, dry and light-proof conditions. This proves that the process has good batch stability and shelf stability, and solves the problem of unstable and easily degraded active ingredients in fermented products in the prior art.

[0023] (6) This invention adopts a one-step solid-state fermentation method, which does not require complex extraction, purification and post-modification processes. The raw materials used are conventional rice and ordinary food-grade precursors. There are no organic solvent residues in the production process, which meets the requirements of food safety and green manufacturing. The resulting product can be used directly as a functional staple food, or it can be further processed into various forms such as rice flour, rice cake, and fermented beverages, which greatly expands the space for industrial chain extension and has the potential for industrial scale-up.

[0024] Furthermore, the fermented rice made from golden ear fungus prepared by this invention can be further processed into diversified food products such as golden ear fungus porridge, golden ear fungus rice noodles, golden ear fungus rice wine, and golden ear fungus rice cake desserts. Attached Figure Description

[0025] Figure 1 These are morphological diagrams of rice fermented with golden ear fungus during the fermentation process; A: Example 2; B: Example 3; C: Comparative Example 1; D: Comparative Example 2; E: Example 4; F: Example 5; G: Comparative Example 3; H: Comparative Example 4; I: Comparative Example 5; J: Comparative Example 6.

[0026] Figure 2 This is a morphological diagram of the fermented rice from golden ear fungus in Example 5.

[0027] Figure 3 This is a morphological diagram of the rice fermented with golden ear fungus in Example 5 after low-temperature drying.

[0028] Figure 4 This is a scanning electron microscope (SEM) image of a cross section of the fermentation product dried at low temperature in Example 5. Detailed Implementation

[0029] Exemplary embodiments of this disclosure will be described below with reference to the accompanying drawings.

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0031] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0032] The following examples of Auricularia auricula-judae ( Tremella aurantialba Purchased from the China Center for Type Microorganisms (CCTCC HF 20081008).

[0033] The detection methods involved in the following embodiments are as follows: Method for calculating the moisture content of fermentation substrate: The moisture content of the fermentation substrate was determined using the atmospheric pressure drying method. A weighing bottle was placed in a drying oven at 105±2℃ and dried to constant weight (m0). Approximately 2 g of sample was weighed and placed in the weighing bottle, and the total weight was accurately measured (m1). The weighing bottle was then placed in a forced-air drying oven at 105±2℃ and dried to constant weight. After removal, it was placed in a desiccator and cooled for 30 min, and then quickly weighed (m2). The formula for calculating the moisture content is: Moisture content (%) = (m1 / m2) m 0) / (m 1) m 2) × 100% Carotenoid detection method: Accurately weigh 200 mg of ground fermented rice flour into a brown tube, add 4 mL of methanol, and sonicate for 2 h. Remove and shake well every 20 min. Take 3 mL of the sonicated solution, centrifuge at 10000 rpm for 10 min, collect the supernatant, and measure its OD450 under a visible spectrophotometer. Construct a standard curve using β-carotene as a standard. The calculated result is expressed as "total carotenoids (as β-carotene equivalent)" in mg / g. The total carotenoids measured by this method include the sum of fat-soluble pigments such as β-carotene, zeaxanthin, and its derivatives produced by *Auricularia auricula-judae* metabolism.

[0034] Protein detection methods should refer to GB 5009.5. 2010; The method for detecting crude polysaccharides is based on GB / T 5009.8. 2008; the method for flavonoid detection refers to GB / T 5009.124 2003; the method for total phenols detection refers to GB / T 8313 2008; The amino acid detection method refers to GB / T 5009.124-2003.

[0035] Primary corn bran liquid culture medium: according to g·L -1 The ingredients are: glucose 20g, magnesium sulfate heptahydrate 2g, potassium dihydrogen phosphate 3g, corn flour 1g, and wheat bran 1g.

[0036] Example 1: Regulation of the amount and timing of addition of β-ionone, a precursor, in Auricularia auricula-judae culture (1) Preparation of Auricularia auricula-judae culture: The preserved Auricularia auricula-judae culture was inoculated on glucose potato agar medium and cultured at 30℃ for 7 days to activate it. Then, the obtained mycelium was cut into 1 cm sections. 2 The chopped material was transferred to seed culture medium, i.e., primary corn bran liquid culture medium, and cultured at 30°C and 150 rpm in a shaker.

[0037] β-ionone addition time: Different groups were set up, and β-ionone was added at 0 h, 24 h, 48 h and 96 h respectively. Then, the culture was continued at 30℃ and 150 rpm in a shaker. The culture was ended at 5~7 days to obtain the primary seed culture of Auricularia auricula-judae.

[0038] β-ionone addition amount: Different groups were set up, and the β-ionone addition amount was set at 0, 10, 25, 50 and 100 ppm for different time periods. Then, the culture was continued at 30℃ and 150 rpm in a shaker. The culture was ended on the 5th to 7th day to obtain the first-level seed culture of Auricularia auricula-judae.

[0039] (2) The obtained first-grade seed liquid of Auricularia auricula-judae was homogenized and crushed by a homogenizer. It was centrifuged at 10,000 rpm for 5 min. The supernatant was tested at 450 nm for carotenoid content. The results are shown in Table 1. When the amount of β-ionone added was 10~50 ppm, the carotenoid content in the bacterial solution was increased. The addition at 48 h was relatively optimal. When the amount of β-ionone added reached 100 ppm, the carotenoid content in the bacterial solution decreased in the early stage of addition.

[0040] Table 1: Carotenoid content in Auricularia auricula-judae seed liquid under different conditions (unit: g / L)

[0041] Example 2: Preparation of rice fermented with 50% (v / w) inoculum from *Auricularia auricula-judae* (1) Preparation of Auricularia auricula seed culture: The preserved Auricularia auricula spawn was inoculated into glucose potato agar medium and cultured at 30℃ for 7 days to activate it. Then, the obtained mycelium was cut into 1 cm sections. 2 The chopped corn bran was transferred to primary corn bran liquid culture medium and cultured at 30°C and 150 rpm for 5-7 days. β-ionone at a concentration of 25 ppm was added at 48 h.

[0042] (2) Preparation of fermentation substrate: Take 30 g of rice from each fermentation container, immerse it in water for 12 h, drain the excess water, test the moisture content to be 30~35%, sterilize at 121℃ for 20 min to obtain fermentation substrate.

[0043] (3) Fermented rice with golden ear fungus: 15 mL of the golden ear fungus liquid obtained in step (1) was inoculated into the fermentation substrate in step (2) to obtain a fermentation system with an inoculation ratio of 50%; the fermentation system was fermented in a 25℃ incubator for 3 days and then transferred to a 30℃ incubator for 4 days, and stirred daily to obtain the fermentation product.

[0044] (4) The fermentation product was dried at 45°C for 48 h. The dried fermentation product is the golden ear fermented rice.

[0045] The fermented rice prepared in this embodiment is an orange-yellow rice with distinct grains, possessing a unique aroma. The grains are intact, non-sticky, and can be directly steamed or cooked. Component analysis of this fermented rice was performed. The results are as follows: the fermented rice contains carotenoids of ≥2.82±0.08 mg / g, protein of ≥98.4±2.85 mg / g, flavonoids of ≥1.36±0.06 mg / g, saponins of ≥10.77±0.15 mg / g, total phenols of ≥2.57±0.11 mg / g, polysaccharides of ≥204.7±1.61 mg / g, total amino acids of 258.5±8.22 mg / g, and starch of 624.8±10.73 mg / g.

[0046] The results showed that by using a high inoculum of 50% combined with two-stage variable temperature fermentation, orange-yellow fermented rice was successfully produced with a carotenoid content of 2.82±0.08 mg / g, achieving efficient enrichment of active ingredients.

[0047] Example 3: Preparation of rice fermented with 30% (v / w) inoculum of Auricularia auricula-judae (1) Preparation of Auricularia auricula seed culture: The preserved Auricularia auricula spawn was inoculated into glucose potato agar medium and cultured at 30℃ for 7 days to activate it. Then, the obtained mycelium was cut into 1 cm sections. 2 The chopped ingredients were transferred to primary corn bran seed culture medium, and β-ionone at a concentration of 25 ppm was added at 48 h. The mixture was then cultured at 30 °C and 150 rpm in a shaker for 5-7 days to prepare primary seed culture of Auricularia aurea.

[0048] (2) Preparation of fermentation substrate: Take 30 g of rice from each fermentation container, immerse it in water for 12 h, drain the excess water, test the moisture content to be 30~35%, sterilize at 121℃ for 20 min to obtain fermentation substrate.

[0049] (3) Fermented rice with golden ear fungus: 10 mL of the golden ear fungus liquid obtained in step (1) was inoculated into the fermentation substrate in step (2) to obtain a fermentation system with an inoculation ratio of 30%; the fermentation system was fermented in a 25℃ incubator for 3 days and then transferred to a 30℃ incubator for 4 days, and stirred daily to obtain the fermentation product.

[0050] (4) The fermentation product was dried at 45°C for 48 h. The dried fermentation product is the golden ear fermented rice.

[0051] The fermented rice from *Auricularia auricula-judae* prepared in this embodiment has a distinct orange-yellow grain appearance, a unique aroma, and the grains are intact, non-sticky, and can be directly steamed or cooked. Component analysis of this fermented rice yielded the following results: The rice contained carotenoids at a concentration of ≥2.73±0.04 mg / g, protein at ≥92.4±2.46 mg / g, flavonoids at ≥1.29±0.02 mg / g, saponins at ≥9.85±0.44 mg / g, total phenols at ≥2.73±0.11 mg / g, and polysaccharides at 193.5±6.49 mg / g. The total amino acid content was 252.5±1.57 mg / g, and the starch content was 668.3±9.15 mg / g.

[0052] The results showed that, with an inoculum size of 30%, fermented rice with a carotenoid content of 2.73 ± 0.04 mg / g was obtained while ensuring good mycelial colonization and metabolism. Both 30% and 50% inoculum sizes consistently achieved the accumulation of highly active ingredients, and the resulting fermented rice grains were intact and non-sticky, making them suitable for direct consumption as a staple food. This verified the stability and applicability of the process to staple foods in industrial production.

[0053] Preparation of Fermented Rice with Auricularia auricula-judae at a Comparative Example 1: 5% (v / w) Inoculum (Low Inoculum) (1) Preparation of Auricularia auricula seed culture: The preserved Auricularia auricula spawn was inoculated into glucose potato agar medium and cultured at 30℃ for 7 days to activate it. Then, the obtained mycelium was cut into 1 cm sections. 2 The chopped ingredients were transferred to primary corn bran seed culture medium, and β-ionone at a concentration of 25 ppm was added at 48 h. The mixture was then cultured at 30 °C and 150 rpm in a shaker for 5-7 days to prepare primary seed culture of Auricularia aurea.

[0054] (2) Preparation of fermentation substrate: Take 30 g of rice from each fermentation container, immerse it in water for 12 h, drain the excess water, test the moisture content to be 30~35%, sterilize at 121℃ for 20 min to obtain fermentation substrate.

[0055] (3) Obtaining fermented rice from golden ear fungus: 1.5 mL of the golden ear fungus liquid obtained in step (1) was inoculated into the fermentation substrate in step (2) to obtain a fermentation system with an inoculation ratio of 5%; the fermentation system was fermented in a 25℃ incubator for 3 days and then transferred to a 30℃ incubator for 4 days, with daily stirring to obtain the fermentation product.

[0056] (4) The fermentation product was dried at 45°C for 48 h. The dried fermentation product is the golden ear fermented rice.

[0057] The fermented rice prepared in this comparative example was light yellow rice with distinct grains. The *Auricularia auricula-judae* fungus showed poor growth, and the aroma was not pronounced, primarily consisting of rice fragrance. Component analysis of this fermented rice yielded the following results: Carotenoids content exceeded 0.82±0.03 mg / g; protein content exceeded 79.8±2.86 mg / g; flavonoid content exceeded 0.96±0.03 mg / g; saponin content exceeded 4.17±0.17 mg / g; total phenol content exceeded 1.84±0.06 mg / g; polysaccharide content exceeded 153.6±4.22 mg / g; total amino acid content exceeded 233.5±8.65 mg / g; and starch content exceeded 698.5±18.17 mg / g.

[0058] The results showed that when the inoculation amount was reduced to 5%, the mycelium could not cover the surface of the rice in a short time due to the low inoculation density, and the final carotenoid content was only 0.82 mg / g.

[0059] Comparative Example 2: Preparation of Fermented Rice from Auricularia auricula-judae with an inoculum size of 50% (v / w) and without the addition of β-ionone precursor. (1) Preparation of Auricularia auricula seed culture: The preserved Auricularia auricula spawn was inoculated into glucose potato agar medium and cultured at 30℃ for 7 days to activate it. Then, the obtained mycelium was cut into 1 cm sections. 2 The chopped ingredients were transferred to a primary corn bran seed culture medium and cultured at 30°C and 150 rpm for 5-7 days to prepare a primary seed culture of Auricularia auricula-judae.

[0060] (2) Preparation of fermentation substrate: Take 30 g of rice from each fermentation container, immerse it in water for 12 h, drain the excess water, test the moisture content to be 30~35%, sterilize at 121℃ for 20 min to obtain fermentation substrate.

[0061] (4) Fermented rice with golden ear fungus: 15 mL of the golden ear fungus liquid obtained in step (1) was inoculated into the fermentation substrate in step (2) to obtain a fermentation system with an inoculation ratio of 5%; the fermentation system was fermented in a 25℃ incubator for 3 days and then transferred to a 30℃ incubator for 4 days, and stirred daily to obtain the fermentation product.

[0062] (4) The fermentation product was dried at 45°C for 48 h. The dried fermentation product is the golden ear fermented rice.

[0063] The fermented rice prepared in this comparative example is characterized by distinct yellow grains and a unique aroma. Component analysis of this fermented rice yielded the following results: Carotenoid content exceeding 2.13±0.05 mg / g, protein content exceeding 115.3±1.34 mg / g, flavonoid content exceeding 1.32±0.06 mg / g, saponin content exceeding 9.17±0.19 mg / g, total phenol content exceeding 2.18±0.05 mg / g, polysaccharide content exceeding 217.7±6.31 mg / g, total amino acid content exceeding 207.4±4.83 mg / g, and starch content exceeding 488.6±18.82 mg / g.

[0064] The results showed that without the addition of β-ionone precursor, carotenoid synthesis was limited due to the lack of metabolic pathway activation signals, with a content of only 2.13±0.05 mg / g, confirming the necessity of precursor induction for activating the synthetic pathway.

[0065] Example 4: Preparation of Fermented Rice with 20% (v / w) Moisture Content from Golden Ear Fungus (1) Preparation of Auricularia auricula seed culture: The preserved Auricularia auricula spawn was inoculated into glucose potato agar medium and cultured at 30℃ for 7 days to activate it. Then, the obtained mycelium was cut into 1 cm sections. 2 The chopped ingredients were transferred to primary corn bran seed culture medium, and β-ionone at a concentration of 25 ppm was added at 48 h. The mixture was then cultured at 30 °C and 150 rpm in a shaker for 5-7 days to prepare primary seed culture of Auricularia aurea.

[0066] (2) Preparation of fermentation substrate: Take 30 g of rice in each fermentation container, add 7.5 mL of water based on the dry weight of the rice, calculate the water content as 20%, sterilize at 121℃ for 20 min to obtain the fermentation substrate.

[0067] (3) Fermented rice with golden ear fungus: 15 mL of the golden ear fungus liquid obtained in step (1) was inoculated into the fermentation substrate in step (2) to obtain a fermentation system with an inoculation ratio of 50%; the fermentation system was fermented in a 25℃ incubator for 3 days and then transferred to a 30℃ incubator for 4 days. The fermentation product was obtained by stirring and turning the fermentation system daily.

[0068] (4) The fermentation product was dried at 45°C for 48 h. The dried fermentation product is the golden ear fermented rice.

[0069] The fermented rice prepared in this embodiment is an orange-yellow rice with distinct grains, possessing a unique aroma. The grains are intact, non-sticky, and can be directly steamed or cooked. Component analysis of this fermented rice yielded the following results: Carotenoid content reached ≥1.38±0.02 mg / g; protein content reached ≥97.6±3.93 mg / g; flavonoid content reached ≥1.29±0.04 mg / g; saponin content reached ≥9.73±0.23 mg / g; total phenolic content reached ≥2.36±0.07 mg / g; polysaccharide content reached ≥183.4±2.81 mg / g; total amino acid content reached ≥232.5±2.02 mg / g; and starch content was 534.3±6.27 mg / g.

[0070] The results showed that when the moisture content was controlled at 20%, although the rice grains were intact, the insufficient water activity limited the mycelial metabolism, resulting in a carotenoid content of only 1.38±0.02 mg / g, which confirmed the inhibitory effect of low moisture content on synthesis.

[0071] Example 5: Preparation of Fermented Rice with 50% (v / w) Moisture Content from Golden Ear Fungus (1) Preparation of Auricularia auricula seed culture: The preserved Auricularia auricula spawn was inoculated into glucose potato agar medium and cultured at 30℃ for 7 days to activate it. Then, the obtained mycelium was cut into 1 cm sections. 2 The chopped ingredients were transferred to primary corn bran seed culture medium, and β-ionone at a concentration of 25 ppm was added at 48 h. The mixture was then cultured at 30 °C and 150 rpm in a shaker for 5-7 days to prepare primary seed culture of Auricularia aurea.

[0072] (2) Preparation of fermentation substrate: Take 30 g of rice in each fermentation container, add 30 mL of water based on the dry weight of the rice, calculate the water content to be 50%, sterilize at 121℃ for 20 min to obtain fermentation substrate.

[0073] (3) Fermented rice with golden ear fungus: 15 mL of the golden ear fungus liquid obtained in step (1) was inoculated into the fermentation substrate in step (2) to obtain a fermentation system with an inoculation ratio of 50%; the fermentation system was fermented in a 25℃ incubator for 3 days and then transferred to a 30℃ incubator for 4 days. The fermentation product was obtained by stirring and turning the fermentation system daily.

[0074] (4) The fermentation product was dried at 45°C for 48 h. The dried fermentation product is the golden ear fermented rice.

[0075] The fermented rice produced in this embodiment is a bright orange-yellow rice with distinct grains, a unique aroma, and intact, non-sticky grains that can be directly steamed or cooked. Figure 4As shown, the hyphae of *Auricularia auricula-judae* have completely penetrated the lattice structure of rice starch granules, forming a dense network structure. This microstructure not only locks in β-carotene but also alters the digestible properties of starch, thus endowing the product with excellent in vitro hypoglycemic activity. The fermented rice from *Auricularia auricula-judae* contains carotenoids of ≥3.03±0.08 mg / g, protein of ≥122.14±0.76 mg / g, saponins of ≥15.78±0.26 mg / g, flavonoids of ≥2.55±0.11 mg / g, total phenols of ≥3.98±0.17 mg / g, polysaccharides of ≥264.7±13.24 mg / g, total amino acids of ≥278.5±8.97 mg / g, and starch of 486.2±16.93 mg / g.

[0076] The results showed that under 50% moisture content and optimal process conditions, the mycelium of Auricularia auricula-judae deeply penetrated the rice matrix, simultaneously achieving synergistic enrichment of carotenoids ≥3.03±0.08 mg / g and polysaccharides ≥264.7±13.24 mg / g, and the rice grains were intact and golden in color.

[0077] Comparative Example 3: Preparation of Fermented Rice with 5% (v / w) Moisture Content from *Auricularia auricula-judae* (1) Preparation of Auricularia auricula seed culture: The preserved Auricularia auricula spawn was inoculated into glucose potato agar medium and cultured at 30℃ for 7 days to activate it. Then, the obtained mycelium was cut into 1 cm sections. 2 The chopped seeds were transferred to primary corn bran seed culture medium and cultured at 30℃ and 150 rpm for 5-7 days. At 48 h, 25 ppm of β-ionone was added to prepare primary seed culture of Auricularia auricula-judae.

[0078] (2) Preparation of fermentation substrate: Take 30 g of rice in each fermentation container, add 1.6 mL of water based on the dry weight of the rice, calculate the water content as 5%, sterilize at 121℃ for 20 min to obtain the fermentation substrate.

[0079] (3) Fermented rice with golden ear fungus: 15 mL of the golden ear fungus liquid obtained in step (1) was inoculated into the fermentation substrate in step (2) to obtain a fermentation system with an inoculation ratio of 50%; the fermentation system was fermented in a 25 ℃ incubator for 3 days and then transferred to a 30 ℃ incubator for 4 days, and stirred daily to obtain the fermentation product.

[0080] (4) The fermentation product was dried at 45°C for 48 h. The dried fermentation product is the golden ear fermented rice.

[0081] The fermentation substrate prepared in this comparative example had low moisture content and was yellowish after sterilization. The fermented product, *Auricularia auricula-judae* fermented rice, was a dark yellow rice with distinct, firm grains. The *Auricularia auricula-judae* fungus showed poor growth and a weak aroma. Component analysis of this fermented rice yielded the following results: Carotenoids content exceeded 0.22±0.01 mg / g; protein content exceeded 87.8±3.25 mg / g; flavonoid content exceeded 0.09±0.01 mg / g; total phenol content exceeded 0.64±0.02 mg / g; polysaccharide content exceeded 147.4±7.13 mg / g; total amino acid content exceeded 255.3±11.75 mg / g; and starch content exceeded 558.4±17.92 mg / g.

[0082] The results showed that when the moisture content was as low as 5%, the substrate water activity was severely insufficient, mycelial growth and metabolism almost stopped, and the carotenoid content plummeted to 0.22±0.01 mg / g, which defined the effective lower limit of the process.

[0083] Comparative Example 4: Preparation of Fermented Rice from Golden Ear Fungus Grown Naturally Without Stirring (1) Preparation of Auricularia auricula seed culture: The preserved Auricularia auricula spawn was inoculated into glucose potato agar medium and cultured at 30℃ for 7 days to activate it. Then, the obtained mycelium was cut into 1 cm sections. 2 The chopped seeds were transferred to primary corn bran seed culture medium and cultured at 30℃ and 150 rpm for 5-7 days. At 48 h, 25 ppm of β-ionone was added to prepare primary seed culture of Auricularia auricula-judae.

[0084] (2) Preparation of fermentation substrate: Take 30 g of rice in each fermentation container, add 30 mL of water based on the dry weight of the rice, calculate the water content to be 50%, sterilize at 121 ℃ for 20 min to obtain fermentation substrate.

[0085] (3) Fermented rice with golden ear fungus: 15 mL of the golden ear fungus liquid obtained in step 1 was inoculated into the fermentation substrate in step 2 to obtain a fermentation system with an inoculation ratio of 50%; the fermentation system was fermented in a 25 ℃ incubator for 3 days and then transferred to a 30 ℃ incubator for 4 days. No artificial stirring or turning was performed during the fermentation process. After natural growth, the fermentation product was obtained.

[0086] (4) The fermentation product was dried at 45°C for 48 h. The dried fermentation product is the golden ear fermented rice.

[0087] The fermented rice prepared in this comparative example was a sticky rice grain with severely clumped pieces and white mycelium covering the surface. Component analysis of this fermented rice yielded the following results: Carotenoids content reached ≥1.94±0.04 mg / g; protein content reached ≥102.3±3.81 mg / g; flavonoid content reached ≥0.93±0.03 mg / g; total phenol content reached ≥1.79±0.07 mg / g; polysaccharide content reached ≥158.4±2.93 mg / g; total amino acid content reached 267.4±8.42 mg / g; and starch content reached 538.1±13.44 mg / g.

[0088] The results showed that when fermentation was left undisturbed, the lack of stirring led to localized oxygen deficiency, resulting in mycelial growth only on the surface. Furthermore, the accumulation of metabolic products inhibited growth, with carotenoid content at only 1.94 ± 0.04 mg / g, and the rice becoming compacted and sticky. This demonstrates the synergistic necessity of physical disturbance and metabolic regulation.

[0089] Comparative Example 5: Preparation of Fermented Rice from Auricularia auricula-judae Fermented at 25℃ Throughout Solid-State Fermentation (1) Preparation of Auricularia auricula seed culture: The preserved Auricularia auricula spawn was inoculated into glucose potato agar medium and cultured at 30℃ for 7 days to activate it. Then, the obtained mycelium was cut into 1 cm sections. 2 The chopped seeds were transferred to primary corn bran seed culture medium and cultured at 30℃ and 150 rpm for 5-7 days. At 48 h, 25 ppm of β-ionone was added to prepare primary seed culture of Auricularia auricula-judae.

[0090] (2) Preparation of fermentation substrate: Take 30 g of rice in each fermentation container, add 30 mL of water based on the dry weight of the rice, calculate the water content to be 50%, sterilize at 121℃ for 20 min to obtain fermentation substrate.

[0091] (3) Obtaining rice fermented with golden ear fungus: 15 mL of the golden ear fungus liquid obtained in step 1 was inoculated into the fermentation substrate in step 2 to obtain a fermentation system with an inoculation ratio of 50%; the fermentation system was fermented in a 25 ℃ incubator for 7 days, and stirred daily to obtain the fermentation product.

[0092] (4) The fermentation product was dried at 45°C for 48 h. The dried fermentation product is the golden ear fermented rice.

[0093] The fermented rice prepared in this comparative example was a sticky rice grain with severely clumped pieces and white mycelium covering the surface. Component analysis of this fermented rice yielded the following results: Carotenoids content reached ≥2.24±0.07 mg / g; protein content reached ≥112.3±4.27 mg / g; flavonoid content reached ≥1.19±0.03 mg / g; total phenol content reached ≥2.79±0.11 mg / g; polysaccharide content reached 228.1±5.93 mg / g; total amino acid content reached 272.4±9.87 mg / g; and starch content reached 488.13±8.37 mg / g.

[0094] The results showed that while mycelial growth was good when the temperature was maintained at 25°C throughout the process, the lack of high-temperature stress signals resulted in a carotenoid synthesis of only 2.24 ± 0.07 mg / g. This demonstrates the key activation effect of variable temperature treatment on secondary metabolic pathways.

[0095] Comparative Example 6: Preparation of Fermented Rice from Auricularia auricula-judae Fermented at 30℃ Throughout Solid-State Fermentation (1) Preparation of Auricularia auricula seed culture: The preserved Auricularia auricula spawn was inoculated into glucose potato agar medium and cultured at 30℃ for 7 days to activate it. Then, the obtained mycelium was cut into 1 cm sections. 2 The chopped seeds were transferred to primary corn bran seed culture medium and cultured at 30℃ and 150 rpm for 5-7 days. At 48 h, 25 ppm of β-ionone was added to prepare primary seed culture of Auricularia auricula-judae.

[0096] (2) Preparation of fermentation substrate: Take 30 g of rice in each fermentation container, add 30 mL of water based on the dry weight of the rice, calculate the water content to be 50%, sterilize at 121℃ for 20 min to obtain fermentation substrate.

[0097] (3) Fermented rice with golden ear fungus: 15 mL of the golden ear fungus liquid obtained in step 1 was inoculated into the fermentation substrate in step 2 to obtain a fermentation system with an inoculation ratio of 50%; the fermentation system was fermented in a 30℃ incubator for 7 days, and stirred daily to obtain the fermentation product.

[0098] (4) The fermentation product was dried at 45°C for 48 h. The dried fermentation product is the golden ear fermented rice.

[0099] The fermented rice prepared in this comparative example was a sticky rice grain with severely clumped and adhered parts, covered with white mycelium. Component analysis of this fermented rice yielded the following results: Carotenoids content reached ≥2.07±0.09 mg / g; protein content reached ≥106.3±3.88 mg / g; flavonoid content reached ≥1.12±0.04 mg / g; total phenol content reached ≥2.83±0.10 mg / g; polysaccharide content reached 233.8±6.92 mg / g; total amino acid content reached 268.5±9.31 mg / g; and starch content reached 493.8±15.58 mg / g.

[0100] The results showed that maintaining a constant temperature of 30℃ throughout the process led to unstable mycelial colonization and metabolic imbalance, with the carotenoid content being only 2.07±0.09 mg / g, further demonstrating the unique advantages of the two-stage variable temperature process in coordinating growth and synthesis.

Claims

1. A method for producing carotenoids from rice through in-situ fermentation of rice by *Auricularia auricula-judae*, characterized in that, Includes the following steps: (1) Take activated auricularia auricula mycelium, inoculate it into primary corn bran liquid culture medium and culture for 5-7 days to obtain auricularia auricula seed liquid, and add 10-50 ppm β-ionone on the 2nd-4th day; (2) Put rice into a fermentation container, add water to adjust the moisture content to 10-60%, sterilize, and obtain the fermentation substrate; (3) Inoculate the fermentation substrate with Auricularia auricula seed liquid, the volume-to-mass ratio of Auricularia auricula seed liquid to the fermentation substrate is 20-50%, first culture at 20-25℃ for 2-4 days, and then raise the temperature to 28-35℃ for 2-4 days; (4) Dry at 45℃ to obtain rice fermented by Auricularia auricula-judae. The rice fermented by Auricularia auricula-judae is orange-yellow, with intact grains and no sticking.

2. The method for producing carotenoids from rice by fermentation with *Auricularia auricula-judae* according to claim 1, characterized in that, The formulation of the primary corn bran liquid culture medium is: based on g·L -1 The formula is as follows: glucose 20-40g, magnesium sulfate heptahydrate 2-3g, potassium dihydrogen phosphate 3-4.5g, corn flour 1-2g, wheat bran 1-2g.

3. The method for producing carotenoids from rice by fermentation with *Auricularia auricula-judae* according to claim 1, characterized in that, The culture temperature in step (1) is 25~30℃ and the rotation speed is 150~180 rpm.

4. A method for producing carotenoids from rice by fermentation with *Auricularia auricula-judae* according to claim 1 or 2, characterized in that, The preparation method of the Auricularia auricula seed liquid in step (1) includes the following steps: ① Strain activation: Inoculate the strain into glucose potato agar medium for activation at a temperature of 28-30℃ for 5-8 days; ② Liquid culture: Take 1 cm of the activated mycelium 2 After being chopped with an inoculation needle, the sample was inoculated into 80 mL of primary corn bran liquid culture medium and cultured at a temperature of 25-30℃ for 5-7 days at a rotation speed of 150-180 rpm. β-ionone was added on the 2nd-4th day at a concentration of 10-50 ppm to obtain the seed culture of Auricularia auricula-judae for inoculation.

5. The method for producing carotenoids from rice by fermentation with *Auricularia auricula-judae* according to claim 1, characterized in that, Step (3) involves stirring during the fermentation process.

6. The method for producing carotenoids from rice by fermentation with *Auricularia auricula-judae* according to claim 5, characterized in that, The stirring treatment time interval is 1 to 3 days.

7. The method for producing carotenoids from rice by fermentation with *Auricularia auricula-judae* according to claim 5, characterized in that, The low-temperature drying time in step (4) is 24~48 h.

8. Rice obtained by the method according to any one of claims 1-7.

9. Food products made from the rice described in claim 8.

10. The food product according to claim 9, characterized in that, This includes golden ear fungus porridge, golden ear fungus rice noodles, golden ear fungus rice wine, or golden ear fungus rice cake.

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