Mucor hy6-19 and its use in preparing extract of jujube seed

Abstract

The application discloses a mucor HY6-19 and application of the mucor HY6-19 in preparation of extract of red date seed, and the mucor HY6-19 is obtained by targeted separation and screening and mutagenesis, and can grow moderately in red date seed powder added with sucrose, produce cellulase with higher activity and other various hydrolytic enzymes, then, the fermented red date seed powder is added with water and kept warm, cellulose, hemicellulose and pectin and other substances in the cell wall are enzymolyzed, the substances combined with cellulose in the cell wall and intracellular substances are dissolved out, the yield of the extract is significantly improved, and the total flavone content is also significantly improved. Before preparation of the extract of the red date seed by the hot water ultrasonic method, the microbial fermentation treatment is added, compared with the direct hot water ultrasonic extraction method, the yield of the extract of the red date seed can be increased by 34.4%, and the total flavone content is increased by 23.3%.

Description

(I) Technical Field

[0001] This invention belongs to the field of biotechnology in the extraction of effective components from plants, specifically involving a strain of Mucor HY6-19 and its application in the preparation of Hovenia dulcis extract. (II) Background Technology

[0002] Hovenia dulcis, Hovenia acerba, and Hovenia trichocarpa are the mature seeds of plants belonging to the genus Hovenia in the family Rhamnaceae. Hovenia trichocarpa is mainly distributed in Zhejiang, Jiangxi, Hubei, Hunan, Guangdong, and Guizhou provinces. Hovenia dulcis is a medicinal and edible herb with the effects of clearing heat and promoting diuresis, and detoxifying alcohol. It is mainly used to treat symptoms such as alcohol poisoning, fever, thirst, vomiting, and difficulty in urination and defecation. There is a folk saying, "Hovenia dulcis can keep you from getting drunk even after a thousand cups of wine." The ancient medical book *Yifang Kao* also records that "Hovenia dulcis (also called chicken spur seed) is more effective in relieving alcohol poisoning than kudzu flower. It should be used for anyone suffering from alcohol poisoning." Hovenia dulcis contains a large number of bioactive components, including polysaccharides, flavonoids, triterpenes, and alkaloids. Modern medical research has also proven that Hovenia dulcis extract can accelerate the metabolism of ethanol in the human body, reduce blood alcohol concentration after drinking, enhance the activity of liver alcohol dehydrogenase, reduce the risk of liver lipid peroxidation, and reduce liver damage caused by ethanol. It has the effects of detoxifying alcohol, quenching thirst and relieving irritability, stopping vomiting, and promoting urination and defecation.

[0003] Currently, there are numerous reports on methods for preparing Hovenia dulcis extract. Based on the solvent used, these methods can be divided into two categories: water extraction and organic solvent extraction. The composition of substances extracted by different solvents varies significantly. Water extracts mainly contain water-soluble substances such as polysaccharides, oligosaccharides, and amino acids; organic solvent extraction primarily uses ethanol or ethyl acetate, with ethanol being the most common, and the extract mainly contains flavonoids, triterpenes, and alkaloids. In addition to these two methods, ultrasonic, microwave, and enzymatic extraction methods can be used to enhance the extraction yield. Ultrasonic-assisted extraction has low equipment requirements and does not significantly increase extraction costs, but its effect on improving extraction yield is not ideal; microwave-assisted extraction is time-efficient and highly effective, but the high internal temperature often damages the structure of the extracted active ingredients; enzymatic extraction uses cellulase, pectinase, or protease to hydrolyze plant materials under suitable conditions, causing cell wall hydrolysis, which facilitates the dissolution of intracellular active ingredients during extraction. Enzymatic hydrolysis not only significantly improves extraction yield, but also operates under mild conditions and does not damage the structure of active ingredients in the extract. Furthermore, enzymatic hydrolysis can reduce the molecular weight of polysaccharides and convert some glycosides into aglycones, resulting in better bioactivity of the extract. Therefore, in recent years, enzymatic hydrolysis has been increasingly used in the extraction of plant active ingredients.

[0004] Microorganisms can produce a variety of enzymes that hydrolyze plant tissues; plant debris and decaying leaves in nature are decomposed by these enzymes. In particular, some molds in nature can produce a variety of enzymes, including cellulase, hemicellulase, ligninase, pectinase, and protease. Therefore, if a suitable mold is used to directly ferment and pre-treat Hovenia dulcis seeds, and the growth rate is controlled to achieve enzyme decomposition of the cell walls without breaking down the active ingredients, the extraction yield can be increased. Compared to enzymatic extraction, the various enzymes produced by microorganisms have a better hydrolytic effect on plant cell walls. Furthermore, polysaccharides are appropriately degraded, resulting in lower molecular weight and increased activity. Some flavonoid glycosides are hydrolyzed, releasing flavonoid aglycones, leading to better bioactivity of the extract.

[0005] To improve the yield and bioactivity of Hovenia dulcis extract, this invention involves microbial fermentation of Hovenia dulcis followed by hot water ultrasonic extraction, which increases the extract yield and significantly increases the total flavonoid content. (III) Summary of the Invention

[0006] The purpose of this invention is to provide a new microbial strain—Mucor HY6-19 and its application in the preparation of Hovenia dulcis extract. By applying microbial fermentation technology to the preparation of Hovenia dulcis extract, the Hovenia dulcis seeds are fermented by microorganisms and then extracted by hot water and ultrasound, resulting in a significant increase in the extract yield and the total flavonoid content.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] This invention provides a novel microbial strain—Mucor sp. HY6-19, deposited at the Guangdong Provincial Center for Microbial Culture Collection, accession number GDMCC No:63391, deposit date April 24, 2023, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province; postcode 510070.

[0009] The *Mucor* HY6-19 strain described in this invention is an excellent strain for fermenting *Hovenia dulcis* seeds, isolated from soil and obtained through screening and mutagenesis. The morphological characteristics of *Mucor* HY6-19 are as follows: When cultured on potato dextrose agar (PDA) plates at 28°C, the initial mycelium is grayish-white; after one day, the gray color deepens slightly, the mycelium becomes longer and fluffy, and a small number of gray spores are produced on the surface. Under an optical microscope, conidiophores are observed to be solitary, densely layered, erect, and all borne terminal sporangia. The sporangia are large, spherical, and grayish-brown, and the sporangiospores are spherical, with a diameter of 4–5 μm. A colony photograph of *Mucor* HY6-19 cultured on PDA plates at 28°C for 2 days is shown below. Figure 1 .

[0010] The nucleotide sequence of the ribosomal DNA internal transcribed spacer (rDNA-ITS) of the *Mucor* HY6-19 is shown in SEQ ID NO.1.

[0011] The present invention also provides an application of the aforementioned *Mucor* sp. HY6-19 in the preparation of *Hovenia dulcis* extract, the method of application being as follows: (1) inoculating *Mucor* sp. HY6-19 spore liquid into *Hovenia dulcis* powder, stirring evenly, and fermenting at 30–32℃ for 52–60h to obtain *Hovenia dulcis* fermented product; (2) adding deionized water to the *Hovenia dulcis* fermented product, stirring evenly, and keeping warm at 35–40℃ for 4–6h, then extracting by hot water ultrasonication and filtering, concentrating the filtrate to obtain *Hovenia dulcis* aqueous extract concentrate; (3) vacuum drying the *Hovenia dulcis* aqueous extract concentrate, pulverizing it, and obtaining *Hovenia dulcis* extract.

[0012] Furthermore, the Hovenia dulcis seeds in step (1) are mature seeds of Hovenia acerba, Hovenia trichocarpa, or Hovenia dulcis plants of the Rhamnaceae family; Hovenia dulcis seed powder is fine powder obtained by drying Hovenia dulcis seeds at 85°C and then pulverizing them through a 60-mesh sieve.

[0013] Further, the preparation method of the *Mucor HY6-19* spore solution in step (1) is as follows: *Mucor HY6-19* spores preserved at low temperature are inoculated onto potato dextrose agar (PDA) plates and cultured at 30°C for 48–72 h. Then, sterile sucrose aqueous solution is added to the culture, and the spores are suspended by stirring with an inoculation loop to obtain the spore solution; preferably, the spore concentration is adjusted to 5 × 10⁻⁶ using sterile sucrose aqueous solution. 6 –9×10 6 The concentration of sucrose in the sterile sucrose aqueous solution is 10–15 g / L, and it is sterilized by autoclaving at 115°C for 15 min. The PDA plate culture medium is a commercially available potato dextrose agar medium (Qingdao Haibo Biotechnology Co., Ltd.), prepared with tap water at a concentration of 46 g / L, with natural pH, and sterilized by autoclaving at 121°C for 15 min.

[0014] Furthermore, the volume of the spore liquid of *Rhizopus HY6-19* used in step (1) is 2–3 mL / g based on the mass of *Hovenia dulcis* powder.

[0015] Further, the preparation method of the aqueous extract concentrate of Hovenia dulcis in step (2) is as follows: add deionized water to the Hovenia dulcis ferment, stir evenly, keep warm at 35–40℃ for 4–6 hours, then transfer to an ultrasonic cleaner at 75–85℃, and ultrasonically extract at 160–200W for 60–80 minutes. After ultrasonic extraction, filter while hot with a 200-mesh filter cloth, and concentrate the filtrate under reduced pressure at 60℃ and -0.1MPa to 1 / 10–1 / 25 of the original volume to obtain the aqueous extract concentrate of Hovenia dulcis. The volume of deionized water added is 20–25 mL / g based on the mass of Hovenia dulcis powder before fermentation.

[0016] Furthermore, the preparation method of the Hovenia dulcis extract in step (3) is as follows: the concentrated aqueous extract of Hovenia dulcis is dried under vacuum at 65°C and –0.1MPa to constant weight, and then pulverized to obtain the Hovenia dulcis extract.

[0017] Compared with existing technologies, the beneficial effects of this invention are mainly reflected in the following aspects: Firstly, this invention targets and isolates microorganisms capable of hydrolyzing the cell walls of *Hovenia dulcis* seeds, and obtains a new microorganism, *Mucor* HY6-19, through mutagenesis. *Mucor* HY6-19 grows moderately in *Hovenia dulcis* powder with added sucrose, producing highly active cellulase and various other hydrolytic enzymes. Subsequently, the fermented *Hovenia dulcis* powder is kept warm with water, and substances such as cellulose, hemicellulose, and pectin in the cell walls are enzymatically hydrolyzed, facilitating the dissolution of substances bound to cellulose in the cell walls and intracellular substances, significantly increasing the extract yield and total flavonoid content. This invention adds microbial fermentation treatment before preparing *Hovenia dulcis* extract using the hot water ultrasonic extraction method, which, compared to directly using the hot water ultrasonic extraction method, increases the extract yield by 34.4% and the total flavonoid content by 23.3%. (iv) Description of the attached drawings

[0018] Figure 1 This is a photograph of the colony morphology of Mucor HY6-19 cultured on a PDA at 28°C for 2 days.

[0019] Figure 2 A standard curve for spectrophotometric determination of total flavonoids (rutin as standard).

[0020] Figure 3 A standard curve for the determination of reducing sugars using the DNS method (glucose as the standard). (V) Detailed Implementation

[0021] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0022] The Hovenia acerba seeds used in this embodiment of the invention are mature seeds of the plant Hovenia acerba, belonging to the Rhamnaceae family; Hovenia acerba seed powder is fine powder obtained by drying Hovenia acerba seeds at 85°C and then pulverizing them through a 60-mesh sieve.

[0023] The yield of the Hovenia dulcis extract described in this embodiment of the invention is calculated according to Formula 1:

[0024]

[0025] Example 1: Isolation and screening of microbial strains for fermenting Hovenia dulcis seeds

[0026] The microbial strains for fermenting Japanese raisin tree seeds were isolated and screened according to the following steps:

[0027] (1) Take 1g of loose, moist soil from a woodland with abundant leaf litter and dilute it with sterile physiological saline at a concentration of 1×10⁻⁶. -4 1×10 -5 1×10 -6 1×10 -7 After dilution, 0.1 mL of the diluted solution was spread onto potato dextrose agar (PDA) plates and incubated at 30°C for 48 h. Colonies of different colors and morphologies were then transferred to fresh PDA plates and incubated at 30°C for 72 h to obtain 10 pure cultured mold strains. The strain numbers are shown in Table 1.

[0028] (2) 10 mL of sterile sucrose solution was added to each of the 10 strains' fresh plate cultures. The spores were suspended by stirring with an inoculation loop. The spore solutions were then transferred to sterile test tubes, and the spore concentration was adjusted with sterile sucrose solution to ensure that the spore solutions of different strains were at a concentration of 5 × 10⁻⁶. 6 –9×10 6 The spore concentration of each strain was within the range of [number] spores / mL. The sterile sucrose aqueous solution had a concentration of 15 g / L and was sterilized by high-pressure steam at 115°C for 15 min.

[0029] (3) Add 5g of Japanese raisin tree powder to 10 150-mL Erlenmeyer flasks that have been dry heat sterilized at 160℃ for 2h, and then add 15mL of the mold spore liquid prepared in step (2) to each flask (the volume is 3mL / g based on the mass of Japanese raisin tree powder). After stirring evenly, seal the Erlenmeyer flasks with 8 layers of gauze and incubate at 30℃ for 60h to obtain Japanese raisin tree ferment.

[0030] (4) In step (3), add 100 mL of deionized water to all the fermented Hovenia dulcis products [material-to-liquid ratio of 1:20 (g:mL)], stir well, and keep warm in a 35℃ water bath for 6 hours. Then transfer to an ultrasonic cleaner at 75℃ and extract with ultrasonic power at 160W for 80 minutes. After ultrasonic extraction, filter the solution while it is still hot through a 200-mesh filter cloth and collect the filtrate.

[0031] (5) The filtrate obtained in step (4) was concentrated under reduced pressure at 60℃ and –0.1MPa to 10mL (1 / 10 of the original volume) to obtain a concentrated aqueous extract of Hovenia dulcis. The concentrated aqueous extract was dried under vacuum at 65℃ and –0.1MPa to constant weight, and then pulverized to obtain Hovenia dulcis extract.

[0032] Starting from step (3), 15 mL of sterile sucrose aqueous solution (concentration 15 g / L) was added to 5 g of Hovenia dulcis powder, and Hovenia dulcis extract was prepared according to steps (3)–(5) as a blank fermentation control without inoculation with mold; starting from step (4), 5 g of Hovenia dulcis was added to 100 mL of deionized water and extracted directly according to steps (4) and (5) to prepare Hovenia dulcis extract as an unfermented control. The yield and total flavonoid content of the Hovenia dulcis extracts fermented with different strains and the control are shown in Table 1.

[0033] Table 1. Extract yield and total flavonoid content of Hovenia dulcis seeds fermented with different strains and control.

[0034]

[0035] As shown in Table 1, the blank fermentation control, which had sterile sucrose solution but no mold spores, showed a 7.38% higher extract yield than the unfermented control due to the growth of a small amount of miscellaneous bacteria, but the total flavonoid content of the extract decreased by 6.09%. After fermentation with most strains, the extract yield of Hovenia dulcis did not significantly increase. Fermentation with strains HY4, HY6, and HY9 increased the extract yield by 12.3%, 13.1%, and 14.8%, respectively. However, the extract fermented with HY6 showed a higher total flavonoid content, increasing by 9.57% compared to the unfermented control (11.5%). Therefore, strain HY6 was selected as the fermentation strain for Hovenia dulcis in this invention.

[0036] The PDA plate culture medium is a ready-made potato dextrose agar medium (Qingdao Haibo Biotechnology Co., Ltd.), prepared with tap water at a concentration of 46 g / L, with natural pH, placed in an Erlenmeyer flask, sealed with 8 layers of gauze, sterilized by high-pressure steam at 121℃ for 15 min, and poured into sterile petri dishes with a diameter of 9 cm before solidification, 15-20 mL per dish.

[0037] The total flavonoid content in the Hovenia dulcis extract was determined by spectrophotometry. The specific method was as follows: the Hovenia dulcis extract was prepared into a 0.5 mg / mL aqueous solution using deionized water. After centrifugation at 5000 r / min for 5 min, 2.5 mL of the supernatant was transferred to a 10-mL graduated test tube. 0.4 mL of 5% sodium nitrite (NaNO2) aqueous solution was added, and the mixture was allowed to stand for 6 min. Then, 0.6 mL of 10% aluminum nitrate [Al(NO3)3] aqueous solution was added, and the mixture was allowed to stand for 6 min. Next, 2 mL of 20% sodium hydroxide (NaOH) aqueous solution was added, and the volume was adjusted to 10 mL with 60% ethanol aqueous solution. The mixture was shaken well and allowed to stand for 15 min. The absorbance (A) at a wavelength of 510 nm was then measured using a spectrophotometer. 510 The total flavonoid concentration in the sample is calculated from the rutin standard curve, and then the total flavonoid mass is obtained by multiplying the concentration by the volume of the sample.

[0038] Construction of the rutin standard curve: Prepare a 0.25 g / L rutin standard solution using a 60% ethanol aqueous solution. Take 0, 0.5, 1.0, 1.5, 2.0, and 2.5 mL of the rutin standard solution into 10-mL graduated test tubes, respectively, and bring the volume to 2.5 mL with 60% ethanol aqueous solution. Add 0.4 mL of 5% sodium nitrite aqueous solution, let stand for 6 min, add 0.6 mL of 10% aluminum nitrate aqueous solution, let stand for 6 min, add 2 mL of 20% sodium hydroxide aqueous solution, and then dilute to 10FeSO4 mL with 60% ethanol aqueous solution. Shake well, let stand for 15 min, and measure the absorbance at 510 nm using a spectrophotometer (A). 510 Plotting rutin concentration on the x-axis, A 510 Plot a standard curve for the ordinate ( Figure 2 ).

[0039] Example 2: Microbial strain mutagenesis and selection for fermenting Japanese raisin tree seeds

[0040] The strain HY6 screened in Example 1 was subjected to mutagenesis breeding to screen strains with excellent fermentation performance. The specific method is as follows:

[0041] (1) Preparation of spore suspension: After activating strain HY6 on PDA agar at 30℃ for 48 h, 5 mL of sterile physiological saline was added, and the spores were suspended by stirring with an inoculation loop. 1 mL of the spore suspension was transferred to an Erlenmeyer flask containing 50 mL of sterile physiological saline (with 20–30 glass beads added), and shaken at room temperature for 15 min. The spore suspension was filtered to remove mycelia (a small wad of fluffy cotton was placed at the bottom of the Erlenmeyer funnel). The spores in the spore suspension were counted under a microscope using a hemocytometer. The spores were then diluted appropriately with sterile physiological saline to adjust the spore count to 1.34 × 10⁻⁶. 7 per mL.

[0042] (2) Mutagenesis: Under red light illumination, 1.5 mL of the above spore solution and a sterile paperclip were placed in six 6 cm diameter petri dishes. The petri dishes were placed on magnetic stirrers and irradiated for 1, 2, 3, 4, 5, and 6 mins respectively at a distance of 30 cm from a 15W UV lamp that had been preheated for 30 min. 0.5 mL of the irradiated spore solution was diluted appropriately with sterile physiological saline, and 0.1 mL was transferred to each dish and spread onto PDA agar plates. The same procedure was performed to dilute and spread un-UV-irradiated spore solution onto plates as a control to calculate the lethality rate. After inoculation, the PDA plates were wrapped in black cloth, inverted, and incubated at 28℃ for 48 h. The colonies on the plates were counted, and the lethality rate was calculated.

[0043] (3) Screening: Colonies with a lethality rate of over 90% on PDA plates were transferred to fresh PDA plates and cultured at 30℃ for 72 h to obtain 50 strains. 10 mL of sterile physiological saline was added to the plate culture of each strain, and the spores were suspended by stirring with an inoculation loop to obtain the spore liquid of each strain. 2.5 mL of the spore liquid of each strain was inoculated into 50 mL of enzyme-producing medium and cultured at 30℃ and 200 r / min for 72 h. The fermentation broth was then filtered using a Buchner funnel, and the filtrate (i.e., crude enzyme solution) was collected. The cellulase activity of the fermentation filtrate of each strain was measured. Fifteen strains with relatively higher enzyme activity than the original strain HY6 were selected. Following the method in Example 1, the spore liquid of these strains was used to inoculate Hovenia dulcis powder for fermentation, followed by hot water ultrasonic extraction. The yield and total flavonoid content of the extracts from Hovenia dulcis fermented by the mutant strains and the control are shown in Table 2.

[0044] Table 2. Extract yield and total flavonoid content of Hovenia dulcis seeds fermented with mutant strains and control strains.

[0045]

[0046] As shown in Table 2, among the 15 screened strains, strain HY6-19 exhibited a cellulase activity of 62.7 U / mL during fermentation, which was 22.3% higher than the 51.3 U / mL of the wild-type strain HY6. After fermenting Hovenia dulcis seeds with this strain, the extract yield was 15.3%, which was 12.5% ​​higher than the 13.6% of the wild-type strain HY6 and 25.4% higher than the 12.2% of the unfermented control. The total flavonoid content in the extract was 13.5%, which was 7.14% higher than the 12.7% of the wild-type strain HY6 and 17.4% higher than the 11.5% of the unfermented control. Therefore, strain HY6-19 was selected as the microbial strain for fermenting Hovenia dulcis seeds in this invention.

[0047] The enzyme-producing culture medium consists of: 50 g / L wheat bran, 6 g / L (NH4)2SO4, 4 g / L peptone, 2 g / L KH2PO4, 1 g / L MgSO4·7H2O, and 0.5 g / L CaCl2, with tap water as the solvent and a pH of 6.0. 50 mL of the enzyme-producing culture medium is placed in a 250 mL Erlenmeyer flask, sealed with eight layers of gauze, and sterilized by autoclaving at 121°C for 20 minutes.

[0048] The cellulase activity assay was performed as follows: 1.5 mL of 10 g / L sodium carboxymethyl cellulose solution (pH 6.0, prepared with 0.2 mol / L phosphate buffer) and 0.5 mL of crude enzyme solution were added to 10 mL graduated test tubes. The tubes were incubated in a 50°C water bath for 30 min, then 3 mL of DNS reagent was added, and the mixture was boiled for 5 min. After cooling under running water, deionized water was added to bring the volume to 10 mL, and the mixture was stirred thoroughly. A crude enzyme solution inactivated by boiling at 100°C for 10 min was used as a reference. The absorbance (A) was measured at 540 nm using a spectrophotometer. 540 ), from the glucose standard curve ( Figure 3 Calculate the glucose concentration in the sample, and then calculate the cellulase activity (U / mL). Cellulase activity is defined as the amount of enzyme required to hydrolyze sodium carboxymethyl cellulose to produce 1 μg of glucose per minute under pH 6.0 and 50℃ conditions; one unit of enzyme activity (U) is defined as this amount of enzyme.

[0049] Cellulase activity is calculated according to Formula 2.

[0050]

[0051] In Formula 2, C: glucose concentration (μg / mL) calculated from the standard curve; V1: enzyme reaction system volume, i.e., 2 mL; T: reaction time, i.e., 30 min; V2: crude enzyme solution volume, i.e., 0.5 mL.

[0052] Construction of the glucose standard curve: Add 0, 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mL of 1 mg / mL standard glucose aqueous solution to seven 10-mL graduated test tubes, respectively. Then add 2.0, 1.8, 1.6, 1.4, 1.2, 1.0, and 0.8 mL of pH 6.0, 0.2 mol / L phosphate buffer to each tube, respectively. Add 3.0 mL of DNS solution to each tube. Boil the mixture in a water bath for 5 minutes, cool under running water, and then dilute to 10 mL with deionized water. Stir well and measure A using a spectrophotometer. 540 Plotting glucose concentration on the x-axis, A 540 Plot a standard curve for the ordinate ( Figure 3 ).

[0053] Preparation of DNS reagent: Add 6.3g of 3,5-dinitrosalicylic acid and 262mL of 2mol / L NaOH aqueous solution to 500mL of hot aqueous solution containing 182g of sodium tartrate, then add 5g of redistilled phenol and 5g of sodium sulfite, stir to dissolve, cool, and add deionized water to make up to 1L. Store in a brown bottle and use after 7 days.

[0054] Example 3: Classification and identification of strain HY6-19

[0055] Strawberry strain HY6-19 was streaked onto PDA plates and cultured at 28°C. Initially, the mycelium was grayish-white; after one day, the gray color deepened slightly, the mycelium became longer and more fluffy, and a small number of gray spores were produced on the surface. Under an optical microscope, conidiophores were observed to be solitary, densely layered, erect, and all bearing terminal sporangia. The sporangia were large, spherical, and grayish-brown, and the sporangiospores were spherical, 4–5 μm in diameter. A colony photograph of *Mucor* HY6-19 cultured on PDA plates at 28°C for 2 days is shown below. Figure 1 .

[0056] The rDNA-ITS nucleotide sequence of strain HY6-19 was determined as shown in SEQ ID NO.1. This sequence was BLAST-aligned using NCBI (National Center for Biotechnology Information, https: / / www.ncbi.nlm.nih.gov). Only two strains showed homology greater than 95%: *Mucor lusitanicus* CBS 108.17 (96.56%) and *Mucor phayaoensis* MFLUCC. Based on the ITS sequence alignment results (21-0044, 95.50%), it can be determined that strain HY6-19 is a Mucor strain. However, HY6-19 cannot yet be classified as a species. Therefore, HY6-19 is classified as (refer to NCBI, http: / / www.ncbi.nlm.nih.gov) a species (Mucor sp.) within the kingdom Fungi, phylum Mucoromycota, subphylum Pezizomycotina, class Mucoromycetes, order Mucorales, family Mucoraceae, and genus Mucor.

[0057] The rDNA-ITS nucleotide sequence of the strain HY6-19 is as follows:

[0058] .

[0059] In summary, microbial strain HY6 was isolated from the soil. After ultraviolet mutagenesis, strain HY6-19, namely Mucor sp. HY6-19, was obtained for fermenting Hovenia dulcis seeds. This strain is deposited at the Guangdong Provincial Microbial Culture Collection Center, accession number GDMCC No:63391, deposit date April 24, 2023, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510070, China.

[0060] Example 4: Application of Mucor HY6-19 in the preparation of Hovenia dulcis extract 1

[0061] The application of *Mucor HY6-19* in the preparation of *Hovenia dulcis* extract can be performed according to the following steps:

[0062] (1) Freeze-dried Mucor HY6-19 spore powder was inoculated onto fresh PDA agar plates and incubated at 30℃ for 72 h. 10 mL of sterile sucrose aqueous solution was added to the petri dish, and the spores were suspended by stirring with an inoculation loop. The spore suspension was transferred to sterile test tubes, and the spore concentration was adjusted to 8.15 × 10⁻⁶ spores with sterile sucrose aqueous solution. 6The concentration of *Mucor HY6-19* spores was measured at 15 g / mL to obtain the spore suspension. The composition and preparation method of the PDA plate culture medium were the same as in Example 1; the concentration of the sterile sucrose aqueous solution was 15 g / L, and it was sterilized by high-pressure steam at 115°C for 15 min.

[0063] (2) 10g of Hovenia dulcis powder was placed in a 250-mL Erlenmeyer flask that had been sterilized by dry heat at 160℃ for 2 hours. Then, 30mL of the Mucor HY6-19 spore solution prepared in step (1) was added (the volume was 3mL / g based on the mass of the Hovenia dulcis powder), and the mixture was stirred evenly. The Erlenmeyer flask was sealed with 8 layers of gauze and incubated at 30℃ for 60 hours to obtain Hovenia dulcis ferment.

[0064] (3) Transfer all the fermented Hovenia dulcis from step (2) into a 500-mL beaker, add 200mL of deionized water [material-to-liquid ratio of 1:20 (g:mL)], stir well, and keep warm in a 35℃ water bath for 6 hours. Then, transfer the beaker into an ultrasonic cleaner at 75℃, and ultrasonically extract at 160W for 80 minutes. Filter while hot through a 200-mesh filter cloth and collect all the filtrate.

[0065] (4) The filtrate prepared in step (3) was concentrated under reduced pressure at 60℃ and –0.1MPa to 10mL (1 / 20 of the original filtrate volume) to obtain a concentrated aqueous extract of Hovenia dulcis. All the concentrated aqueous extract was placed in a clean petri dish and dried under vacuum at 65℃ and –0.1MPa to constant weight, then pulverized to obtain Hovenia dulcis extract.

[0066] Following the steps described above, 1.51g of extract was obtained from 10g of Japanese raisin tree fruit, with a yield of 15.1% and a total flavonoid content of 13.6%.

[0067] Comparative Example 1: Preparation of Hovenia dulcis extract using conventional hot water ultrasonic method (compared to Example 4)

[0068] (1) Place 10g of Hovenia dulcis powder in a 500-mL beaker, add 200mL of deionized water [solid-to-liquid ratio 1:20 (g:mL)], stir well, and keep warm in a 35℃ water bath for 6 hours. Then, transfer the beaker to an ultrasonic cleaner at 75℃ and extract with ultrasonic power at 160W for 80 minutes. Filter while hot through a 200-mesh filter cloth and collect the filtrate.

[0069] (2) The filtrate prepared in step (1) was concentrated under reduced pressure at 60℃ and –0.1MPa to 10mL (1 / 20 of the original filtrate volume) to obtain a concentrated aqueous extract of Hovenia dulcis. All the concentrated aqueous extract was placed in a clean petri dish and dried under vacuum at 65℃ and –0.1MPa to constant weight, then pulverized to obtain Hovenia dulcis extract.

[0070] Following the steps described above, 1.16g of extract was obtained from 10g of Japanese raisin tree fruit, with a yield of 11.6% and a total flavonoid content of 11.2%.

[0071] Comparing the results of Example 4 and Comparative Example 1, it can be seen that: before the hot water ultrasonic extraction of Hovenia dulcis, the addition of Mucor HY6-19 fermentation treatment increased the yield of the extract from 11.6% to 15.1%, an increase of 30.2%, and the total flavonoid content in the extract from 11.2% to 13.6%, an increase of 21.4%.

[0072] Example 5: Application of Mucor HY6-19 in the preparation of Hovenia dulcis extract 2

[0073] The application of *Mucor HY6-19* in the preparation of *Hovenia dulcis* extract can be performed according to the following steps:

[0074] (1) PDA plate spores of *Mucor HY6-19* stored at 4℃ were inoculated onto fresh PDA plate medium and incubated at 30℃ for 64 h. 10 mL of sterile sucrose aqueous solution was added to the culture dish, and the spores were suspended by stirring with an inoculation loop. The spore suspension was then transferred to sterile test tubes, and the spore concentration was adjusted to 7.75 × 10⁻⁶ using sterile sucrose aqueous solution. 6 The concentration of *Mucor HY6-19* spores was determined to be 12.5 g / L, and the culture medium was sterilized by autoclaving at 115°C for 15 min. The PDA plate culture medium composition and preparation method were the same as in Example 1; the concentration of the sterile sucrose aqueous solution was 12.5 g / L, and it was autoclaved at 115°C for 15 min.

[0075] (2) 10g of Hovenia dulcis powder was placed in a 250-mL Erlenmeyer flask that had been sterilized by dry heat at 160℃ for 2h. Then, 25mL of the Mucor HY6-19 spore solution prepared in step (1) was added (the volume was 2.5mL / g based on the mass of the Hovenia dulcis powder), and the mixture was stirred evenly. The Erlenmeyer flask was sealed with 8 layers of gauze and incubated at 32℃ for 56h to obtain Hovenia dulcis ferment.

[0076] (3) Transfer all the fermented Hovenia dulcis from step (2) into a 500-mL beaker, add 225mL of deionized water [material-to-liquid ratio of 1:22.5 (g:mL)], stir well, and keep warm in a 37.5℃ water bath for 5 hours. Then, transfer the beaker into an ultrasonic cleaner at 80℃ and extract with ultrasonic power at 180W for 70 minutes. Filter while hot through a 200-mesh filter cloth and collect all the filtrate.

[0077] (4) The filtrate prepared in step (3) was concentrated under reduced pressure at 60℃ and –0.1MPa to 10mL (1 / 22.5 of the original filtrate volume) to obtain a concentrated aqueous extract of Hovenia dulcis. All the concentrated aqueous extract was placed in a clean petri dish and dried under vacuum at 65℃ and –0.1MPa to constant weight, then pulverized to obtain Hovenia dulcis extract.

[0078] Following the steps described above, 1.64g of extract was obtained from 10g of Japanese raisin tree fruit, with a yield of 16.4% and a total flavonoid content of 14.3%.

[0079] Comparative Example 2: Preparation of Hovenia dulcis extract using conventional hot water ultrasonic method (compared to Example 5)

[0080] (1) Place 10g of Hovenia dulcis powder in a 500-mL beaker, add 225mL of deionized water [solid-to-liquid ratio 1:22.5 (g:mL)], stir well, and keep warm in a 37.5℃ water bath for 5h. After that, transfer the beaker to an ultrasonic cleaner at 80℃, and extract with ultrasonic 180W for 70min. Filter while hot through a 200-mesh filter cloth and collect all the filtrate.

[0081] (2) The filtrate prepared in step (1) was concentrated under reduced pressure at 60℃ and –0.1MPa to 10mL (1 / 22.5 of the original filtrate volume) to obtain a concentrated aqueous extract of Hovenia dulcis. All the concentrated aqueous extract was placed in a clean petri dish and dried under vacuum at 65℃ and –0.1MPa to constant weight, then pulverized to obtain Hovenia dulcis extract.

[0082] Following the steps described above, 1.22g of extract was obtained from 10g of Japanese raisin tree fruit, with a yield of 12.2% and a total flavonoid content of 11.6%.

[0083] Comparing the results of Example 5 and Comparative Example 2, it can be seen that: before the hot water ultrasonic extraction of Hovenia dulcis, the addition of Mucor HY6-19 fermentation treatment increased the yield of the extract from 12.2% to 16.4%, an increase of 34.4%, and the total flavonoid content in the extract from 11.6% to 14.3%, an increase of 23.3%.

[0084] Example 6: Application of Mucor HY6-19 in the preparation of Hovenia dulcis extract 3

[0085] The application of *Mucor HY6-19* in the preparation of *Hovenia dulcis* extract can be performed according to the following steps:

[0086] (1) PDA plate spores of *Mucor HY6-19* stored at 4℃ were inoculated onto fresh PDA plate culture medium and incubated at 30℃ for 48 h. 10 mL of sterile sucrose aqueous solution was added to the culture dish, and the spores were suspended by stirring with an inoculation loop. The spore suspension was transferred to sterile test tubes, and the spore concentration was adjusted to 5.80 × 10⁻⁶ using sterile sucrose aqueous solution. 6 The concentration of *Mucor HY6-19* spores was measured at 10 g / mL to obtain the spore suspension. The composition and preparation method of the PDA plate culture medium were the same as in Example 1; the concentration of the sterile sucrose aqueous solution was 10 g / L, and it was sterilized by high-pressure steam at 115°C for 15 min.

[0087] (2) 10g of Hovenia dulcis powder was placed in a 250-mL Erlenmeyer flask that had been sterilized by dry heat at 160℃ for 2h. Then, 20mL of the Mucor HY6-19 spore solution prepared in step (1) was added (the volume was 2mL / g based on the mass of the Hovenia dulcis powder), and the mixture was stirred evenly. The Erlenmeyer flask was sealed with 8 layers of gauze and incubated at 32℃ for 52h to obtain Hovenia dulcis ferment.

[0088] (3) Transfer all the fermented Hovenia dulcis from step (2) into a 500-mL beaker, add 250mL of deionized water [material-to-liquid ratio of 1:25 (g:mL)], stir well, and keep warm in a 40℃ water bath for 4 hours. Then, transfer the beaker into an ultrasonic cleaner at 85℃, and ultrasonically extract at 200W for 60 minutes. Filter while hot through a 200-mesh filter cloth and collect all the filtrate.

[0089] (4) The filtrate prepared in step (3) was concentrated under reduced pressure at 60℃ and –0.1MPa to 10mL (1 / 25 of the original filtrate volume) to obtain a concentrated aqueous extract of Hovenia dulcis. All the concentrated aqueous extract was placed in a clean petri dish and dried under vacuum at 65℃ and –0.1MPa to constant weight, then pulverized to obtain Hovenia dulcis extract.

[0090] Following the steps described above, 1.69g of extract was obtained from 10g of Japanese raisin tree fruit, with a yield of 16.9% and a total flavonoid content of 14.5%.

[0091] Comparative Example 3: Preparation of Hovenia dulcis extract using conventional hot water ultrasonic method (compared with Example 6)

[0092] (1) Place 10g of Hovenia dulcis powder in a 500-mL beaker, add 250mL of deionized water [solid-to-liquid ratio 1:25 (g:mL)], stir well, and keep warm in a 40℃ water bath for 4 hours. Then, transfer the beaker to an ultrasonic cleaner at 85℃ and extract with 200W ultrasonic extraction for 60 minutes. Filter while hot through a 200-mesh filter cloth and collect all the filtrate.

[0093] (2) The filtrate prepared in step (1) was concentrated under reduced pressure at 60℃ and –0.1MPa to 10mL (1 / 25 of the original filtrate volume) to obtain a concentrated aqueous extract of Hovenia dulcis. All the concentrated aqueous extract was placed in a clean petri dish and dried under vacuum at 65℃ and –0.1MPa to constant weight, then pulverized to obtain Hovenia dulcis extract.

[0094] Following the steps described above, 1.27g of extract was obtained from 10g of Japanese raisin tree fruit, with a yield of 12.7% and a total flavonoid content of 12.1%.

[0095] Comparing the results of Example 6 and Comparative Example 3, it can be seen that: before the hot water ultrasonic extraction of Hovenia dulcis, the addition of Mucor HY6-19 fermentation treatment increased the yield of the extract from 12.7% to 16.9%, an increase of 33.1%, and the total flavonoid content in the extract from 12.1% to 14.5%, an increase of 19.8%.

Claims

1. Mucor sp. HY6-19, deposited at Guangdong Provincial Center for Microbial Culture Collection, accession number GDMCCNo:63391, deposited on April 24, 2023, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510070, China.

2. The use of the *Mucor* HY6-19 of claim 1 in the preparation of *Hovenia dulcis* extract.

3. The application as described in claim 2, characterized in that, The method of application is as follows: (1) Inoculate the spore liquid of Mucor HY6-19 into the Hovenia dulcis powder, stir evenly, and ferment at 30–32℃ for 52–60h to obtain Hovenia dulcis fermented product; (2) Add deionized water to the Hovenia dulcis fermented product, stir evenly, keep warm at 35–40℃ for 4–6h, extract by hot water ultrasonic extraction, filter, concentrate the filtrate to obtain Hovenia dulcis water extract concentrate; (3) Vacuum dry the Hovenia dulcis water extract concentrate, pulverize it, and obtain Hovenia dulcis extract.

4. The application as described in claim 3, characterized in that, In step (1), the Hovenia dulcis powder is fine powder made by drying Hovenia dulcis seeds at 85°C and then pulverizing them through a 60-mesh sieve.

5. The application as described in claim 3, characterized in that, The preparation method of the HY6-19 spore solution in step (1) is as follows: HY6-19 spores preserved at low temperature are inoculated on PDA plate culture medium and cultured at 30℃ for 48–72 h. Then, sterile sucrose aqueous solution is added to the culture, and the spores are suspended by stirring with an inoculation loop to obtain the spore solution.

6. The application as described in claim 5, characterized in that, The concentration of sucrose in the sterile sucrose aqueous solution is 10–15 g / L, and the concentration of the spore solution is 5 × 10⁻⁶ g / L. 6 –9×10 6 per mL.

7. The application as described in claim 3, characterized in that, The volume of spore liquid of *Rhizopus HY6-19* used in step (1) is 2–3 mL / g based on the mass of *Hovenia dulcis* powder.

8. The application as described in claim 3, characterized in that, The preparation method of the aqueous extract concentrate of Hovenia dulcis in step (2) is as follows: Deionized water is added to the Hovenia dulcis ferment, and after stirring evenly, it is kept at 35–40℃ for 4–6 hours. Then, it is transferred to an ultrasonic cleaner at a water temperature of 75–85℃ and ultrasonically extracted at 160–200W for 60–80 minutes. After ultrasonic extraction, it is filtered while hot using a 200-mesh filter cloth. The filtrate is concentrated under reduced pressure at 60℃ and -0.1MPa to 1 / 10–1 / 25 of the original volume to obtain the aqueous extract concentrate of Hovenia dulcis.

9. The application as described in claim 3 or 8, characterized in that, The volume of deionized water added is 20–25 mL / g based on the mass of the Hovenia dulcis powder before fermentation.

10. The application as described in claim 3, characterized in that, The preparation method of the Hovenia dulcis extract in step (3) is as follows: the concentrated aqueous extract of Hovenia dulcis is dried under vacuum at 65°C and –0.1MPa to constant weight, and then pulverized to obtain the Hovenia dulcis extract.

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