Method for solid state fermentation of tartary buckwheat by using morchella for high yield of triterpenoid active components

Abstract

The invention belongs to the technical field of food deep processing, and provides a method for solid-state fermentation of tartary buckwheat with high yield of triterpenoid active components by using morchella. The preparation method comprises the following steps: inoculating a morchella fermentation liquid into sterilized tartary buckwheat, carrying out constant-temperature fermentation culture at 24-30 DEG C for 15-25 days, and drying at 60 DEG C for 18 hours, so as to obtain the morchella solid-state fermented tartary buckwheat with high yield of triterpenoid active ingredients, the sterilized tartary buckwheat is prepared by the following steps: soaking 50g of tartary buckwheat in purified water with the volume 4 times that of the tartary buckwheat for 24 hours, draining, and sterilizing for 1 hour by high-pressure steam at 121 DEG C; the inoculum size of the morchella fermentation liquor is 3-7 mL. The four triterpenoid components are analyzed by using non-targeted metabonomics, the four triterpenoid components are all in an up-regulation trend in a tartary buckwheat solid-state fermentation process by using morchella, the up-regulation trend of beta-citraulin and ganoderic acid is most obvious, and relative quantitative values are respectively up-regulated by 404.44% and 195.42% when the tartary buckwheat is fermented for 20 days; in addition, the two triterpenoid components are important intermediates in the fungal cell proliferation process, and up-regulation of response values of the two triterpenoid components also reflects continuous proliferation of fungal cells in the fermentation process.

Description

Technical Field

[0001] This invention belongs to the field of food deep processing technology, specifically relating to a method for solid-state fermentation of buckwheat with morel mushrooms to produce high levels of triterpenoid active ingredients. Background Technology

[0002] Buckwheat ( Fagopyrum tataricum (L.) Gaertn. Tartary buckwheat, also known as buckwheat, is a dicotyledonous plant belonging to the Polygonaceae family. It is renowned for its strong adaptability, cold tolerance, and short growth cycle, and is one of my country's traditional coarse grain crops. my country began cultivating buckwheat as early as the Western Zhou Dynasty, and today, my country still holds an important position in buckwheat cultivation and export. Buckwheat is not only rich in protein, fat, and dietary fiber, but also contains various vitamins, effectively meeting the body's nutritional needs. More importantly, as a crop with both medicinal and edible uses, buckwheat contains many active ingredients lacking in other coarse grains, such as flavonoids, D-chiral inositol, and polyphenols. The content of flavonoids is significantly higher than in other coarse grains, giving it a unique advantage in functional value. Studies have shown that flavonoids have significant positive effects in improving inflammatory diseases, hypertension, and anti-cancer properties. These bioactive components make buckwheat widely recognized as an excellent functional food ingredient. With people's increasing emphasis on healthy eating, the market prospects for buckwheat are becoming increasingly broad, thus earning it the reputation of a green food for the 21st century.

[0003] As people pay increasing attention to the nutritional and functional components of buckwheat, the variety of foods developed from buckwheat is gradually increasing, including buckwheat vinegar, buckwheat wine, buckwheat tea, and buckwheat noodles. Among these, fermented buckwheat products are particularly abundant, and there is considerable research on this topic. For example, using buckwheat flour to replace wheat flour in the production of soybean paste has shown that buckwheat soybean paste has superior flavor, texture, and nutritional quality compared to traditional soybean paste. Liquid fermentation of buckwheat flour has been used to increase the total flavonoid content of buckwheat vinegar. However, buckwheat has a relatively rough texture and a certain bitterness, making its taste a challenge in product development.

[0004] Solid-state fermentation technology not only effectively enhances the nutritional components of tartary buckwheat but also improves its functional activity, providing a new approach for developing high-quality tartary buckwheat foods. However, solid-state fermentation of tartary buckwheat involves a variety of strains, including bacteria, fungi, and macroed edible fungi, while research on solid-state fermentation of tartary buckwheat using medicinal and edible fungi is relatively limited.

[0005] Morel mushrooms ( Morehella esculenta Also known as morel mushroom, it belongs to the Ascomycota (Ascomycota phylum). AscomycotinaMorel mushrooms are a globally renowned wild medicinal and edible fungus. They are extremely rich in nutrients, containing crude protein, fat, amino acids, alkaloids, polyphenols, polysaccharides, and various minerals and trace elements, giving them exceptional nutritional value and earning them the title of "vegetarian meat." Modern medical research has further revealed the various health benefits of morel mushrooms, including anti-fatigue, anti-radiation, and immune-enhancing properties, making them increasingly popular in the health food industry. Although research on morel mushrooms is gradually increasing, studies on solid-state fermentation of morel mushrooms and buckwheat are still relatively limited, leaving ample room for future research. Summary of the Invention

[0006] This invention provides a method for solid-state fermentation of buckwheat with morel mushrooms to produce high levels of triterpenoid active ingredients.

[0007] This invention is achieved by the following technical solution: a method for solid-state fermentation of tartary buckwheat with morel mushrooms to produce high levels of triterpenoid active ingredients, wherein morel mushroom fermentation broth is inoculated into sterilized tartary buckwheat, fermented at a constant temperature of 24-30℃ for 15-25 days, and dried at 60℃ for 18 hours, which is the solid-state fermented tartary buckwheat with morel mushrooms to produce high levels of triterpenoid active ingredients; wherein: the sterilized tartary buckwheat is: 50g of tartary buckwheat soaked in 4 times its volume of purified water for 24 hours, drained, and sterilized by high-pressure steam at 121℃ for 1 hour; the inoculation volume of morel mushroom fermentation broth is 3-7mL.

[0008] Morel mushroom fermentation broth was inoculated into sterilized tartary buckwheat, fermented at a constant temperature of 27℃ for 20 days, and dried at 60℃ for 18 hours to produce solid-state fermented tartary buckwheat with high production of triterpenoid active ingredients; wherein, the inoculation volume of morel mushroom fermentation broth was 5 mL.

[0009] The triterpenoid active ingredients are lanosterol, 4,4-dimethyl-5α-cholest-8,24-dien-3β-ol, β-limonene, and ganoderic acid Jb.

[0010] The triterpenoid active ingredients are β-citrin and ganoderic acid Jb. After 20 days of fermentation, their relative quantitative values ​​were increased by 404.44% and 195.42%, respectively.

[0011] The morel mushrooms used in this invention were purchased from the China Industrial Microbial Culture Collection Center, with the strain number CICC14033.

[0012] This invention employs non-targeted metabolomics to analyze four triterpenoid components during the solid-state fermentation of tartary buckwheat by four edible and medicinal fungi. The results showed that all four triterpenoid components exhibited an upregulation trend during the solid-state fermentation of tartary buckwheat by *Morchella esculenta*, with β-citrin and ganoderic acid Jb showing the most significant upregulation, increasing by 404.44% and 195.42%, respectively, at 20 days of fermentation. The other two triterpenoid components are important intermediates in the fungal cell proliferation process, and their upregulation also reflects the continuous proliferation of fungal cells during fermentation. At 10 days of fermentation, the fold change (FC) of β-citrin and ganoderic acid Jb in *Morchella esculenta* was significantly higher than that in *Auricularia auricula-judae*, *Ganoderma lucidum*, and *Hericium erinaceus*, reaching 3.36 and 1.87, respectively, indicating that *Morchella esculenta* has a stronger accumulation capacity for β-citrin and ganoderic acid Jb compared to these fungi. Attached Figure Description

[0013] Figure 1 The effect of fermentation time on the response values ​​of four triterpenoids during solid-state fermentation of buckwheat by morel mushrooms; Figure 2 The effect of solid-state fermentation of different strains for 10 days on the response value of β-limonin in buckwheat; Figure 3 The effect of solid-state fermentation of different strains for 10 days on the Jb response value of ganoderic acid in buckwheat; Figure 4 This is the secondary spectrum of β-citrin; Figure 5 This is the secondary spectrum of ganoderic acid Jb. Detailed Implementation

[0014] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and all materials publicly cited herein and cited by them are incorporated herein by reference.

[0016] Equivalent technologies of the specific embodiments described herein that are readily apparent to those skilled in the art through routine experimentation are included in this application.

[0017] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all standard laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from regular biochemical reagent stores.

[0018] This invention employs untargeted metabolomics to analyze small molecule chemical components during solid-state fermentation. In untargeted metabolomics, the response value of a substance is usually positively correlated with its actual content in the sample. In the same batch and the same sample matrix, for the same compound, samples with higher content usually produce higher response values ​​(all metabolomics data in this experiment came from the same detection batch).

[0019] I. Materials and Methods 1. The main experimental materials and reagents are shown in Table 1. The experimental instruments and equipment are shown in Table 2.

[0020] Table 1: Main Experimental Materials and Reagents Table 2: Test Instruments and Equipment 2. Strain Screening: Strain Cultivation and Activation: Black fungus, Ganoderma lucidum, morel, and Hericium erinaceus were inoculated onto potato dextrose agar (PDA) slant medium, respectively. Black fungus and Hericium erinaceus were incubated at 25℃ for 10 days, while Ganoderma lucidum and morel were incubated at 27℃ for 10 days. Different strains were inoculated using a 0.8 cm agar shovel. 2 The bacterial blocks were inoculated into sterilized PDA liquid culture medium, shaken evenly, and placed in a shaker to be continuously activated at the above-mentioned culture temperature for 48 hours. The culture was then stored at 4℃ for later use.

[0021] 3. Preparation of buckwheat samples from solid-state fermentation: Weigh 50g of buckwheat and place it in an Erlenmeyer flask. Soak in 4 times its volume of purified water for 24 hours, then drain. Autoclave at 121℃ for 1 hour, then cool to room temperature. In a clean bench, use a sterile pipette to inject 5mL of activated bacterial strains into each Erlenmeyer flask, shake well, and incubate at 27℃. Dry at 60℃ for 18 hours, then ultrafinely pulverize and pass through an 80-mesh sieve. Send the samples to Beijing Biomarker Biotechnology Co., Ltd. for non-targeted metabolomics analysis.

[0022] 4. Metabolite extraction: Weigh 50 mg of sample and add 1000 μL of extraction buffer containing internal standard (methanol acetonitrile water volume ratio = 2:2:1 (v:v:v), internal standard concentration 20 mg / L), vortex mix for 30 seconds. Then add steel balls, grind in a 45 Hz grinder for 10 min, sonicate for 10 min (ice-water bath), and then analyze.

[0023] 5. Chromatographic conditions: HPLC conditions: Waters Acquity UPLC HSS T3 column (1.8 μm 2.1 × 100 mm); column temperature 40℃; flow rate 0.3 mL / min; injection volume 2 μL. Mobile phase A was 0.1% formic acid aqueous solution / %, and B was 0.1% formic acid acetonitrile / %. Gradient elution process: 0-5.5 min, A: 95%, B: 5%; 5.5-9.0 min, A: 50%, B: 50%; 9.0-10.5 min, A: 5%, B: 95%; 10.5-12 min, A: 95%, B: 5%.

[0024] Mass spectrometry conditions: Samples were detected using electrospray ionization (ESI) in both positive and negative ion modes. The capillary voltage was 2500V for positive ion mode and -2000V for negative ion mode; the ion source temperature was 100℃, and the mass-to-nuclear ratio (m / z) acquisition range was 50-1200.

[0025] 6. Data Processing: Raw data collected using MassLynx V4.2 was processed using Progenesis QI software, including peak extraction and peak alignment. Based on Progenesis QI software, identification was performed using the online METLIN database, public databases, and the BGI database, while also identifying theoretical fragments.

[0026] II. Experimental Results and Analysis 1. Changes in triterpenoid components during solid-state fermentation of buckwheat with morel mushrooms: Triterpenoids are a class of terpenoid compounds composed of 30 carbon atoms, widely found in plants and traditional Chinese medicines, possessing various biological activities such as anti-inflammatory, anti-tumor, and immunomodulatory effects. Non-targeted metabolomics analysis revealed changes in triterpenoid components, such as… Figure 1 As shown, during the solid-state fermentation of buckwheat by morel mushrooms, the relative quantitative values ​​of various triterpenoid components in the sample showed an increasing trend with the extension of fermentation time.

[0027] The changes in the response values ​​of four triterpenoids during the solid-state fermentation of buckwheat by morel mushrooms are as follows: Figure 1As shown, β-limonene is a carotenoid commonly found in the peel of citrus plants. It belongs to the lutein family. Lutein generally has functional activities such as improving visual function and reducing the risk of cardiovascular disease[1], and shows health potential in terms of antioxidation[2]. There are currently no reports on its presence in buckwheat, morel mushrooms and their fermentation products. Its relative quantitative value reaches the highest at 20 days of fermentation. During the entire solid-state fermentation process, the relative quantitative value of ganoderic acid Jb increases continuously with the extension of fermentation time, reaching the maximum at 25 days. There are no reports on its presence in buckwheat, morel mushrooms and their fermentation products. Ganoderic acid Jb is a type II ganoderic acid. Due to the conjugated double bond on its four-ring skeleton, it has higher antitumor activity than type I ganoderic acid which has only one double bond on its four rings[3]. Lanosterol is a precursor of ergosterol in fungal cell membranes. 4,4-Dimethyl-5α-cholest-8,24-dien-3β-ol is an intermediate in the biosynthesis of sterols. The increase in the relative quantitative values ​​of the above substances indicates that the solid-state fermentation of buckwheat by morel mushrooms involves not only the continuous proliferation of morel mushroom cells but also the biosynthesis of active substances such as β-limonin and ganoderic acid Jb. Combined with the results of in vitro antioxidant activity assays, it can be concluded that this fermentation process can effectively improve the antioxidant and other biological activities of the fermentation products. The secondary spectrum of β-limonin is shown below. Figure 4 As shown, the secondary spectrum of ganoderic acid Jb is as follows. Figure 5 As shown.

[0028] 2. Effects of different microbial strains on the Jb response values ​​of β-limonin and ganoderic acid in tartary buckwheat: The metabolomics results of products from solid-state fermentation of tartary buckwheat by four microbial strains (Auricularia auricula-judae, Ganoderma lucidum, Hericium erinaceus, and Morchella esculenta) for 10 days were analyzed. Figure 2 As shown, solid-state fermentation of Auricularia auricula-judae, Ganoderma lucidum and morel mushrooms all significantly increased the relative quantitative value of β-limonin in tartary buckwheat, especially morel mushrooms (FC>2.0, P<0.05), which increased the relative quantitative value by 236.02%.

[0029] The metabolomics results of the products from 10 days of solid-state fermentation of tartary buckwheat were analyzed using four fungal strains: Auricularia auricula-judae, Ganoderma lucidum, Hericium erinaceus, and Morchella esculenta. The results are as follows: Figure 3 As shown, the relative quantitative values ​​of ganoderic acid Jb in the solid-state fermentation products of Auricularia auricula-judae, Ganoderma lucidum and Hericium erinaceus did not change significantly, while the ganoderic acid Jb in the fermentation products of Morchella esculenta was significantly upregulated (FC>1.5, P<0.05), with the relative quantitative value increasing by 87.25% compared to before fermentation.

[0030] This invention employs non-targeted metabolomics to analyze four triterpenoid components during the solid-state fermentation of tartary buckwheat by four edible and medicinal fungi. The results showed that all four triterpenoid components exhibited an upregulation trend during the solid-state fermentation of tartary buckwheat by *Morchella esculenta*, with β-citrin and ganoderic acid Jb showing the most significant upregulation, increasing by 404.44% and 195.42%, respectively, at 20 days of fermentation. The other two triterpenoid components are important intermediates in the fungal cell proliferation process, and their upregulation also reflects the continuous proliferation of fungal cells during fermentation. At 10 days of fermentation, the fold change (FC) of β-citrin and ganoderic acid Jb in *Morchella esculenta* was significantly higher than that in *Auricularia auricula-judae*, *Ganoderma lucidum*, and *Hericium erinaceus*, reaching 3.36 and 1.87, respectively, indicating that *Morchella esculenta* has a stronger accumulation capacity for β-citrin and ganoderic acid Jb compared to these fungi.

[0031] 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 or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

[0032] References: [1] Tian Jiamei, Yao Yanqiang, Luo Qinxiang, et al. Changes in carotenoid components and their contents in sweet potatoes at different developmental stages [J / OL]. Modern Food Science and Technology, 1-11 [2025-11-19]. [2]Ma, G., Zhang, L., Kato, M., Yamawaki, K., Kiriiwa, Y., Yahata,M.,&Nakamura, Y. (2015). Effect of blue and red LED light irradiation on β-citraurin accumulation in the flavedo of citrus fruits. Journal ofAgricultural and Food Chemistry, 63(13), 3434-3442. [3]Yuan, W., Jiang, C., Wang, Q., Fang, Y., Wang, J.,&Wang, M., etal. (2022).Biosynthesis of mushroom-derived type ii ganoderic acids byengineered yeast.Nature Communications,13。

Claims

1. A method for solid-state fermentation of buckwheat with morel mushrooms to produce high levels of triterpenoid active ingredients, characterized in that: Morel mushroom fermentation broth is inoculated into sterilized tartary buckwheat, fermented at a constant temperature of 24-30℃ for 15-25 days, and then dried at 60℃ for 18 hours to produce solid-state fermented tartary buckwheat with high production of triterpenoid active ingredients. The sterilized tartary buckwheat is prepared by soaking 50g of tartary buckwheat in 4 times its volume of purified water for 24 hours, draining the water, and then sterilizing it with high-pressure steam at 121℃ for 1 hour. The inoculation volume of morel mushroom fermentation broth is 3-7mL.

2. The method for solid-state fermentation of buckwheat with morel mushrooms to produce high levels of triterpenoid active ingredients according to claim 1, characterized in that: Morel mushroom fermentation broth was inoculated into sterilized tartary buckwheat, fermented at a constant temperature of 27℃ for 20 days, and dried at 60℃ for 18 hours to produce solid-state fermented tartary buckwheat with high production of triterpenoid active ingredients; wherein, the inoculation volume of morel mushroom fermentation broth was 5 mL.

3. The method for solid-state fermentation of buckwheat with morel mushrooms to produce high levels of triterpenoid active ingredients according to claim 2, characterized in that: The triterpenoid active ingredients are lanosterol, 4,4-dimethyl-5α-cholest-8,24-dien-3β-ol, β-limonene, and ganoderic acid Jb.

4. The method for solid-state fermentation of buckwheat with morel mushrooms to produce high levels of triterpenoid active ingredients according to claim 3, characterized in that: The triterpenoid active ingredients are β-citrin and ganoderic acid Jb. After 20 days of fermentation, their relative quantitative values ​​were increased by 404.44% and 195.42%, respectively.

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