Rhizopus arrhizus jhk31 and application thereof in assisted extraction of malve diospyros flavonoids

Abstract

The application discloses a Rhizopus arrhizus JHK31 and application thereof in assisted extraction of malveflavone, and belongs to the technical field of biological medicine. The Rhizopus arrhus JHK31 has a preservation number of CCTCC NO: M2023722, and the extraction rate of malveflavone can be improved by fermenting malve with the Rhizopus arrhizus JHK31. The flavone extraction rate can be significantly improved to 3.65% by the biological fermentation pretreatment of malve, which is much higher than the flavone extraction rate 2.61% of the same batch of unfermented malve, the flavone extraction rate after fermentation is significantly improved by 39.85% compared with the flavone extraction rate before fermentation, and the fermentation pretreatment can improve the antioxidant performance of the flavone. The malveflavone after fermentation has shorter extraction time, less solvent consumption, milder extraction conditions, higher extraction efficiency, and is more conducive to large-scale production.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a Rhizopus spp. JHK31 and its application in the flavonoid-assisted extraction of Hibiscus rosa-sinensis. Background Technology

[0002] Golden hibiscus (Abelmoschus manihot, syn.:Hibiscus manihot), a unique wild vegetable and traditional Chinese medicine, is an annual herb belonging to the Malvaceae family and the Abelmoschus genus. It experienced extinction before being rediscovered in 2003, and research on the plant is relatively limited. Golden hibiscus is rich in flavonoids, trace elements, polysaccharides, and other beneficial bioactive components and nutrients. Its high content of beneficial components, wide applicability, and good stability make it suitable for meeting people's growing health needs.

[0003] Flavonoids, as plant secondary products widely found in nature, have long been a subject of research for scholars. Their physiological activities, such as antibacterial and anti-inflammatory properties, antioxidant effects, ability to improve obesity, and prevention of cardiovascular and cerebrovascular diseases, have made them crucial in pharmaceutical raw materials and human health. Furthermore, their application as additives in food and cosmetics has become a new development direction with broad market potential. Golden sunflower is one of the plants with the highest flavonoid content, exceeding that of raw materials used in flavonoid production such as soybeans and ginkgo by several times. The highest content and the richest variety of flavonoids are found in golden sunflower flowers, with a total flavonoid content reaching 228.19 mg / g.

[0004] Plant-derived natural active substances are typically encapsulated by cell walls. Due to the tough structure, complex composition, and high mechanical strength of plant cell walls, they act as a natural barrier to these active substances, making them difficult to break down and resulting in low absorption and utilization rates. This severely hinders the transfer of active substances and solvents. Commonly used mechanical pulverization techniques, even ultrafine pulverization, only achieve a 15% breakage rate of the cell walls of traditional Chinese medicine, and the release rate of effective components is less than 40%, thus restricting the rapid development of the industry. Furthermore, traditional extraction methods for flavonoids, such as hot water reflux extraction, solvent extraction, and acid (alkali) precipitation, require large amounts of solvent, high extraction temperatures, and long reaction times, failing to meet the demands of green and low-temperature sustainable industry development. Novel extraction methods, such as ultrasound-assisted extraction, are time-efficient but consume high energy per unit volume and may alter the molecular structure of active ingredients. Microwave-assisted extraction is time-saving, efficient, and pollution-free, but requires sophisticated equipment and is difficult to scale up. Ultrafine grinding technology-assisted extraction significantly improves the extraction rate of active substances, but requires specialized equipment and incurs high costs. Enzyme-assisted extraction struggles to determine the optimal enzyme type, hydrolysis temperature, time, pH, and concentration. Therefore, effectively breaking down the cell walls of traditional Chinese medicine and improving the release rate of active ingredients, finding more efficient extraction methods for Hibiscus mutabilis flavonoids, has become a crucial opportunity to drive the rapid development of the traditional Chinese medicine industry. Summary of the Invention

[0005] The purpose of this invention is to provide a Rhizopus spp. JHK31 and its application in the flavonoid-assisted extraction of Hibiscus rosa-sinensis, in order to solve the problems existing in the prior art. This invention significantly improves the flavonoid extraction rate by pre-treating Hibiscus rosa-sinensis with bio-fermentation. Furthermore, the flavonoid extraction time of Hibiscus rosa-sinensis after fermentation is shorter, the amount of solvent used is less, the extraction conditions are milder, and the extraction efficiency is higher, which is more conducive to large-scale production.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a Rhizopus arrhizus JHK31, which was deposited at the China Center for Type Culture Collection (CCTCC) at Wuhan University, China on May 10, 2023, with accession number CCTCC NO: M 2023722.

[0008] The present invention also provides a method for improving the flavonoid extraction rate of sunflower, including the step of fermenting sunflower using the aforementioned Rhizopus japonicus JHK31.

[0009] Furthermore, the specific steps include:

[0010] Add water to the dried and pulverized golden sunflower powder until the moisture content is 30-80%, then add ammonium sulfate and glucose to obtain a fermentation medium. Then, inoculate the fermentation medium with the Rhizopus oryzae JHK31 and ferment to obtain fermented golden sunflower. Finally, extract the product using the pharmacopoeia method.

[0011] Furthermore, the amounts of ammonium sulfate and glucose added are 0.2 wt% and 2 wt%, respectively; the water content is 50 wt%.

[0012] Furthermore, the inoculum amount of Rhizopus spp. JHK31 is 7 wt%; the fermentation conditions are a fermentation temperature of 28°C, an initial fermentation pH of 6, and a fermentation time of 60 h.

[0013] Furthermore, the extraction is performed using an alcohol extraction method, and the extraction conditions include: an extraction time of 2 hours, an extraction temperature of 70°C, an ethanol concentration of 60%, and a material-to-liquid ratio of 1 g: 40 mL.

[0014] The present invention also provides an application of the aforementioned Rhizopus spp. JHK31 in the flavonoid-assisted extraction of Hibiscus rosa-sinensis.

[0015] The present invention also provides the application of the aforementioned Rhizopus spp. JHK31 in improving the antioxidant properties of the flavonoid extract of Hibiscus rosa-sinensis.

[0016] The present invention discloses the following technical effects:

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] a. This invention uses a pressure screening method to screen high-performance strains, constructs a fermentation strain library, and combines different strains with different Chinese medicines for fermentation. At the same time, fermentation optimization is carried out during the fermentation process to achieve coupled control of water and air temperature during fermentation. The resulting fermented golden sunflower is instantly soluble in water, and the effective components can be fully released.

[0019] b. The *Rhizopus spp.* enzyme JHK31 used in this invention is a dedicated fermentation strain for *Rhizopus spp.*, with accession number CCTCC NO: M2023722. Solid-state fermentation of *Rhizopus spp.* with *Rhizopus spp.* utilizes enzymes produced by *Rhizopus spp.* during its growth to degrade macromolecules in the cell wall, such as cellulose, hemicellulose, and pectin. This specifically promotes the dissolution of flavonoids from the cells. In the flavonoid extraction process, it not only overcomes the cell's anti-degradation barrier but also the mass transfer resistance of the cell wall and intercellular matrix.

[0020] c. Through pretreatment with bio-fermentation of *Hymenochloa crus-galli*, the flavonoid extraction rate can be significantly increased to 3.65%, which is much higher than the 2.61% flavonoid extraction rate of the same batch of unfermented *Hymenochloa crus-galli*. The flavonoid extraction rate after fermentation is significantly higher than that before fermentation by 39.85%. At the same time, it was found that the flavonoids after fermentation have better antioxidant properties than those before fermentation.

[0021] d. The extraction time for flavonoids from fermented golden sunflower is shorter, the amount of solvent used is less, the extraction conditions are milder, and the extraction efficiency is higher, which is more conducive to large-scale production. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 Colony morphology and microscopic morphology of JHK31, a root enzyme;

[0024] Figure 2 The mycelial growth status during the fermentation of *Hippophae rhamnoides* using the root enzyme JHK31;

[0025] Figure 3 The effect of different bacterial strains fermenting *Hippophae rhamnoides* on flavonoid extraction rate;

[0026] Figure 4 The effect of different fermentation times on the flavonoid extraction rate of *Hippophae rhamnoides*.

[0027] Figure 5 The effect of different initial fermentation pH on the flavonoid extraction rate of *Hippophae rhamnoides*.

[0028] Figure 6 The effect of different fermentation moisture contents on the flavonoid extraction rate of *Hippophae rhamnoides*.

[0029] Figure 7 The effect of different fermentation temperatures on the flavonoid extraction rate of *Hippophae rhamnoides*.

[0030] Figure 8 The effect of different inoculum amounts on the flavonoid extraction rate of *Hippophae rhamnoides*.

[0031] Figure 9 The effect of different nitrogen source (ammonium sulfate) addition amounts on the flavonoid extraction rate of *Hippophae rhamnoides*.

[0032] Figure 10 The effect of different amounts of carbon source (glucose) added on the flavonoid extraction rate of *Hippophae rhamnoides*.

[0033] Figure 11 The effect of extraction time on the flavonoid content of sunflower before and after fermentation;

[0034] Figure 12 The effect of extraction temperature on the flavonoid content of sunflower before and after fermentation;

[0035] Figure 13 The effect of ethanol concentration on the flavonoid content of sunflower before and after fermentation;

[0036] Figure 14 The effect of the solid-liquid ratio on the flavonoid content of sunflower before and after fermentation;

[0037] Figure 15 To purify and obtain the cell morphology of single strains A1, A2, A3, and A4;

[0038] Figure 16 To determine the in vitro DPPH free radical scavenging capacity of flavonoids from the alcohol extract of *Hippophae rhamnoides*.

[0039] Figure 17 To determine the in vitro hydroxyl radical scavenging capacity of flavonoids from the alcohol extract of *Hippophae rhamnoides*;

[0040] Figure 18 To determine the in vitro ABTS free radical scavenging ability of flavonoids from the alcohol extract of *Hippophae rhamnoides*.

[0041] Figure 19 To determine the total reducing power of flavonoids in the alcohol extract of *Hippophae rhamnoides* in vitro;

[0042] Figure 20 This study aimed to determine the antioxidant lifespan of flavonoids extracted from *Hymenochloa crus-galli* in vivo against *C. elegans*. Detailed Implementation

[0043] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0044] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0045] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0046] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0047] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0048] Example 1: Strain Screening

[0049] 1. Initial screening of bacterial strains

[0050] The purchased sunflower flowers (purchased from Yulin Xinbang Pharmaceutical Co., Ltd.) were dried at 65℃ to constant weight, crushed, and sieved (40 mesh). No sterilization was required. 10g of the powder was added to a sterile fermentation tank, and then sterile water was added until the moisture content reached 50% to form a fermentation medium. The tank was sealed and placed in a constant temperature incubator (28℃) for fermentation culture for 4-12 days. During the fermentation process, the growth of the colonies was observed continuously.

[0051] Under aseptic conditions, after culturing, single colonies of different morphologies were picked and placed into pre-prepared PDA medium, sealed with sterile sealing film, and further cultured at 28°C for 4–12 days. The growth status of the strains was observed and recorded.

[0052] 2. Strain purification

[0053] Under aseptic conditions, the strains with different morphologies obtained from the initial screening were streaked onto fresh PDA medium and cultured at 28°C for 4–12 days. Growth was observed and recorded, and this process was repeated until single colonies were purified. Colony and cell morphology were observed, and representative strains were selected for physiological and biochemical identification tests. The strains were then preserved in an aseptic environment for further identification. Four strains were isolated and purified, such as… Figure 15 They are named A1, A2, A3, and A4.

[0054] 3. Strain selection

[0055] Strains A1, A2, A3, and A4 (bacterial concentration 10) 8 (CFU / ml) was inoculated at 7% of the mass of *Hippophae rhamnoides* onto fermentation medium containing *Hippophae rhamnoides* flowers for fermentation culture. The flavonoid extraction rate was used as the response value. Samples were taken every 12 hours and dried in a 65℃ oven to constant weight to obtain fermented *Hippophae rhamnoides*. Extraction was performed on the fermented *Hippophae rhamnoides* samples according to the pharmacopoeia method. The dried fermented *Hippophae rhamnoides* powder was passed through a 40-mesh sieve. 1g of fermented *Hippophae rhamnoides* powder was accurately weighed, and 40ml of 60% ethanol was added. The mixture was heated under reflux for 2 hours, cooled, filtered, and placed in a 50ml volumetric flask. The residue was washed with 60% ethanol, and the washings were added to the same volumetric flask. The solution was diluted to the mark with 60% ethanol, shaken well, and the total flavonoid content in the sample was determined. Experimental results are as follows: Figure 3 As shown in Table 1, strain A4 produced the highest flavonoid content in fermented sunflower under the conditions of 28℃, 60% moisture content, and 60h fermentation, with a flavonoid extraction rate of 3.24%. Therefore, strain A4 was selected as the dedicated strain for the bio-directional cell wall disruption of sunflower. Figure 2 The image shows the growth status of *Hymenochloa crus-galli* mycelium fermented by strain A4.

[0056] Table 1. Effects of different bacterial strains on the flavonoid content of fermented golden sunflower.

[0057] A1 2.45 2.48 2.54 2.68 2.88 2.74 2.78 2.76 A2 2.37 2.33 2.66 2.79 3.12 3.07 2.94 2.85 A3 2.51 2.54 2.68 2.72 2.95 2.98 3.04 3.05 A4 2.48 2.62 2.84 3.02 3.24 3.23 3.22 3.08

[0058] 4. Identification of A4 strains

[0059] a. Observation of morphology, colony and hyphae morphology

[0060] The colony morphology of strain A4 on PDA plates was observed using the spot-inoculation method. The isolated and purified strain was cultured in PDA medium at a constant temperature of 28℃, and the morphological and color changes during colony growth were observed. The hyphal morphology was observed and photographed under a microscope using the slide culture method.

[0061] Colony morphology observation: The mycelium is relatively dense, initially white, spreading outwards from creeping hyphae. The hyphae are loosely distributed and grow rapidly. After 48 hours, it almost covers the entire plate. The hyphae are grayish-white and white spores begin to appear at the tips of the hyphae. On the 3rd day, the mycelium is grayish-brown or even black. The sporangia turn black after maturing.

[0062] Microscopic morphological observation: Under the microscope, the hyphae are well-developed, slender, and unseptate. The conidia grow in elliptical clusters with swollen tips and sporangia. The sporangia are round, the columella is round, and the sporangiophores are branched. The sporangiospores are densely packed inside the sporangiospores. The sporangiospores are elliptical to nearly round. The rhizoids are root-like and well-developed. The mycelium contains chlamydospores. Figure 1 The colony morphology and microscopic morphology of strain A4 are shown. Based on the above results and with reference to the Handbook of Fungal Identification and Fungal Taxonomy, it is preliminarily identified as Rhizopus.

[0063] 5. Preservation of microbial strains

[0064] The strain was identified as Rhizopus arrhizus JHK31, with accession number CCTCCNO: M 2023722, and was deposited at the China Center for Type Culture Collection on May 10, 2023, at Wuhan University, China.

[0065] Example 2: Optimization of the fermentation process of golden sunflower

[0066] 1. Based on the fermentation medium of Example 1, the fermentation conditions were optimized, with the flavonoid extraction rate as the response value. Single-factor experiments were conducted to optimize fermentation time, initial pH, culture temperature, inoculum size, moisture content, carbon source addition, and nitrogen source addition.

[0067] Using sunflowers from the same origin and batch as experimental materials, and using sunflowers as the sole culture medium, after sterilization, 10g of sunflower powder was taken, added to sterile water, and mixed evenly to obtain a water-containing sunflower culture medium. Ammonium sulfate and glucose were then added, along with the highly efficient cell-wall-breaking strain JHK31 of sunflower (bacterial concentration 10). 8 The mixture (cfu / ml) was thoroughly mixed and allowed to ferment statically in a constant temperature incubator. After fermentation, a sample was taken, spread evenly on a stainless steel tray, and dried in an oven at 65℃ to obtain fermented golden hibiscus. Extraction was performed according to the pharmacopoeia method. The dried fermented golden hibiscus powder was passed through a 40-mesh sieve. 1g of the fermented golden hibiscus powder was accurately weighed, and 40ml of 60% ethanol was added. The mixture was heated under reflux for 1 hour, cooled, filtered, and placed in a 50ml volumetric flask. The residue was washed with 60% ethanol, and the washings were added to the same volumetric flask. The solution was diluted to the mark with 60% ethanol, shaken well, and the total flavonoid content in the sample was determined. The total flavonoid content in the fermented golden hibiscus samples was determined by sampling every 6 hours.

[0068] The experiment was conducted sequentially according to the following parameters:

[0069] (1) Using fermentation time (12h, 24h, 36h, 48h, 60h, 72h, 84h, 96h) as the single research parameter, fermentation temperature 28℃, inoculum amount 7%, water content 60%, initial pH 5, ammonium sulfate 0.1%, and glucose 2%, the effect of different fermentation times on flavonoid extraction rate was determined. The experimental results are as follows: Figure 4 .

[0070] (2) Using 0.1 mol / L sodium dihydrogen phosphate and 0.1 mol / L disodium hydrogen phosphate to adjust the initial pH of fermentation (3, 4, 5, 6, 7, 8) as the single research parameter, the fermentation time was 60 h, the fermentation temperature was 28℃, the inoculum quantity was 7%, the water content was 60%, the ammonium sulfate was 0.1%, and the glucose was 2%. The effect of different initial pH values ​​on the flavonoid extraction rate was determined. The experimental results are as follows: Figure 5 .

[0071] (3) Using moisture content (30%, 40%, 50%, 60%, 70%, 80%) as the single research parameter, fermentation time 60 h, fermentation temperature 28℃, inoculum size 7%, initial pH 5, ammonium sulfate 0.1%, and glucose 2%, the effect of different moisture contents on flavonoid extraction rate was determined. The experimental results are as follows: Figure 6 .

[0072] (4) Using fermentation temperature (20℃, 24℃, 28℃, 32℃, 36℃, 40℃) as the single research parameter, fermentation time 60 h, inoculum amount 7%, water content 60%, initial pH 5, ammonium sulfate 0.1%, and glucose 2%, the effect of different fermentation temperatures on flavonoid extraction rate was determined. The experimental results are as follows: Figure 7 .

[0073] (5) Using inoculum size (3%, 5%, 7%, 10%, 15%, 20%) as a single research parameter, fermentation time 60 h, fermentation temperature 28℃, moisture content 60%, initial pH 5, ammonium sulfate 0.1%, and glucose 2%, the effect of different inoculum sizes on flavonoid extraction rate was determined. The experimental results are as follows: Figure 8 .

[0074] (6) Using the amount of nitrogen source (ammonium sulfate) added (0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%) as the single research parameter, with a fermentation time of 60 h, a fermentation temperature of 28 °C, an inoculum size of 7%, a moisture content of 60%, an initial pH of 5, and a glucose content of 2%, the effect of ammonium sulfate addition on the flavonoid extraction rate was determined. The experimental results are as follows: Figure 9 .

[0075] (7) Using the amount of carbon source (glucose) added (0.5%, 1%, 2%, 3%, 4%, 5%) as the single research parameter, with a fermentation time of 60 h, a fermentation temperature of 28 °C, an inoculum size of 7%, a moisture content of 60%, an initial pH of 5, and 0.1% ammonium sulfate, the effect of glucose addition on flavonoid extraction rate was determined. The experimental results are as follows: Figure 10 .

[0076] Based on the above experiments, the optimal fermentation conditions for strain JHK31 are 28℃, 7wt% inoculum, initial fermentation pH 6, 0.2wt% ammonium sulfate, 2wt% glucose, and 50wt% water content for 60h, under which the flavonoid extraction rate is the highest.

[0077] Example 3: Comparison of flavonoid content before and after fermentation of *Hippophae rhamnoides* under different extraction conditions

[0078] 1. Preparation of fermented golden sunflower: Take 10g of sterilized golden sunflower powder and ferment it with strain JHK31. Fermentation conditions: 28℃, inoculum 7%, initial fermentation pH 6, ammonium sulfate 0.2%, glucose 2%, water content 50%, fermentation time 60h. After fermentation, take a sample, spread it evenly on a stainless steel tray, and dry it in an oven at 65℃ to obtain fermented golden sunflower.

[0079] 2. Extraction conditions: Using the flavonoid extraction rate as the response value, the extraction conditions of the pharmacopoeia method were slightly adjusted. Four factors were selected: material-to-liquid ratio, extraction time, ethanol volume fraction, and extraction temperature. Under the basic conditions of 1:40, 2h, 60%, 80℃, and one extraction, the changes in flavonoid content before and after fermentation of *Hippophae rhamnoides* were determined.

[0080] (1) Take 1g of fermented and unfermented golden hibiscus powder respectively. Using extraction time (1h, 1.5h, 2h, 2.5h, 3h) as the single variable, with a material-to-liquid ratio of 1:40, ethanol concentration of 60%, and extraction temperature of 80℃, determine the change in flavonoid content before and after fermentation. The experimental results are as follows: Figure 11 .

[0081] (2) Take 1g of fermented and unfermented golden hibiscus powder respectively. Using extraction temperature (50℃, 60℃, 70℃, 80℃, 90℃) as the single variable, extraction time 2h, material-to-liquid ratio 1:40, and ethanol concentration 60%, determine the change in flavonoid content before and after fermentation. The experimental results are as follows: Figure 12 .

[0082] (3) Take 1g of fermented and unfermented golden hibiscus powder respectively. Using ethanol concentration (40%, 50%, 60%, 70%, 80%) as the single variable, the extraction time is 2h, the material-to-liquid ratio is 1:40, and the extraction temperature is 80℃. Measure the change in flavonoid content before and after fermentation. The experimental results are as follows: Figure 13 .

[0083] (4) Take 1g of fermented and unfermented golden hibiscus powder respectively. Using the material-to-liquid ratio (1:30, 1:35, 1:40, 1:45, 1:50 (g / ml)) as the single variable, the extraction time is 2h, the ethanol concentration is 60%, and the extraction temperature is 80℃. The changes in flavonoid content before and after fermentation are measured. The experimental results are as follows: Figure 14 .

[0084] The results showed that under different extraction conditions, the flavonoid content after fermentation was generally higher than that before fermentation, proving the feasibility of the experiment. Using the optimized extraction conditions, the flavonoid extraction rate could be significantly increased to 3.65%, far exceeding the 2.61% flavonoid extraction rate of the same batch of unfermented *Hibiscus rosa-sinensis*. The flavonoid extraction rate after fermentation was significantly increased by 39.85% compared to the flavonoid extraction rate before fermentation (see Table 2).

[0085] Table 2

[0086] Before fermentation 2.66％ 2.51％ 2.66％ 2.61％ After fermentation 3.71％ 3.54％ 3.70％ 3.65％

[0087] Example 4: Determination of the in vitro antioxidant activity of flavonoids from the ethanol extract of *Hippophae rhamnoides*

[0088] The unfermented holly extract extracted under preferred conditions in Example 3 was named UEE (Unfermented Ethanol Extract), and the fermented holly extract was named FEE (Fermented Ethanol Extract). Vitamin C was used as a positive control in the following experiments.

[0089] Determination of 1DPPH free radical scavenging ability

[0090] Take 2.5 ml of sample solutions with concentrations of 0.2, 0.4, 0.6, 0.8, and 1.0 mg / ml into test tubes, add 2.5 ml of DPPH solution, mix well, and incubate at room temperature in the dark for 30 min. Measure the absorbance of the solution at 517 nm. The formula for the scavenging rate of DPPH free radicals is:

[0091]

[0092] In the formula, A2 is the absorbance of the sample experimental group; A1 is the absorbance of the control group with distilled water instead of DPPH solution; and A0 is the absorbance of the blank group with a mixture of distilled water and DPPH solution.

[0093] Experimental results are as follows Figure 16 As shown in the figure, when the concentration of the flavonoid extract from *Hippophae rhamnoides* increased from 0.2 mg / ml to 1.0 mg / ml, the DPPH free radical scavenging rate of fermented *Hippophae rhamnoides* flavonoids increased from 25.12% to 88.43%, showing a linear increase. When the concentration reached 1.0 mg / ml, its antioxidant effect reached its maximum, being 0.93 times that of ascorbic acid. Furthermore, at the same flavonoid concentration, the DPPH free radical scavenging rate of fermented *Hippophae rhamnoides* flavonoids was higher than that of unfermented fermented *Hippophae rhamnoides* flavonoids, indicating that fermentation effectively increases the antioxidant properties of flavonoids.

[0094] Determination of 2-hydroxyl radical scavenging capacity

[0095] Take 2.5 mL of sample solutions with concentrations of 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL into test tubes, respectively. Add 2.5 mL each of 6 mmol / L FeSO4 solution and H2O2 solution sequentially. Shake well and let stand for 10 min. Then add 2.5 mL of 6 mmol / L salicylic acid, shake well, and let stand at room temperature in the dark for 30 min. Measure the absorbance at 510 nm. The formula for the scavenging rate of hydroxyl radicals is:

[0096]

[0097] In the formula, A2 is the absorbance of the experimental sample group; A1 is the absorbance of the control group with distilled water instead of salicylic acid; and A0 is the absorbance of the blank group with distilled water instead of the sample solution.

[0098] Experimental results are as follows Figure 17 As shown in the figure, the extract of flavonoids from *Hippophae rhamnoides* has a certain ability to scavenge hydroxyl radicals. Moreover, the scavenging rate of hydroxyl radicals increases linearly with the increase of concentration. At different concentrations, the scavenging rate of hydroxyl radicals by fermented *Hippophae rhamnoides* flavonoids is always greater than that by unfermented *Hippophae rhamnoides* flavonoids. When the concentration is 1.0 mg / ml, the scavenging rates of hydroxyl radicals by fermented and unfermented *Hippophae rhamnoides* flavonoids reach the maximum values ​​of 57% and 52%, respectively.

[0099] Determination of 3ABTS free radical scavenging ability

[0100] Mix equal volumes of 7 mmol / L ABTS solution and 14 mmol / L persulfate solution thoroughly, and incubate in the dark for 24 hours. Adjust the absorbance to 0.7 ± 0.02 with distilled water to obtain the working solution. Take 2.5 ml of sample solutions with concentrations of 0.2, 0.4, 0.6, 0.8, and 1.0 mg / ml into test tubes, add 2.5 ml of the ABTS working solution, mix thoroughly, and incubate at room temperature in the dark for 15 minutes. Measure the absorbance of the solution at 734 nm.

[0101]

[0102] In the formula, A2 is the absorbance of the sample experimental group; A1 is the absorbance of the control group with distilled water instead of ABTS working solution; and A0 is the absorbance of the blank group with distilled water instead of sample solution.

[0103] Experimental results are as follows Figure 18 As shown in the figure, golden hibiscus flavonoids have an effect on ABTS. + The free radical scavenging rate was positively correlated with the mass concentration, and at the same concentration, the scavenging rate of fermented *Hippophae rhamnoides* flavonoids was greater than that of unfermented *Hippophae rhamnoides* flavonoids. When the mass concentration of *Hippophae rhamnoides* flavonoids was 1 mg / ml, the scavenging rate of fermented *Hippophae rhamnoides* flavonoids against ABTS was significantly higher. +The free radical scavenging rate reached a maximum of 92%.

[0104] 4. Determination of total reducing power

[0105] Take 1.0 ml of sample solutions with concentrations of 0.2, 0.4, 0.6, 0.8, and 1.0 mg / ml into test tubes, respectively. Add 2.5 ml each of phosphate buffer (pH 6.6) and 1% potassium ferricyanide solution sequentially. Mix well and incubate at 50°C for 20 minutes. Then add 2.5 ml of 10% trichloroacetic acid, mix well, and centrifuge at 3000 rpm for 10 minutes. Take 2.5 ml of the supernatant and place it in a test tube. Add 0.5 ml of 0.1% ferric chloride solution and 2.5 ml of distilled water, shake well, and let stand for 10 minutes. Take an appropriate amount and measure the absorbance at 700 nm. VC is used as a positive control. Total reducing power formula:

[0106] Total reducing capacity = A2 - A1;

[0107] In the formula, A2 is the absorbance of the sample experimental group; A1 is the absorbance of the blank group with distilled water replacing the sample solution.

[0108] Experimental results are as follows Figure 19 As shown in the figure, the total antioxidant capacity of the two types of golden hibiscus flavonoid extracts increased with the increase of the mass concentration of the extract. When the flavonoid mass concentration was 1 mg / ml, the total antioxidant capacity of both reached the maximum, and the total antioxidant capacity of fermented golden hibiscus flavonoids was higher than that of unfermented golden hibiscus flavonoids.

[0109] Determination of the antioxidant lifespan of flavonoids extracted from 5 golden sunflower alcohol extracts against Caenorhabditis elegans in vivo

[0110] Nematode lifespan experiment

[0111] To synchronize nematodes, lysed eggs were hatched overnight and then evenly added to plates in each group. The plates were incubated at 20°C for 48 hours to obtain synchronized nematodes that had grown to the L4 stage. The experiment consisted of a control group and an experimental group. The control group was treated with OP50 bacterial suspension without sample extract spread on the culture medium. The experimental group received 1.0 mg / mL of unfermented holly extract (UEE) and fermented holly extract (FEE) after filtration sterilization, and then mixed with E. coli OP50 bacterial suspension (OD). 600=1) Mix equal volumes, and spread 100 μL onto NGM plates to feed nematodes (all culture media contain 0.25 mg / mL Fudr). Each group contains 30 nematodes, initially counted as day 0. Every 24 hours, transfer the nematodes to a new plate corresponding to the experimental group, recording the daily survival and mortality counts until all nematodes die. Record the number of dead and missing nematodes in each plate. Nematodes that die abnormally due to climbing or damage caused by handling errors can be considered missing. Repeat the experiment three times, and finally calculate the average lifespan using statistical data to plot a survival curve.

[0112] like Figure 20 As shown, compared with the control group, feeding *C. elegans* with both unfermented and fermented *Haloxylon ammodendron* flavonoid extract shifted the lifespan curve to the right, with average lifespans extended by 14.87% and 20.55%, respectively, indicating that a certain concentration of *Haloxylon ammodendron* flavonoid extract can effectively delay nematode aging. Fermented *Haloxylon ammodendron* flavonoid extract (FEE) was even more effective in extending the lifespan of *C. elegans*, exceeding that of unfermented *Haloxylon ammodendron* flavonoid extract (UEE) by 4.93%. The maximum lifespans of nematodes in the control group, unfermented group, and fermented group reached 19 days, 22 days, and 24 days, respectively. Compared with the control group, the maximum lifespans of nematodes in the unfermented and fermented groups were extended by 15.79% and 26.32%, respectively.

[0113] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A type of Rhizopus with few roots ( Rhizopus arrhizus JHK31, characterized in that, It was deposited on May 10, 2023 at the China Center for Type Culture Collection, located at Wuhan University, China, with accession number CCTCC NO: M 2023722.

2. A method for improving the extraction rate of flavonoids from *Hippophae rhamnoides*, characterized in that, The step includes fermenting golden sunflower using Rhizopus japonicus JHK31 as described in claim 1.

3. The method according to claim 2, characterized in that, Specifically, the following steps are included: Water was added to the dried and pulverized golden sunflower powder until the moisture content was 30-80%. Ammonium sulfate and glucose were then added to obtain a fermentation medium. Rhizopus japonicus JHK31 was then inoculated into the fermentation medium for fermentation to obtain fermented golden sunflower. Finally, extraction was performed.

4. The method according to claim 3, characterized in that, The amounts of ammonium sulfate and glucose added are 0.2 wt% and 2 wt%, respectively; the water content is 50 wt%.

5. The method according to claim 3, characterized in that, The inoculum size of Rhizopus spp. JHK31 was 7 wt%; the fermentation conditions were a fermentation temperature of 28 °C, an initial fermentation pH of 6, and a fermentation time of 60 h.

6. The method according to claim 3, characterized in that, The extraction was performed using an alcohol extraction method, with the following extraction conditions: extraction time of 2 hours, extraction temperature of 70 °C, ethanol concentration of 60%, and a solid-liquid ratio of 1 g: 40 mL.

7. The application of Rhizopus septemlobus JHK31 as described in claim 1 in improving the antioxidant properties of Hibiscus mutabilis flavonoid extract.

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