A method for improving the extraction rate and antioxidant activity of bamboo leaf flavonoids based on sequential fermentation

Abstract

The application discloses a method for improving the extraction rate and antioxidant activity of bamboo leaf flavones based on sequential fermentation, and belongs to the technical field of microbial fermentation. The method comprises the following steps: (1) inoculating a green Trichoderma spore suspension into sterilized bamboo leaf powder to perform first-stage fermentation; (2) after the first-stage fermentation is completed, inoculating an Aspergillus niger spore suspension into the fermentation system to perform second-stage fermentation; and (3) after the fermentation is completed, extracting bamboo leaf flavones from the fermentation product by using a solvent extraction method. The sequential fermentation method is used to make the green Trichoderma and the Aspergillus niger fully exert their enzymatic advantages in respective suitable stages, so that the overall degradation efficiency of the bamboo leaf cell wall structure is improved, and the extraction efficiency and biological function are simultaneously improved. The solid-state fermentation method has the characteristics of simple operation and mild process, and provides a feasible technical scheme for the high-value utilization of bamboo leaf resources.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, specifically to a method for improving the extraction rate of bamboo leaf flavonoids and enhancing their antioxidant activity based on the sequential fermentation of Trichoderma viride and Aspergillus niger. Background Technology

[0002] Bamboo leaf flavonoids are a natural organic mixture extracted from bamboo leaves, containing various flavonoid active ingredients with antioxidant and anti-inflammatory biological functions. Bamboo leaf flavonoids are widely used as natural active ingredients in health products and food additives; however, their extraction technology faces challenges such as low extraction rates and safety concerns. For example, due to the dense cell wall structure of bamboo leaves and the high content of cellulose, hemicellulose, and lignin, the release of flavonoids is limited during conventional solvent extraction, resulting in low extraction efficiency. Traditional methods also suffer from high energy consumption, high costs, and potential environmental pollution.

[0003] To improve the utilization rate of plant active ingredients, microbial fermentation technology is widely used in the pretreatment process of plant raw materials. Existing research shows that microorganisms can secrete a variety of degradation enzymes during fermentation, such as cellulase, pectinase and β-glucosidase, thereby destroying the plant cell wall structure and promoting the release of active ingredients (Zhao Bingnan et al. Research progress on the application of microbial fermentation technology in the extraction of plant active ingredients. Pharmaceutical Research, 2025, 44(7): 696-702.).

[0004] Currently, most microbial fermentation technologies for plant raw materials employ single-strain fermentation. However, different microorganisms exhibit significant differences in enzyme composition and functional characteristics. Single-strain fermentations often only function at specific stages or targeting specific substrates, making it difficult to achieve deep degradation of complex plant cell wall structures. Furthermore, single-strain fermentation still has limitations in improving the composition, structure, and functional activity of plant bioactive components.

[0005] To overcome the aforementioned problems, sequential fermentation technology has been gradually developed in recent years. This technology introduces microorganisms with different enzyme system advantages at different fermentation stages, allowing each strain to exert its maximum effect at the appropriate stage, achieving graded processing and synergistic transformation of plant raw materials. Compared with simultaneous inoculation or single-strain fermentation, sequential fermentation has significant advantages in substrate utilization efficiency, depth of structural degradation, and transformation of active ingredients.

[0006] However, existing research on microbial fermentation of bamboo leaf raw materials is still limited, especially lacking technical solutions that utilize sequential fermentation technology to systematically improve the extraction rate of bamboo leaf flavonoids and enhance their anti-inflammatory activity. Therefore, it is essential to develop a bamboo leaf processing method based on sequential inoculation fermentation to achieve efficient extraction and functional enhancement of bamboo leaf flavonoids. Summary of the Invention

[0007] The purpose of this invention is to discover microorganisms suitable for sequential fermentation of bamboo leaves, and to provide a bamboo leaf processing method based on sequential inoculation fermentation. By leveraging the synergistic effect of enzyme systems secreted by different microorganisms at different fermentation stages, the extraction rate of bamboo leaf flavonoids is improved, and a bamboo leaf flavonoid extract with enhanced physiological activity is obtained.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: On one hand, the present invention provides a microbial ensemble for bamboo leaf fermentation treatment, including *Trichoderma viride* (… Trichoderma viride ) and Aspergillus niger ( Aspergillus niger ), which is used in a sequential manner for the fermentation of bamboo leaf raw materials.

[0009] Specifically, *Trichoderma viride* is inoculated during the early stage of fermentation to initially degrade the cell wall structure of bamboo leaves, making the cell wall structure looser. *Aspergillus niger* is inoculated during the later stage of fermentation to further decompose the bamboo leaf matrix after initial degradation by *Trichoderma viride*, thereby promoting the full release of flavonoids.

[0010] Studies have shown that, compared with inoculating Trichoderma viride alone, inoculating Aspergillus niger alone, or inoculating Aspergillus niger first and then Trichoderma viride, the treatment method of inoculating Trichoderma viride first and then Aspergillus niger for fermentation of bamboo leaf raw materials is more conducive to improving the extraction rate of bamboo leaf flavonoids.

[0011] Secondly, the present invention provides a method for sequential fermentation of bamboo leaf raw materials based on the aforementioned microbial combination to improve the extraction rate and antioxidant activity of bamboo leaf flavonoids, the method comprising the following steps: (1) The green fungus ( Trichoderma viride The bacterial suspension was inoculated into sterilized bamboo leaf powder for the first stage of fermentation; (2) After the first stage of fermentation is completed, Aspergillus niger is inoculated into the fermentation system. Aspergillus niger The bacterial suspension is used for the second stage of fermentation; (3) After fermentation, bamboo leaf flavonoid extract was obtained from the fermentation product by solvent extraction.

[0012] This invention utilizes *Trichoderma viride* for the first stage of fermentation of bamboo leaves and *Aspergillus niger* for the second stage. This sequential fermentation method avoids competitive inhibition between different microorganisms in the same fermentation stage, allowing *Trichoderma viride* and *Aspergillus niger* to fully exert their enzymatic advantages at their respective optimal stages, thereby improving the overall degradation efficiency of the bamboo leaf cell wall structure. The entire fermentation process takes 48–96 hours, and the fermentation time ratio of the two microorganisms needs to be controlled between 1:2 and 2:1.

[0013] In this invention, the bamboo leaf raw material is pretreated before sequential fermentation. The pretreatment includes: pulverizing the bamboo leaf raw material to obtain bamboo leaf powder, and then sterilizing it. The method for preparing the bamboo leaf powder includes: washing, drying, pulverizing, and passing the bamboo leaf raw material through a 40-80 mesh sieve to obtain bamboo leaf powder.

[0014] In this invention, *Trichoderma viride* and *Aspergillus niger* are cultured to the logarithmic growth phase for sequential fermentation. Preferably, the culture method includes: inoculating *Trichoderma viride* or *Aspergillus niger* into PDB liquid medium and incubating at 30°C and 150 °C. rpm Cultured under oscillating conditions until the logarithmic growth phase.

[0015] In step (1), the green Trichoderma suspension is mixed with sterilized bamboo leaf powder for the first stage of static fermentation.

[0016] Preferably, the concentration of live *Trichoderma viride* in the logarithmic growth phase of the *Trichoderma viride* suspension is 10. 4 ~10 8 CFU / mL; the bacterial suspension and bamboo leaf powder were mixed at a liquid-to-solid ratio of 0.5~2 mL:1 g.

[0017] More preferably, the concentration of live *Trichoderma viride* in the logarithmic growth phase of the *Trichoderma viride* suspension is 10. 6 CFU / mL; the bacterial suspension and bamboo leaf powder were mixed at a liquid-to-solid ratio of 1 mL:1 g.

[0018] As a preferred option, the conditions for the first stage of fermentation are: temperature 25-35℃ and time 24-48 h.

[0019] In step (2), after the first stage of fermentation is completed, the Aspergillus niger suspension is inoculated into the same fermentation system for the second stage of static fermentation.

[0020] Preferably, the concentration of live Aspergillus niger in the logarithmic growth phase in the Aspergillus niger suspension is 10. 4 ~10 8 CFU / mL; the bacterial suspension and bamboo leaf powder were mixed at a liquid-to-solid ratio of 0.5~2 mL:1 g.

[0021] More preferably, the concentration of live Aspergillus niger in the logarithmic growth phase of the Aspergillus niger suspension is 10. 6 CFU / mL; the bacterial suspension and bamboo leaf powder were mixed at a liquid-to-solid ratio of 0.5 mL:1 g.

[0022] As a preferred option, the conditions for the second stage of fermentation are: temperature 25-35℃ and time 24-48 h.

[0023] As a specific embodiment of the present invention, the first stage is to use a concentration of 106 A CFU / mL suspension of *Trichoderma viride* was mixed with bamboo leaf powder at a liquid-to-solid ratio of 1 mL:1 g and fermented at 30°C for 24 h. In the second stage, a concentration of 10... 6 A CFU / mL Aspergillus niger suspension was added to the fermentation system at a liquid-to-solid ratio of 0.5 mL:1 g with bamboo leaf powder, and fermented at 30°C for 48 h.

[0024] As a specific embodiment of the present invention, *Trichoderma viride* ( Trichoderma viride The strain used was CICC 13038; Aspergillus niger ( Aspergillus niger The strain used was CICC 41586. Both strains can be used in food production.

[0025] After sequential fermentation, bamboo leaf flavonoid extract was obtained from the fermentation product using solvent extraction. Preferably, the solvent used for solvent extraction was an ethanol-water mixture, wherein the volume percentage of ethanol was 60% to 90%.

[0026] More preferably, the volume percentage of ethanol in the ethanol-water mixed solvent is 70%.

[0027] Preferably, the fermentation product and solvent are mixed at a ratio of 1 g: 25 mL and treated with ultrasound at a power of 100 W for 15-20 min.

[0028] Thirdly, this invention provides a bamboo leaf flavonoid extract prepared by the above method. Research in this invention shows that treating bamboo leaf raw materials using the above sequential fermentation method can not only effectively promote the release of flavonoids and increase the extraction rate of bamboo leaf flavonoids, but also promote the enrichment of highly active ingredients and enhance the antioxidant and anti-inflammatory activities of the extract.

[0029] Fourthly, this invention provides the application of the aforementioned bamboo leaf flavonoid extract in the preparation of antioxidant or anti-inflammatory products. The products may be, but are not limited to, health foods for antioxidant purposes or pharmaceuticals or functional ingredients for anti-inflammatory purposes.

[0030] The beneficial effects of this invention are as follows: (1) Significantly improved extraction efficiency: Through sequential fermentation, the cell wall structure of bamboo leaves is graded and synergistically degraded, and the extraction rate of bamboo leaf flavonoids is increased from about 20 mg / g in the traditional method to about 50 mg / g, which significantly improves the extraction efficiency.

[0031] (2) Environmental friendliness: The fermentation conditions are mild and there is no need for high-temperature heating, thus reducing the energy consumption of extraction.

[0032] (3) Enhanced bioactivity: Improved composition of bamboo leaf flavonoids and enhanced functional activity.

[0033] (4) Simple operation and mild process: The preparation and fermentation process of the microbial agent is mild, has good repeatability, can be scaled up, reduces the cost of bamboo leaf processing, and is suitable for the food and functional raw material fields.

[0034] (5) Comprehensive utilization of resources: The fermentation residue is safe and non-toxic, and the cell structure of bamboo leaves is more loose than that of the initial stage, which can be converted into organic fertilizer or animal feed, realizing the full utilization of resources. Attached Figure Description

[0035] Figure 1 This describes the process flow for sequential fermentation extraction of flavonoids from bamboo leaves.

[0036] Figure 2 The effect of different fermentation methods on the flavonoid extraction rate of bamboo leaves.

[0037] Figure 3 Results of DPPH free radical scavenging experiments on bamboo leaf flavonoids extracted by different fermentation methods.

[0038] Figure 4 Results of ABTS free radical scavenging experiments on bamboo leaf flavonoids extracted by different fermentation methods.

[0039] Figure 5 The results of cytotoxicity experiments on bamboo leaf flavonoids extracted from bamboo leaves by different fermentation methods are shown. (A) Bamboo leaf flavonoids extracted by solid-state fermentation inoculated with Aspergillus niger; (B) Bamboo leaf flavonoids extracted by solid-state fermentation inoculated with Trichoderma viride; (C) Bamboo leaf flavonoids extracted by sequential fermentation of two strains; (D) Bamboo leaf flavonoids extracted by mixed fermentation of two strains; (E) Quercetin; and (F) Rutin.

[0040] Figure 6 The results of testing the antioxidant capacity of bamboo leaf flavonoids extracted from bamboo leaves by different fermentation methods on cells.

[0041] Figure 7 Results of tests on the anti-inflammatory effects of bamboo leaf flavonoids extracted from bamboo leaves treated with different fermentation methods on cells.

[0042] Figure 8 The composition of four C-glycoside flavonoids in bamboo leaf flavonoids extracted from bamboo leaves by different fermentation methods. Detailed Implementation

[0043] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0044] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0045] The raw materials, reagents, and bacterial strains involved in the following examples: The bamboo leaves used are from moso bamboo (…). Phyllostachys edulis The bamboo leaves were harvested from Anji County, Zhejiang Province. The processing method was as follows: after harvesting, the bamboo leaves were dried naturally and then pulverized, passing through a 40-mesh sieve to obtain uniform bamboo leaf powder. The obtained bamboo leaf powder was stored in a dry environment for subsequent experimental use.

[0046] Trichoderma viride ( Trichoderma viride Purchased from the China Industrial Microbial Culture Collection Center, strain number CICC 13038; Aspergillus niger ( Aspergillus niger Purchased from the China Industrial Microbial Culture Collection Center, strain number CICC 41586.

[0047] PDB medium was commercially available potato dextrose medium (purchased from Qingdao Haibo Biotechnology Co., Ltd.). The medium was prepared with distilled water at a concentration of 35 g / L without additional pH adjustment. After preparation, it was autoclaved at 121℃ for 20 min, cooled, and then dispensed into sterile culture tubes.

[0048] Quercetin (CAS: 117-39-5), Rutin (CAS: 153-18-4), Vitexin (CAS: 3681-93-4), Isovitexin (CAS: 38953-85-4), Orientin (CAS: 28608-75-5), and Isoorientin (CAS: 4261-42-1).

[0049] Example 1: Inoculation and Fermentation Microbial Culture and Preparation of Inoculum This embodiment aims to illustrate the cultivation method of Trichoderma viride and Aspergillus niger and the preparation process of the inoculant, as detailed below: Trichoderma viride and Aspergillus niger were inoculated into PDB liquid medium, respectively, and incubated at 30°C and 150°C. rpm Under shaking conditions, the bacterial culture was incubated. After incubation, the bacterial count was performed using a hemocytometer or plate count method. Subsequent fermentation used bacterial cultures in the logarithmic growth phase, diluted with sterile physiological saline to a cell concentration of 1×10⁻⁶. 6 CFU / mL available for use.

[0050] Example 2: Method for sequential fermentation treatment of bamboo leaves using *Trichoderma viride* and *Aspergillus niger* This embodiment aims to illustrate the process flow of treating bamboo leaves using sequential fermentation of Trichoderma viride and Aspergillus niger, as detailed below: (1) Raw material preparation: Take bamboo leaf powder that has been washed, naturally dried and crushed to a 40-mesh sieve, and place it in a drying container for later use.

[0051] (2) First stage fermentation: Weigh bamboo leaf powder and place it in a 250 mL Erlenmeyer flask. Autoclave at 121℃ for 20 min, cool to room temperature, and then inoculate with *Trichoderma viride*. The concentration of the *Trichoderma viride* suspension was 10... 6 CFU / mL, the mass-to-volume ratio of bamboo leaf powder to bacterial suspension was 1:1 ( w / v The first stage of fermentation was carried out at 30°C for 24 hours.

[0052] (3) Second stage fermentation: After the first stage fermentation is completed, inoculate with a suspension of Aspergillus niger without changing the fermentation system. The concentration of the suspension is 10. 6 The concentration of CFU / mL was determined by a 2:1 mass-to-volume ratio of bamboo leaf powder to bacterial suspension. After inoculation, fermentation was continued at 30°C for 48 hours to complete the sequential fermentation process.

[0053] The specific process is as follows: Figure 1 As shown.

[0054] The following compares different fermentation methods for treating leaves: Solid-state fermentation: Take 20 g of bamboo leaf powder and place it in a 250 mL Erlenmeyer flask, then add 20 mL of the *Trichoderma viride* suspension prepared in Example 1 (live bacteria concentration of 10). 6 The mixture was stirred thoroughly. The Erlenmeyer flask was sealed with breathable sealing film and statically fermented at 30°C for 24 hours. After fermentation, a small amount of white mycelium was visible on the surface of the bamboo leaf powder. Without changing the fermentation system, 10 mL of the Aspergillus niger suspension prepared in Example 1 (viable cell concentration of 10 CFU / mL) was added. 6 (CFU / mL). The Erlenmeyer flasks were sealed with breathable sealing film and statically fermented at 30℃ for 48 hours. After fermentation, black mycelium was visible on the surface of the bamboo leaf powder.

[0055] Ingredient: Bamboo leaf powder is extracted directly without fermentation.

[0056] Blank group: Bamboo leaf powder was mixed with an equal amount of sterile physiological saline and placed under the same fermentation conditions as the fermentation group before extraction.

[0057] Single-strain fermentation group (AN): fermented by inoculating Aspergillus niger alone under the same fermentation conditions.

[0058] Single-strain fermentation group (TV): fermentation of Trichoderma viride under the same fermentation conditions.

[0059] Mixed fermentation group (MI): Under the same fermentation conditions, Trichoderma viride and Aspergillus niger were mixed at a ratio of 1:1 (M11), 2:1 (M21), and 1:2 (M12) of bacterial culture volume, respectively, and then inoculated for fermentation.

[0060] Sequential fermentation groups: Fermentation was carried out according to the solid-state fermentation procedure described above. The group inoculated with Trichoderma viride first and then Aspergillus niger was designated as the TV-AN group, and the group inoculated with Aspergillus niger first and then Trichoderma viride was designated as the AN-TV group.

[0061] Example 3: Extraction of flavonoids from bamboo leaves after fermentation This embodiment aims to extract flavonoids from bamboo leaves treated with different fermentation methods in Example 2, and calculate the total flavonoid content in the extract, as follows: 1. Extraction Add 500 mL of 70% ethanol solution (solid-liquid ratio 1:25 g / mL) to the fermentation product, then place it in an ultrasonic cleaner and extract it by ultrasonic extraction at 100 W power for 15 min. After extraction, filter the product using a Buchner funnel, collect the filtrate, and determine the total flavonoid content.

[0062] 2. The total content of bamboo leaf flavonoids in the extract was determined by the aluminum nitrate-sodium nitrite colorimetric method. The specific procedure is as follows: Dilute the bamboo leaf flavonoid extract appropriately with deionized water to ensure its concentration falls within the linear range of the standard curve. Place 1 mL of the diluted sample solution into a 10 mL stoppered colorimetric tube, and sequentially add 0.3 mL of 5% sodium nitrite solution, 0.3 mL of 10% aluminum nitrate solution, and 4 mL of 4% sodium hydroxide solution. Then, bring the volume to 10 mL with deionized water. After mixing thoroughly, allow the mixture to stand for 15 min. Using the corresponding blank reagent as a reference, measure the absorbance at a wavelength of 510 nm. Plot a standard curve using rutin as a standard. The extraction yield of bamboo leaf flavonoids is calculated using the following formula: Extraction yield = flavonoid mass (mg) / bamboo leaf powder mass (g).

[0063] 3. Experimental Results Flavonoid extraction yield as follows Figure 2As shown, the single-strain fermentation group of *Aspergillus niger* (AN), the single-strain fermentation group of *Trichoderma viride* (TV), the mixed fermentation group of two strains (M11), and the sequential fermentation treatment group (TV-AN, first inoculated with *Trichoderma viride* and then inoculated with *Aspergillus niger*) all significantly promoted flavonoid extraction. Among them, the TV-AN group showed the most outstanding performance, and analysis of variance (ANOVA) confirmed that its extraction rate was significantly higher than that of other single treatment groups. This indicates that the sequential fermentation method is more effective in promoting flavonoid release and improving the extraction rate of flavonoids from bamboo leaves.

[0064] Example 4: Determination of the in vitro antioxidant capacity of sequential fermentation products This embodiment aims to evaluate the effect of bamboo leaf flavonoid extracts extracted from different fermentation groups (Control group, Aspergillus niger fermentation group (AN), Trichoderma viride fermentation group (TV), 1:1 mixed fermentation group of two strains (MI), and sequential fermentation group first inoculated with Trichoderma viride and then inoculated with Aspergillus niger (SI)) on in vitro antioxidant activity through DPPH and ABTS free radical scavenging experiments.

[0065] 1. Determination of DPPH free radical scavenging ability DPPH powder was dissolved in anhydrous ethanol to a concentration of 0.2 mmol / L. Quercetin and rutin at a concentration of 5 mg / L were used as positive controls. Lyophilized bamboo leaf flavonoid powder extracted using different fermentation methods was prepared into sample solutions with a concentration of 10 mg / L. 1 mL of sample solution or positive control solution was mixed with 1 mL of DPPH solution, and the mixture was reacted in the dark for 30 min. The absorbance was measured at 517 nm. The DPPH free radical scavenging rate of each sample was calculated using the following formula: DPPH radical scavenging rate (%) = [1 - (As-Ab / Ac)] × 100%; Where As is the absorbance value of the sample solution and DPPH solution, Ab is the absorbance value of the sample solution and blank solvent, and Ac is the absorbance value of the blank solvent and DPPH solution.

[0066] 2. ABTS Scavenging Rate Determination Prepare an ABTS stock solution with an absorbance of 0.700 ± 0.002 at 734 nm. Quercetin and rutin at a concentration of 5 mg / L were used as positive controls. Lyophilized bamboo leaf flavonoid powder extracted using different fermentation methods was prepared into a sample solution with a concentration of 10 mg / L. Mix 0.5 mL of the sample solution or positive control solution with 2 mL of ABTS stock solution, react in the dark for 20 min, and measure the absorbance at 734 nm. Calculate the ABTS free radical scavenging rate of each sample using the following equation: ABTS radical scavenging rate (%) = [1 - (As - Ab / Ac)] × 100%; Where As is the absorbance value of the sample solution and DPPH solution, Ab is the absorbance value of the sample solution and blank solvent, and Ac is the absorbance value of the blank solvent and DPPH solution.

[0067] 3. Experimental Results Experimental results are as follows Figure 3 and Figure 4 As shown, in samples with the same extract concentration, the sequential fermentation group exhibited higher DPPH and ABTS free radical scavenging ability, indicating that this method can not only increase the flavonoid extraction yield, but also significantly enhance the antioxidant activity of the extract.

[0068] Example 5: Cytotoxicity evaluation of sequential fermentation products This embodiment aims to evaluate the cellular safety of bamboo leaf flavonoid extracts extracted from different fermentation groups in Example 3.

[0069] 1. Experimental Methods (1) Cell culture: HaCaT cells were selected and cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2.

[0070] (2) Treatment method: Bamboo leaf flavonoid extract powder from different fermentation groups was prepared into solutions with concentrations of 1, 5, 10, 20, and 50 mg / L, respectively. Cells were seeded in 6-well plates (approximately 1×10⁻⁶ cells / well). 5 After culturing for 24 h, different concentrations of bamboo leaf flavonoid extract solution were added, and incubation continued for another 24 h.

[0071] (3) CCK-8 detection: Add 10 μL of CCK-8 reagent to each well, incubate in the dark for 30 min, and then measure the absorbance at a wavelength of 450 nm.

[0072] (4) Cell viability is calculated using the following formula: Cell viability (%) = (As - Ab) / (A0 - Ab) × 100%; Where As represents the absorbance value of the wells with different concentrations of sample solution; Ab represents the absorbance value of the blank wells without cells; and A0 represents the absorbance value of the wells without sample solution.

[0073] 2. Experimental Results like Figure 5 As shown, within the experimental concentration range, the cell survival rate did not decrease significantly with different extraction methods, indicating that the flavonoid extract obtained by sequential fermentation has no cytotoxicity and has good biosafety.

[0074] Example 6: Evaluation of the cellular antioxidant and anti-inflammatory capabilities of sequential fermentation products This embodiment aims to evaluate the effect of bamboo leaf flavonoid extracts extracted from different fermentation groups in Example 3 on the antioxidant capacity of cells by measuring SOD activity and MDA content.

[0075] 1. Experimental Methods (1) Cell treatment: HaCaT cells were seeded in 6-well plates and cultured in DMEM complete culture medium (containing 10% fetal bovine serum) for 12 h before being divided into groups, including blank group, model group, positive control group and drug treatment group.

[0076] Control group: No free fatty acids (FFA) were used to induce inflammation, and no active ingredients were added; Model: 2 mM FFA was added to induce inflammation, without any added active ingredients; Positive control group: 2 mM FFA was added to induce inflammation, and 5 mg / L quercetin was added as the positive control group. 5 mg / L rutin was added as the positive control group for oxyglycoside compounds.

[0077] Drug administration group: 2 mM FFA was added to induce inflammation, and then a sample solution with a concentration of 10 mg / L was prepared by adding bamboo leaf flavonoid freeze-dried powder extracted from different fermentation groups in Example 3 (Trichoderma viride fermentation (TV), Aspergillus niger fermentation (AN), mixed fermentation of two bacteria (MI) and sequential fermentation (SI)).

[0078] Each group should have at least three parallel groups.

[0079] (2) SOD activity assay: Following the instructions of the SOD kit, the SOD activity in the cell lysate was measured using the xanthine oxidase method. The absorbance was measured at a wavelength of 550 nm, and the SOD activity (U / 10) was calculated. 4 cells).

[0080] (3) MDA content determination: The MDA content was determined by the thiobarbituric acid (TBA) method. The absorbance was measured at a wavelength of 532 nm, and the MDA content was calculated based on the standard curve.

[0081] 2. Experimental Results like Figure 6 and Figure 7 As shown, compared with the model group, the degree of oxidative damage and the level of inflammatory factors in the cells of the sequential fermentation group were significantly lower, and the antioxidant and anti-inflammatory effects were better than those of other groups, indicating that the bamboo leaf flavonoids extracted by the sequential fermentation method described in this invention have stronger antioxidant protection.

[0082] Example 7: Analysis of flavonoid components in sequential fermentation products This embodiment aims to analyze the effect of sequential fermentation on the composition of flavonoids in bamboo leaves using UPLC-MS / MS technology.

[0083] 1. Experimental Methods (1) Sample preparation and instrumentation: Ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry (UPLC–MS / MS) was used to perform qualitative and quantitative analysis of carbon glycoside flavonoids in the sample.

[0084] The flavonoid extract obtained in Example 3 was filtered through a 0.22 μm organic filter membrane before being injected into the instrument for analysis. A Waters ACQUITY Premier UPLC system was used, coupled with a Xevo TQ-XS triple quadrupole mass spectrometer detector (Waters, USA). The injection volume was 1 μL.

[0085] (2) Chromatographic conditions: RP18 (2.1 mm × 100 mm, 1.7 μm) was used, with mobile phase A being 0.1% formic acid aqueous solution and mobile phase B being acetonitrile. The column temperature was 40℃ and the flow rate was 0.30 mL / min. The gradient elution program is shown in Table 1.

[0086] Table 1. High Performance Liquid Chromatography Elution Procedure

[0087] (3) Mass spectrometry conditions: Electrospray ionization (ESI) source was used, positive ion mode was used, capillary voltage was about 2.5 kV, and the scanning range was 100-1000 m / z. Multiple reaction monitoring (MRM) mode was used for detection. The parent ion / daughter ion pairs (m / z), cone voltage and collision energy of each target compound are shown in Table 2.

[0088] Table 2. Qualitative Spectroscopic Information of C-glycoside flavonoids

[0089] (4) Data analysis: Qualitative analysis of four C-glycoside flavonoids, namely vitexin, isovitexin, sennain and isosennain, was performed based on mass spectrometry fragment information. Quantitative analysis of the four C-glycoside flavonoids was performed based on the retention time and peak area of ​​the standard.

[0090] 2. Experimental Results like Figure 8 As shown, the content of isopropanol in the sequential fermentation group was significantly higher than that in other groups, indicating that this method can not only effectively promote flavonoid release and increase the extraction rate of bamboo leaf flavonoids, but also promote the enrichment of highly active ingredients, thereby enhancing the antioxidant activity of flavonoid extracts.

[0091] Comparative Example 1 This comparative example will include Trichoderma longifolia ( Trichoderma longibrachiatum CICC 40340) and extended penicillin ( Penicillium expansum (CICC 41063) was applied to solid-state fermentation, and the fermentation process and conditions were the same as those described in the single-strain fermentation group in Example 2. The method for extracting bamboo leaf flavonoids was the same as that in Example 3.

[0092] The results showed that *Trichoderma longifolia* (CICC 40340) extracted flavonoids from bamboo leaves at a rate of 25.67 ± 1.67 mg / g, while *Penicillium expansum* (CICC 41063) extracted flavonoids from bamboo leaves at a rate of 30.46 ± 0.80 mg / g. Although this was a slight improvement compared to the control group (around 20 mg / g), the promoting effect was far less than that of *Trichoderma viride* CICC 13038 (around 46 mg / g) and *Aspergillus niger* CICC 41586 (around 45 mg / g).

[0093] in conclusion: This invention significantly improves the extraction efficiency of bamboo leaf flavonoids and enhances the functional activity of the flavonoid extract through sequential fermentation of *Trichoderma viride* and *Aspergillus niger*. The sequentially fermented extract shows significant enhancement in DPPH free radical scavenging capacity and cellular antioxidant indicators such as SOD and MDA. In other words, this method simultaneously achieves a dual improvement in extraction efficiency and biological function. Furthermore, the solid-state fermentation method is simple to operate and has a mild process, providing a feasible technical solution for the high-value utilization of bamboo leaf resources. Those skilled in the art can adjust the parameters according to actual needs, and such adjustments all fall within the protection scope of this invention.

Claims

1. A method for improving the extraction rate and antioxidant activity of bamboo leaf flavonoids based on sequential fermentation, characterized in that, Includes the following steps: (1) The green fungus ( Trichoderma viride The bacterial suspension was inoculated into sterilized bamboo leaf powder for the first stage of fermentation; (2) After the first stage of fermentation is completed, Aspergillus niger is inoculated into the fermentation system. Aspergillus niger The bacterial suspension is used for the second stage of fermentation; (3) After fermentation, bamboo leaf flavonoid extract was obtained from the fermentation product by solvent extraction.

2. The method as described in claim 1, characterized in that, In step (1), the method for preparing the bamboo leaf powder includes: the bamboo leaf raw material is washed, dried, crushed, and passed through a 40-80 mesh sieve to obtain bamboo leaf powder.

3. The method as described in claim 1, characterized in that, In step (1), the concentration of live Trichoderma viride in the logarithmic growth phase in the Trichoderma viride suspension is 10. 4 ~10 8 CFU / mL; the bacterial suspension and bamboo leaf powder were mixed at a liquid-to-solid ratio of 0.5~2 mL:1 g.

4. The method as described in claim 1 or 3, characterized in that, In step (1), the conditions for the first stage of fermentation are: temperature 25-35℃ and time 24-48 h.

5. The method as described in claim 1, characterized in that, In step (2), the concentration of live Aspergillus niger in the logarithmic growth phase in the Aspergillus niger suspension is 10. 4 ~10 8 CFU / mL; the bacterial suspension and bamboo leaf powder were mixed at a liquid-to-solid ratio of 0.5~2 mL:1 g.

6. The method as described in claim 1 or 5, characterized in that, In step (2), the conditions for the second stage of fermentation are: temperature 25-35℃ and time 24-48 h.

7. The method as described in claim 1, characterized in that, In step (3), the solvent used for solvent extraction is an ethanol-water mixed solvent, wherein the volume percentage of ethanol is 60%~90%.

8. The method as described in claim 7, characterized in that, The fermentation product and solvent were mixed at a ratio of 1 g: 25 mL and treated with ultrasound at 100 W for 15-20 min.

9. The bamboo leaf flavonoid extract prepared by the method according to any one of claims 1-8.

10. The use of the bamboo leaf flavonoid extract as described in claim 9 in the preparation of antioxidant or anti-inflammatory products.

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