Aspergillus niger, fermentation liquor, enzyme preparation and application of aspergillus niger in preparation of alpha-monoglucosyl hesperidin

Abstract

The invention discloses aspergillus niger, fermentation liquor, an enzyme preparation and application of the aspergillus niger in preparation of alpha-monoglucosyl hesperidin. The preservation number of the aspergillus niger is CGMCC (China General Microbiological Culture Collection Center) No.42150. When the enzyme preparation obtained by fermenting the aspergillus niger is used for producing the alpha-monoglucosyl hesperidin, hesperetin can be rapidly separated into precipitate, the precipitate can be effectively removed only by simple solid-liquid separation, and then the high-purity alpha-monoglucosyl hesperidin can be ensured through simple purification, so that the yield of the alpha-monoglucosyl hesperidin is improved. And the whole preparation process is simpler and more feasible.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a black mold, fermentation broth, enzyme preparation and application thereof in preparation of α-glucosyl hesperidin (α-GH). BACKGROUND

[0002] Hesperidin is a dihydroflavonol glycoside extracted from young citrus fruits, and its structure is shown below. Many studies have reported the diverse biological properties of hesperidin, for example, as a food raw material, it can reduce body fat, enhance bone density, protect the nervous system, regulate immunity, and have anti-diabetic, anti-hypertensive and anti-hyperlipidemic effects (doi: 10.3390 / nu12051488); as a cosmetic raw material, it can have anti-inflammatory and antioxidant effects, and can enhance microvascular circulation, improve dark circles, brighten dull skin, and improve skin edema. In addition, some health problems, such as allergies (hay fever, sinusitis), hemorrhoids, varicose veins, symptoms related to menopausal changes, and premenstrual syndrome, can also be treated with hesperidin (doi: 10.34297 / AJBSR.2024.24.003250). However, the solubility of hesperidin in water is only 0.02 g / L, and its clinical application is often hindered by its poor solubility in water and limited bioavailability in vivo.

[0003]

[0004] Hesperidin

[0005] In 1989, a patent of Hayashibara Co., Ltd. (JP3060227B2) disclosed a method for converting hesperidin into glycosylated hesperidin, and the solubility of the produced α-glucosyl hesperidin (α-GH, structure shown below) was more than 200 g / L, greatly improving the water solubility of hesperidin. This method uses cyclodextrin glycosyltransferase as a catalyst, maltodextrin as a sugar donor, converts hesperidin into polyglucosyl hesperidin, and then hydrolyzes most of the glucose groups to form α-GH through saccharifying enzyme, and finally purifies α-GH through resin. This process includes two steps of enzyme treatment and one step of resin purification, and the final product contains both α-GH and hesperidin.

[0006]

[0007] α-glucosyl hesperidin (α-GH)

[0008] In 1996, a patent (JP3833775B2) jointly applied by Lin Yuan and Toyo Sugar Refining Co., Ltd. disclosed a method for preparing stabilized a-GH, which is characterized by not precipitating for a long time. After obtaining polyglucosyl hesperidin, the method converts polyglucosyl hesperidin into a-GH by adding glucosidase, and then converts hesperidin into 7-O-β-monoglucosyl hesperetin by adding a-L-rhamnosidase. 7-O-β-monoglucosyl hesperetin has good solubility, and the hesperidin solution containing it will not precipitate for a long time.

[0009] In 1997, a patent (JP3833811B2) jointly applied by Lin Yuan and Toyo Sugar Refining Co., Ltd. disclosed a method for preparing high-purity a-GH. After obtaining polyglucosyl hesperidin, the method continues to add glucosidase (to convert polyglucosyl hesperidin into a-GH), a-L-rhamnosidase (to convert hesperidin into 7-O-β-monoglucosyl hesperetin), and β-D-glucosidase (to convert 7-O-β-monoglucosyl hesperetin into hesperetin). Only two substances are left in the obtained reaction product, one is a-GH, and the other is hesperetin. Next, the reaction product is purified using a resin to remove impurities such as sugars, and then the purified product is dissolved in 80°C methanol and placed at room temperature for a period of time. Only a-GH will crystallize and precipitate, and the purity of the final a-GH can reach 98%. This process includes two steps of enzyme treatment, one step of resin purification, and one step of recrystallization.

[0010] The above three processes are the mainstream processes for preparing a-GH from hesperidin, which are widely used by researchers around the world. For example, patent application CN104862362A uses preparative high-performance liquid chromatography to separate high-purity a-GH, making the product purity reach 98.5%, but the production cost is extremely high; CN110982865A uses Escherichia coli to produce a cyclodextrin glycosyltransferase from alkaliphilic Bacillus, and uses the enzyme to catalyze the glycosylation of hesperidin, with a maximum conversion rate of only 88%; CN114350730A proposes to use saccharifying enzyme produced by Aspergillus oryzae to convert oligoglucosyl hesperidin into a-GH, in order to avoid converting a-GH into hesperidin substrate; CN117701465A proposes that a-GH can be converted from oligoglucosyl hesperidin by a-L-glucosidase produced by Bacillus licheniformis B1 without excessive hydrolysis; CN117925756A proposes to use Escherichia coli to ferment a β-glucosidase from a source of the genus Zygosaccharomyces, to hydrolyze unconverted hesperidin into hesperetin, and finally purify a-GH by resin.

[0011] The above patents all improve the Japanese Hayashino and Oriental Sugar process, but to ensure a certain purity, complete production of alpha-GH always needs at least two steps of enzyme treatment and one resin purification, and involves the input of multiple enzymes, and the overall process is relatively complex.

[0012] In January 2025, the European Commission issued Regulation (EU) 2025 / 167, approving glucose-based hesperidin as a new food product for the market, which can be applied to soft drinks, energy drinks, dietary supplements and other categories, and it is expected that the market demand for alpha-GH will enter a period of rapid expansion. Therefore, developing a lower-cost alpha-GH production process has significant economic value. SUMMARY

[0013] The technical problem to be solved by the present application is to overcome the defects of the existing alpha-monoglucosyl hesperidin (alpha-GH) production process, such as complex process and high cost, and to provide a Aspergillus niger, a fermentation broth, an enzyme preparation and their applications in preparing alpha-monoglucosyl hesperidin. When the enzyme preparation obtained by fermentation of the Aspergillus niger is used for alpha-GH production, it can promote the rapid precipitation of hesperetin, which can be effectively removed by simple solid-liquid separation, and then high-purity alpha-GH can be obtained by simple purification.

[0014] To achieve the above object, the technical scheme adopted by the present application is as follows.

[0015] The present application provides a Aspergillus niger, and the preservation number of the Aspergillus niger is CGMCC No.42150 (Aspergillus niger HW16).

[0016] Based on citrus samples, the present application accidentally found that the strain Aspergillus niger HW16 can efficiently degrade hesperidin and then prepare high-purity alpha-GH after repeated enrichment and screening. Research has found that the strain can produce glucoamylase, alpha-L-rhamnosidase and beta-D-glucosidase at the same time after simple fermentation, and more surprisingly, the fermentation broth or enzyme preparation using the strain can make other degradation products such as hesperetin precipitate directly after hydrolysis to obtain alpha-GH without special operation, and then high-purity alpha-GH can be obtained by simple solid-liquid separation (such as centrifugation) and purification, which simplifies the operation and significantly reduces the production cost.

[0017] The present application also provides a fermentation broth, which is obtained by fermentation of the Aspergillus niger as described above.

[0018] In the present application, the fermentation conditions can be selected according to the conventional Aspergillus niger fermentation conditions in the art.

[0019] In some embodiments, in the fermentation, the fermentation medium comprises the following components in percentage by mass: 1.0%-5.0% corn starch, 0.1%-2.0% yeast extract, 0.1%-2.0% peptone, 0.1%-2.0% ammonium sulfate, 0.1%-1.0% potassium dihydrogen phosphate, 0.01%-0.10% calcium chloride, and 0.001%-0.10% magnesium sulfate; for example, 1.0% corn starch, 0.5% yeast extract, 2.0% peptone, 0.5% ammonium sulfate, 0.2% potassium dihydrogen phosphate, 0.05% calcium chloride, and 0.01% magnesium sulfate.

[0020] In some embodiments, in the fermentation, the inoculation amount is 1%-10%, in percentage of the volume ratio of the seed liquid containing the Aspergillus niger and the fermentation medium.

[0021] In some embodiments, in the fermentation, the fermentation temperature is 25-30°C.

[0022] In some embodiments, in the fermentation, the fermentation time is 24-72h.

[0023] In some embodiments, in the fermentation, the fermentation is carried out under aeration.

[0024] The present application also provides an enzyme preparation, which is extracted from the fermentation broth as described above.

[0025] In the present application, the enzyme preparation comprises glucoamylase, α-L-rhamnosidase, β-D-glucosidase, and active substances that can cause the degradation product of hesperidin, hesperetin, to be precipitated.

[0026] In the present application, the extraction conditions can be selected according to the conventional conditions for extracting enzymes from fermentation broth.

[0027] In some embodiments, the extraction comprises the following steps: the fermentation broth is centrifuged, and the supernatant is obtained by filtration.

[0028] In some preferred embodiments, in the extraction, the centrifugation is carried out at a speed of 4000-10000 rpm.

[0029] In some preferred embodiments, in the extraction, the centrifugation is carried out for 5min-30min, for example, 10min.

[0030] In some preferred embodiments, in the extraction, the filtration is carried out using filter cloth, filter paper, or filter membrane, and the pore size of the filter membrane is, for example, 0.22μm.

[0031] In some preferred embodiments, the extraction further comprises a step of concentration after the filtration; the concentration is preferably performed using a membrane, for example with a pore size of 3 KDa. After the concentration, a step of mixing the obtained concentrated solution with excipients can also be included; the excipients can include, for example, glycerol, potassium sorbate and methylparaben, and can specifically be 30% (v / v) glycerol, 0.2% (m / v) potassium sorbate and 0.1% (m / v) methylparaben, the percentages being the mass or volume of each component per volume of the resulting mixture.

[0032] The present application also provides use of the Aspergillus niger as described above, the fermentation broth as described above, or the enzyme preparation as described above in the preparation of a- monoglucosyl hesperidin.

[0033] Specifically, the preparation of a-monoglucosyl hesperidin can comprise the following steps:

[0034] Under the action of the fermentation broth or the enzyme preparation, the hesperidin glycosylation product is hydrolyzed, followed by solid-liquid separation, and the supernatant is obtained; the hesperidin glycosylation product includes polyglucosyl hesperidin and unreacted hesperidin.

[0035] Specifically, the hesperidin glycosylation product can be prepared by a method comprising the following steps:

[0036] Under the action of a glycosyltransferase, a reaction substrate containing hesperidin and a glycosyl donor is subjected to glycosylation reaction, and the hesperidin glycosylation product is obtained.

[0037] In some preferred embodiments, the glycosyl donor includes one or more of maltodextrin, β-cyclodextrin and starch, for example maltodextrin.

[0038] In some preferred embodiments, the mass ratio of the hesperidin to the glycosyl donor is 1: (2-5), for example 1:4.

[0039] In some preferred embodiments, the pH of the reaction substrate is 7.5-10.5, for example 8.0. The pH can be adjusted by a pH adjuster such as sodium hydroxide in the art.

[0040] In some preferred embodiments, the glycosyltransferase is a cyclodextrin glycosyltransferase, for example a β-cyclodextrin glycosyltransferase.

[0041] In some preferred embodiments, the glycosyltransferase can be used in the form of the glycosyltransferase itself, a fermentation broth containing the glycosyltransferase, or a glycosyltransferase enzyme solution.

[0042] The fermentation broth containing glycosyltransferase is preferably derived from Paenibacillus silvae, and the accession number of the Paenibacillus silvae is, for example, CGMCC No. 35445.

[0043] The amount of the fermentation broth containing glycosyltransferase or the glycosyltransferase enzyme solution can be routinely selected according to the need.

[0044] In some embodiments, when the glycosyltransferase is used in the form of a fermentation broth containing glycosyltransferase or a glycosyltransferase enzyme solution, the mass ratio of the hesperidin to the "fermentation broth containing glycosyltransferase or glycosyltransferase enzyme solution" is, for example, 1:2, based on 300 U / mL of enzyme activity of the glycosyltransferase.

[0045] In some embodiments, when the glycosyltransferase is used in the form of a fermentation broth containing glycosyltransferase or a glycosyltransferase enzyme solution, the amount of the hesperidin to the "fermentation broth containing glycosyltransferase or glycosyltransferase enzyme solution" is, for example, 5 g:3 mL, based on 2182.8 U / mL of enzyme activity of the glycosyltransferase.

[0046] In some embodiments, the fermentation broth containing glycosyltransferase is obtained by fermentation of the Paenibacillus silvae, and the glycosyltransferase enzyme solution is obtained by further centrifugation of the fermentation broth. The fermentation medium comprises, for example, the following components in percentage by mass: 1.0% wheat starch, 0.5% yeast extract, 1.0% peptone, 0.3% potassium dihydrogen phosphate, 0.05% calcium chloride, and 0.01% magnesium sulfate. The fermentation is performed, for example, at 35°C under aeration for 72 hours. The centrifugation is performed, for example, at a speed of 10,000 rpm.

[0047] In some preferred embodiments, the temperature of the glycosylation reaction is 45-75°C, for example, 55-60°C.

[0048] In some preferred embodiments, the time of the glycosylation reaction is 1-40 h, for example, 30 h.

[0049] In some preferred embodiments, the glycosylation reaction further comprises a step of inactivation. The inactivation is preferably performed by heating at 85-100°C for 10-30 min, for example, heating at 90°C for 30 min.

[0050] In some embodiments, the glycosylation product of hesperidin comprises 86%-88% polyglucosyl hesperidin and 12%-14% unreacted hesperidin; the percentages are the mass percentages of each component in the glycosylation product of hesperidin.

[0051] In some preferred embodiments, the mass ratio of the hesperidin glycosylation product to the enzyme preparation is (80-120): 1, for example 100: 1, based on the total enzyme activity of the enzyme preparation.

[0052] In some preferred embodiments, the pH of the hydrolysis reaction is 4.0-6.0, preferably 4.5-5.5, for example 5.0.

[0053] In some preferred embodiments, the temperature of the hydrolysis reaction is 40-60°C, preferably 50-55°C.

[0054] In some preferred embodiments, the time of the hydrolysis reaction is 1-3h.

[0055] In some preferred embodiments, the hydrolysis reaction is further followed by a step of inactivation; the inactivation is preferably performed by heating at pH 3.0, 85-100°C for 10-30min, for example by heating at pH 3.0, 90°C for 30min.

[0056] In some preferred embodiments, the solid-liquid separation is performed by centrifugation or filtration.

[0057] In some preferred embodiments, the centrifugation is performed at a speed of 4000-10000rpm.

[0058] In some preferred embodiments, the centrifugation is performed for a time of 5-30min, for example 10min.

[0059] In some preferred embodiments, the solid-liquid separation is further followed by a step of purification. The purification is preferably ultrafiltration purification or resin purification.

[0060] In some preferred embodiments, the ultrafiltration purification is performed using an ultrafiltration membrane, for example with a pore size of 150-200Da.

[0061] In some preferred embodiments, the ultrafiltration purification is performed using pure water, for example by adding equal volume of pure water.

[0062] In some preferred embodiments, the ultrafiltration purification is further followed by drying, for example by spray drying.

[0063] In some preferred embodiments, the resin purification is performed using HP-10 resin.

[0064] In some preferred embodiments, the resin purification comprises the following steps: loading, washing with water, and eluting with ethanol.

[0065] In some preferred embodiments, the resin purification is further followed by concentration and / or drying; the concentration is for example vacuum concentration, and the drying is for example spray drying.

[0066] The above-mentioned preferred conditions can be combined arbitrarily to obtain preferred examples of the present application.

[0067] The reagents and raw materials used in the present application are commercially available.

[0068] The positive progress effect of the present application is that:

[0069] The Aspergillus niger HW16 provided by the present application can produce high-activity glucose amylase, alpha-L-rhamnosidase and beta-D-glucosidase simultaneously through simple fermentation, and the strain is easy to cultivate, stable in character and suitable for industrial production.

[0070] Further, when the enzyme preparation obtained by fermentation is used for the production of alpha-GH, the polyglucosyl hesperidin can be hydrolyzed into alpha-GH within 3 hours, and the unreacted hesperidin in the hesperidin glycosylation product can be degraded into hesperetin; more importantly, the enzyme preparation can promote the rapid precipitation of hesperetin, which can be effectively removed through simple solid-liquid separation, and then high-purity alpha-GH can be obtained through simple purification.

[0071] Compared with the existing alpha-GH production process, the overall process of the present application is simpler, the cost and energy consumption are lower, it is suitable for large-scale production, and has significant economic benefits. Biological material preservation information

[0072] The Aspergillus niger of the present application has been preserved in the China General Microbiological Culture Collection Center (CGMCC) on July 30, 2025, the preservation address is No. 3, Beichen West Road, Chaoyang District, Beijing, the postcode is 100101, the preservation number is CGMCC No. 42150, the culture name is HW16, and the classification name is Aspergillus niger.

[0073] The Paenibacillus silvae involved in the present application has been preserved in the China General Microbiological Culture Collection Center (CGMCC) on July 30, 2025, the preservation address is No. 3, Beichen West Road, Chaoyang District, Beijing, the postcode is 100101, the preservation number is CGMCC No. 35445, the culture name is HW253, and the classification name is Paenibacillus silvae. BRIEF DESCRIPTION OF DRAWINGS

[0074] Figure 1 The morphology of the colony of the strain Aspergillus niger HW16 in the YPD culture medium on the surface of the present application is shown in Example 1.

[0075] Figure 2 A micrograph of a colony of Aspergillus niger HW16 in YPD medium.

[0076] Figure 3 A micrograph of a spore head of Aspergillus niger HW16.

[0077] Figure 4 A micrograph of a spore head of Aspergillus niger HW16.

[0078] Figure 5 The results of the effect of pH on the activity of the enzyme preparation obtained from the fermentation of Aspergillus niger HW16 in Example 2.

[0079] Figure 6 The results of the effect of temperature on the activity of the enzyme preparation obtained from the fermentation of Aspergillus niger HW16 in Example 2.

[0080] Figure 7 The results of the high performance liquid chromatography analysis of the products obtained after treating hesperidin (top) and the fermentation broth containing β-cyclodextrin glycosyltransferase with the enzyme preparation obtained from the fermentation of Aspergillus niger HW16 in Example 3.

[0081] Figure 8 The results of the high performance liquid chromatography analysis of the products obtained after treating polyglucosyl hesperidin and hesperidin with the enzyme preparation obtained from the fermentation of Aspergillus niger HW16 for 1 hour (top) and 3 hours (bottom) in Example 3.

[0082] Figure 9 The results of the high performance liquid chromatography analysis of the products obtained after treating polyglucosyl hesperidin and hesperidin with the enzyme preparation obtained from the fermentation of Aspergillus niger HW16 for 1 hour (top) and 3 hours (bottom) in Example 3.

[0083] Figure 10 The results of the high performance liquid chromatography analysis of the products obtained after treating polyglucosyl hesperidin and hesperidin with the enzyme preparation obtained from the fermentation of Aspergillus niger HW16 for 1 hour (top) and 3 hours (bottom) in Example 3.

[0084] Figure 11 The results of the high performance liquid chromatography analysis of the products obtained after treating polyglucosyl hesperidin and hesperidin with the enzyme preparation obtained from the fermentation of Aspergillus niger HW16 for 1 hour (top) and 3 hours (bottom) in Example 3.

[0085] Figure 12The results of high performance liquid chromatography analysis of the product obtained by treating the glucose amylase and hesperidinase in Comparative Example 3. DETAILED DESCRIPTION

[0086] The present application is further illustrated by the following examples without thereby limiting the present application to the examples described. The experimental methods in the following examples, for which specific conditions are not mentioned, are selected according to the conventional methods and conditions, or according to the instructions of the commercial products.

[0087] In the following examples and comparative examples, the quantitative determination of enzyme activity uses the following method:

[0088] (1) Glucose amylase activity determination

[0089] Reagent preparation: 6.3 g of 3,5-dinitrosalicylic acid (DNS) is dissolved in 300 ml of distilled water, and 21 g of sodium hydroxide is dissolved in 400 ml of distilled water. Then the two are slowly mixed dropwise and continuously stirred. Then 182 g of potassium sodium tartrate tetrahydrate, 5 g of phenol, and 5 g of anhydrous sodium sulfite are sequentially added, and continuously stirred in a 55°C water bath until the solution is clear and transparent. Finally, make up to 1000 ml with distilled water, and store in a brown bottle. After standing for 5-7 days, use.

[0090] Enzyme activity determination: 2% (m / v) starch solution is prepared as a substrate using 100 mM phosphate buffer at pH 6.0 as a solvent, 500 μl of appropriately diluted enzyme solution is mixed with 500 μl of substrate solution, and the mixture is reacted at 55°C for 10 minutes. 1 ml of DNS reagent is added and mixed, heated in boiling water for 10 minutes, cooled to room temperature 25°C, and the absorbance is measured at a wavelength of 540 nm using a spectrophotometer. At the same time, 1 ml of a glucose solution prepared at a concentration of 0.1 mg / ml to 0.5 mg / ml is mixed with 1 ml of DNS reagent, heated in a boiling water bath for 10 minutes, and then cooled to room temperature, and the absorbance at a wavelength of 540 nm is measured, and a standard curve is prepared.

[0091] Enzyme activity definition: the amount of enzyme required to release 1 μmol of reducing sugar per minute from a 1% starch solution is defined as one enzyme activity unit (U).

[0092] (2) α-L-rhamnosidase activity determination

[0093] 0.2% hesperidin substrate solution is prepared using 100 mM citric acid buffer at pH 5.0 as a solvent, 0.5 ml of the substrate solution is preheated at 55°C for 5 minutes, 0.5 ml of α-L-rhamnosidase solution is added, and the mixture is reacted at 55°C for 10 minutes, and then inactivated in a boiling water bath. The residual amount of hesperidin is detected by HPLC under the following conditions: Shimadzu high performance liquid chromatography system, chromatographic column: Gemini ®C18 (250 mm x 4.6 mm, 5 μm), mobile phase volume ratio of methanol: water = 55:45, detection wavelength of 280 nm, column temperature of 40 °C, injection volume of 10 μl, flow rate of 1 ml / min, analysis time of 30 min)

[0094] Enzyme activity definition: the amount of enzyme required to consume 1 μg naringin per minute from a standard solution of naringin with a concentration of 0.1% (m / v) is one enzyme activity unit (U).

[0095] (3) β-D-glucosidase activity determination

[0096] Take 1.2 ml of citric acid buffer with a concentration of 100 mM pH 5.0, 0.4 ml of 10 mM p-NPG (p-nitrophenol-β-D-glucoside); then add 0.4 ml of appropriately diluted enzyme solution, react at 55 °C for 10 min, add 2 ml of 0.5 M sodium carbonate solution, and measure the absorbance value at 405 nm wavelength.

[0097] Enzyme activity definition: the amount of enzyme required to generate 1 μmol of p-nitrophenol per minute from a standard solution of p-NPG with a concentration of 2 mM is one enzyme activity unit (U).

[0098] (4) β-cyclodextrin glycosyltransferase activity determination

[0099] β-cyclodextrin glycosyltransferase has four activities of hydrolysis, disproportionation, coupling and cyclization. In this paper, only the cyclization activity is determined to mark the activity of β-cyclodextrin glycosyltransferase, and the determination method is as follows:

[0100] Prepare 2% (m / v) maltodextrin in 100 mM phosphate buffer with pH 6.0 as a substrate solution. Take 0.500 ml of 2% maltodextrin and 0.500 ml of appropriately diluted enzyme solution to react at 55 °C for 10 min. Then add 3.5 ml of 30 mM NaOH solution to terminate the reaction, and add 0.5 ml of 0.02% (m / v) phenolphthalein solution (phenolphthalein is dissolved in 5 mM sodium carbonate solution as solvent), mix well and react at room temperature for 15 min, and measure the absorbance at 540 nm wavelength. At the same time, prepare 0.5 g / L~1.5 g / L β-cyclodextrin solution, take 1 ml of each and mix with 3.5 ml of 30 mM NaOH solution and 0.5 ml of 0.02% phenolphthalein solution, react at room temperature for 15 min, and then measure the absorbance at 540 nm wavelength, and draw a standard curve.

[0101] Enzyme activity definition: the amount of enzyme required to generate 1 μmol of β-cyclodextrin per minute from 1% maltodextrin is one enzyme activity unit (U).

[0102] The analysis of hesperidin, polyglucosyl hesperidin, a-GH, 7-O-β-monoglucosyl hesperetin and hesperetin and other substances in the following examples and comparative examples all use high performance liquid chromatography, and the specific operation is as follows:

[0103] The analysis conditions are: Shimadzu high performance liquid chromatography system, the chromatographic column is Gemini ® C18 (250 mm x 4.6 mm, 5 μm), the mobile phase is acetonitrile: water: acetic acid = 200:800:0.1 by volume ratio, the detection wavelength is 280 nm, the column temperature is 40°C, the injection amount is 10 μl, the flow rate is 1 ml / min, the analysis time is 30 min, and the elution time program is shown in Table 1:

[0104] Table 1

[0105]

[0106] Example 1: Isolation and identification of strain Aspergillus niger HW16

[0107] (1) Enrichment and isolation of strains

[0108] A number of samples of naturally growing citrus fruits, fallen fruits, leaves, humus and soil near citrus trees were collected from citrus orchards in Pujiang County, Sichuan Province, and an appropriate amount of physiological saline was added to each sample, and the samples were shaken for 10 minutes to obtain sample suspensions. 1 ml of each sample suspension was added to 100 ml of enrichment medium, and the mixture was cultured at 30°C and 100 rpm for 10 days. The composition of the enrichment medium is (by mass concentration): 1.0% hesperidin glycoside mixture (including 12% hesperidin and 88% polyglucosyl hesperidin), 0.5% yeast extract, 1.0% peptone, 0.5% ammonium sulfate, 0.2% potassium dihydrogen phosphate, 0.05% calcium chloride, and 0.01% magnesium sulfate. Before use, sterilize at 121°C for 30 minutes.

[0109] The medium containing 1.0% hesperidin glycoside mixture is a clear solution. After 10 days of enrichment, the enrichment material that produces a precipitate is selected. After appropriate dilution, it is spread on sterile solid culture plates, and the solid culture plates have the same composition as the enrichment medium, except that 1.5% agar powder is added. After incubation at 30°C for 3 days, the strains with different colony morphologies are inoculated into test tubes containing 4 ml of enrichment medium, and the mixture is incubated at 30°C and 160 rpm for 3 days. Observe whether a precipitate is produced in the medium in the test tube. The strains that have a high ability to degrade hesperidin glycoside mixture are transferred to 50 ml of enrichment medium in a flask, and the mixture is incubated at 30°C and 160 rpm for 3 days. After centrifugation, the ability of each strain to produce a precipitate is compared. The more precipitate produced after fermentation, the higher the activity of the microorganism in converting hesperidin to hesperetin.

[0110] After repeated enrichment screening, the present application found that the strain Aspergillus niger HW16 has high efficiency in degrading hesperidin. The colony morphology of the strain Aspergillus niger HW16 on YPD medium is as follows: after 2 days of culture, the colony is flat and yellow, closely adhering to the culture medium, with white to light yellow villi on the edge and surface, and yellow mycelium inside the culture medium, and the colony as a whole presents dense wrinkle lines without obvious odor. After 3 days of culture, black spores are produced on the surface of the colony. Under a microscope, the strain contains spore stalks and spore heads (see Figures 1-4 ).

[0111] (2) Molecular biology identification of the strain Aspergillus niger HW16.

[0112] The strain Aspergillus niger HW16 was cultured in the above enrichment medium, and the bacterial body precipitate was collected by centrifugation at 5000 rpm for 5 minutes. The DNA of the bacterial body was extracted using a commercial DNA extraction kit (Qingke Biology), and the ITS gene sequence of the strain was determined. By searching and comparing on NCBI, the consistency of the ITS gene sequence of the strain with that of Aspergillus niger CBS 513.88 was 100%.

[0113] Example 2: Preparation and property determination of Aspergillus niger HW16 fermentation broth and enzyme preparation

[0114] (1) Preparation of Aspergillus niger HW16 fermentation broth and enzyme preparation

[0115] ① Fermentation medium (in mass concentration) composition:

[0116] 1.0% corn starch, 0.5% yeast extract, 2.0% peptone, 0.5% ammonium sulfate, 0.2% potassium dihydrogen phosphate, 0.05% calcium chloride, 0.01% magnesium sulfate.

[0117] ② Preparation of fermentation broth

[0118] The medium components were dissolved in water in the proportions stated, sterilized at 121°C for 30 minutes, and cooled to 30°C. A colony of Aspergillus niger HW16 was picked into the fermentation medium and incubated at 30°C for 20 hours with sufficient agitation and aeration to serve as a seed culture. The seed culture was inoculated into the fermentation medium at a volume ratio of 10% and incubated at 30°C for 72 hours with sufficient agitation and aeration to obtain a fermentation broth. The fermentation broth obtained contained hesperetin glycoside hydrolyzing enzymes, including glucoamylase, α-L-rhamnosidase, and β-D-glucosidase.

[0119] Table 2 Activity of three glycoside hydrolyzing enzymes in the fermentation broth of HW16

[0120]

[0121] ③ Preparation of crude enzyme solution and refined enzyme solution (containing hesperetin glycoside hydrolyzing enzymes):

[0122] The fermentation broth was centrifuged at 10,000 rpm for 10 minutes, and the precipitate was discarded to obtain a fermentation broth centrifugate. The fermentation broth was then filtered through a 0.22 μm filter to remove the bacterial cells and insoluble components in the fermentation broth, and the supernatant obtained was the crude enzyme solution.

[0123] The crude enzyme solution was further concentrated and purified by membrane filtration technology. The molecular pore size of the membrane used was 3 KDa, and the permeate side was discarded. The concentrated side was the refined enzyme solution.

[0124] ④ Determination of comprehensive enzyme activity of the refined enzyme solution

[0125] As described above, the enzyme solution obtained by fermentation of the strain Aspergillus niger HW16 has three glycosidase activities. In order to facilitate application in production, the present application further provides a unified activity index, i.e., comprehensive enzyme activity. The specific determination method is as follows:

[0126] A 0.1 mol / L phosphate buffer was prepared at pH 6.0, and 2% of a hesperetin glycoside mixture (containing 12% of hesperidin and 88% of polyglucosyl hesperidin) was added as a substrate solution.

[0127] 500 μl of the substrate solution was added to 500 μl of the appropriately diluted enzyme solution, and the mixture was reacted at 55°C for 10 minutes. Then, 1 ml of DNS reagent was added, and the mixture was heated in boiling water for 10 minutes. After cooling to room temperature, the absorbance was measured at a wavelength of 540 nm.

[0128] Enzyme activity definition: the amount of enzyme required to produce 1 μmol of reducing sugar per minute from a 1% concentration of a hesperetin glycoside mixture is one enzyme activity unit (U).

[0129] After the activity of the refined enzyme solution was determined, the refined enzyme solution was uniformly diluted to an enzyme solution diluent with a comprehensive activity of 42900±30U / ml.

[0130] Preparation of liquid enzyme preparation

[0131] The liquid enzyme preparation was obtained by adding 30% (v / v) glycerol, 0.2% (m / v) potassium sorbate and 0.1% (m / v) nipagin methyl ester to the enzyme solution diluent with a comprehensive enzyme activity of 42900±30U / ml, and the enzyme preparation had a comprehensive enzyme activity of 30000U / ml and could be stored at 4°C for more than 3 months.

[0132] (2) Measurement of optimal reaction pH of liquid enzyme preparation

[0133] ① Preparation of buffer solution and preparation of different substrate solutions:

[0134] 0.1 mol / L citric acid-sodium citrate buffer was used to prepare the buffer solution with a pH of 3.0 to 5.5;

[0135] 0.1 mol / L sodium dihydrogen phosphate-disodium hydrogen phosphate was used to prepare the buffer solution with a pH of 6.0 to 7.5;

[0136] 2% hesperidin glycoside mixture (including 12% hesperidin and 88% polyglucosyl hesperidin) was dissolved in the above buffer solutions as the substrate solution.

[0137] ② Measurement of relative enzyme activity:

[0138] 500μl of the above substrate solution with different pH was added to 500μl of appropriately diluted enzyme preparation, and the reaction was carried out at 55°C for 30 minutes, then 1ml of DNS reagent was added, heated in boiling water for 10 minutes, cooled to room temperature, and the absorbance was measured at 540nm with a colorimetric cell. The enzyme preparation inactivated by boiling for 10 minutes was used as the negative control for each pH gradient test group. The enzyme activity of the test group with the highest absorbance was taken as 100% to calculate the relative enzyme activity of each pH gradient, and three parallel samples were set for each test group. The results are shown in Figure 5 .

[0139] The results showed that the enzyme had the highest activity under weakly acidic conditions, and the optimal reaction pH range was 4.5 to 5.5.

[0140] (3) Measurement of optimal reaction temperature of liquid enzyme preparation

[0141] 0.1 mol / L citric acid-sodium citrate buffer was used to prepare the buffer solution with a pH of 5.0, and 2% hesperidin glycoside mixture (including 12% hesperidin and 88% polyglucosyl hesperidin) was added as the substrate solution.

[0142] Substrate solution 500 μl was added to 500 μl of appropriately diluted enzyme preparation, and the reaction was carried out at 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C and 65°C respectively for 30 minutes, then 1 ml of DNS reagent was added, heated in boiling water for 10 minutes, cooled to room temperature, and the absorbance was measured at 540 nm with a cuvette. The enzyme preparation inactivated by boiling for 10 minutes was used as a negative control for each temperature gradient test group. The relative enzyme activity of each temperature gradient was calculated based on the enzyme activity of the test group with the highest absorbance as 100%, and three parallel samples were set for each test group. The results are shown in Table 1. Figure 6 .

[0143] The results show that the enzyme has certain heat resistance, and the optimal reaction temperature range is 50°C to 55°C.

[0144] Example 3: Production of α-GH using liquid enzyme preparation containing β-cyclodextrin glycosyltransferase and enzyme solution obtained from Aspergillus niger HW16

[0145] (1) Preparation of liquid enzyme preparation containing β-cyclodextrin glycosyltransferase

[0146] Paenibacillus silvae HW253 was fermented in a fermentation medium (1.0% wheat starch, 0.5% yeast extract, 1.0% peptone, 0.3% potassium dihydrogen phosphate, 0.05% calcium chloride, 0.01% magnesium sulfate) at 35°C under sufficient aeration for 72 hours. The fermentation broth was centrifuged at 10,000 rpm, and the obtained supernatant was a crude enzyme solution containing high-activity β-cyclodextrin glycosyltransferase.

[0147] The above crude enzyme solution was further concentrated and purified by membrane filtration technology. The molecular pore size of the membrane used was 10 KDa, and the concentrated side was the refined enzyme solution.

[0148] After appropriate dilution of the refined enzyme solution, 30% (v / v) glycerol, 0.2% (m / v) potassium sorbate and 0.1% (m / v) nipagin methyl ester were added to obtain a liquid enzyme preparation. The liquid enzyme preparation can be stored at 4°C for more than 3 months. The activity of the enzyme preparation is calibrated by the β-cyclodextrin production activity, and the determination method is as described above, with a requirement of 300 U / ml.

[0149] (2) Glycosylation reaction of hesperidin

[0150] Take 5 g of hesperidin (purchased from Sichuan Qingyi Pure) and 20 g of maltodextrin, add sodium hydroxide, adjust the solution to alkaline (pH 13), at this time the hesperidin will be better dissolved, then adjust the pH of the substrate solution to 8.0, and then add the liquid enzyme preparation obtained in step (1) to 10 g, and react at 60°C for 30 hours, and finally sterilize at 90°C for 30 minutes. The hesperetin glycoside reactant (including 12% unreacted hesperidin and 88% polyglucosyl hesperidin) can be obtained.

[0151] (3) Hydrolysis reaction of polyglucosyl hesperidin and hesperidin

[0152] Take the Aspergillus niger HW16 liquid enzyme preparation described in Example 2 and mix it with the above hesperetin glycoside reactant (liquid enzyme preparation to hesperetin glycoside reactant mass ratio of 1:100), adjust the pH to 5.0, and stir at 55°C for 3 hours. After hydrolysis is complete, adjust the pH to 3.0, and heat to 90°C for 30 minutes to inactivate the enzyme. At this time, a significant precipitate is observed, and the hydrolysis solution is centrifuged at 10,000 rpm for 10 minutes to obtain the supernatant, which contains α-GH, glucose and a small amount of oligoglucose.

[0153] (4) Analysis of the hydrolysis process

[0154] Take the hesperidin substrate solution, the final product of the hesperidin glycosylation reaction obtained in step (2), the reaction liquid after 1 hour and 3 hours of hydrolysis of polyglucosyl hesperidin and hesperidin, respectively, centrifuge and dilute for high performance liquid analysis, and the results are as follows Figure 7 and Figure 8 .

[0155] First, under the action of β-cyclodextrin glycosyltransferase, hesperidin is converted into polyglucosyl hesperidin. After 1 hour of action of the Aspergillus niger HW16 liquid enzyme preparation, the polyglucosyl hesperidin is converted into α-GH, while the unreacted hesperidin is first converted into 7-O-β-monoglucosyl hesperidin, and then further degraded into hesperidin, at this time the reaction liquid is still clear, and hesperidin has not precipitated. After 3 hours of reaction, hesperidin precipitates, and the supernatant after centrifugation of the reaction liquid is analyzed by liquid chromatography, and the flavonoid component is only α-GH.

[0156] (5) Purification of α-GH using ultrafiltration technology

[0157] The hydrolysis supernatant obtained in step (3) above was concentrated using an ultrafiltration membrane with a molecular weight cut-off of 200 Da. During the concentration process, an equal volume of pure water was added to rinse the membrane. After concentration, glucose and maltose were removed on the permeate side and discarded, and the concentrated side contained high concentrations of a-GH and a small amount of oligomeric glucose. The concentrated solution was spray dried to obtain a product with a purity of 95% a-GH.

[0158] (6) Purification of a-GH using a resin

[0159] The hydrolysis supernatant obtained in step (3) above was treated using HP-10 resin. The amount of resin used was 300 g / L of supernatant, and the loading flow rate was 0.5 BV / h. After loading was complete, the resin was washed with pure water at a flow rate of 2 BV / h for 2 BV. Elution was then performed using a 60% ethanol solution at a flow rate of 1 BV / h. This resulted in a solution of a-GH, which was spray dried after vacuum concentration to remove the ethanol to obtain a product with a purity of 98% a-GH.

[0160] Example 4: Production of a-GH using a cyclodextrin glycosyltransferase and an enzyme preparation from Aspergillus niger HW16

[0161] (1) Hesperidin glycosylation reaction

[0162] Hesperidin (5 g, purchased from Sichuan Qingyi) and maltodextrin (20 g) were mixed, and sodium hydroxide was added to adjust the solution to alkaline (pH 13), at which point the hesperidin was well dissolved. The pH of the substrate solution was then adjusted to 9.5, and the volume was made up to 97 ml. Cyclodextrin glycosyltransferase (Amano enzyme preparation a-Amylase G "Amano" L, which has cyclodextrin glycosyltransferase activity, with an enzyme activity of 2182.8 U / mL) was added at 3 ml, and the reaction was performed at 55°C for 30 hours. Finally, the solution was sterilized at 90°C for 30 minutes. This resulted in a hesperetin glycoside reaction product (containing 14% unreacted hesperidin and 86% polyglucosyl hesperidin).

[0163] (2) Hydrolysis reaction of polyglucosyl hesperidin and hesperidin

[0164] The liquid enzyme preparation of Aspergillus niger HW16 described in Example 2 was mixed with the hesperetin glycoside reaction product described above (liquid enzyme preparation:hesperetin glycoside reaction product mass ratio of 1:100), and the pH was adjusted to 5.0. The mixture was stirred at 55°C for 3 hours. After the hydrolysis was complete, the pH was adjusted to 3.0, and the temperature was increased to 90°C to inactivate the enzyme. At this point, a significant precipitate was observed, and the hydrolysis solution was centrifuged at 10,000 rpm for 10 minutes to obtain the supernatant, which contained a-GH, glucose, and a small amount of oligomeric glucose.

[0165] (3) Purification of a-GH by ultrafiltration

[0166] The hydrolysis supernatant obtained in step (2) above was concentrated using an ultrafiltration membrane with a molecular weight cut-off of 200 Da. During the concentration process, an equal volume of purified water was added to rinse. After concentration, glucose and maltose were both in the permeate side and discarded, and the concentrated side contained high concentration of a-GH and a small amount of oligomeric glucose. The concentrated solution was spray dried to obtain a-GH. The product was analyzed by HPLC, and the results are shown in Table 3. Figure 9 A product with a-GH purity of 95% was obtained.

[0167] (4) Purification of a-GH by resin

[0168] The hydrolysis supernatant obtained in step (2) above was treated using HP-10 resin. The amount of resin used was 300 g / L of the hydrolysis solution, and the loading flow rate was 0.5 BV / h. After loading, the resin was washed with purified water at a flow rate of 2 BV / h, and the washing volume was 2 BV. After washing, the resin was eluted with 60% ethanol solution at a flow rate of 1 BV / h, and the elution volume was 2 BV. A high-purity a-GH solution was obtained. The sample was vacuum concentrated to remove ethanol and then spray dried to obtain a high-purity glucosyl hesperidin product. The product was analyzed by HPLC, and the results are shown in Table 4. Figure 9 A product with a-GH purity of 98% was obtained.

[0169] As shown in Examples 3 and 4 above, in addition to the fermentation broth of Paenibacillus sp. HW253 (CGMCC No. 35445), a commercially available cyclodextrin glycosyltransferase can also be used to prepare high-purity a-GH after glucosyl hesperidin is prepared.

[0170] Comparative Example 1: Production of a-GH using enzyme broth obtained by fermentation of Aspergillus niger CICC 2279

[0171] (1) Preparation of enzyme broth (containing glucose amylase)

[0172] Fermentation medium (in mass percentage): 1.0% corn starch, 0.5% yeast extract, 2.0% peptone, 0.5% ammonium sulfate, 0.2% potassium dihydrogen phosphate, 0.05% calcium chloride, 0.01% magnesium sulfate.

[0173] The medium components were dissolved in water in the proportions indicated, sterilized at 121 °C for 30 minutes, and cooled to 30 °C. A colony of strain CICC 2279 was picked into the fermentation medium and incubated at 30 °C for 20 hours with vigorous agitation and aeration as a seed culture. The seed culture of A. niger CICC 2279 was inoculated into the cooled fermentation medium at a volume ratio of 10% and incubated at 30 °C for 72 hours with vigorous agitation and aeration to obtain the fermentation broth.

[0174] The fermentation broth was centrifuged at 10,000 rpm for 10 minutes, and the precipitate was discarded to obtain the fermentation broth supernatant. The fermentation broth was then filtered through a 0.22 μm filter to remove the bacterial cells and insoluble components, and the resulting supernatant was the enzyme solution (crude enzyme solution). The enzyme solution was treated by centrifugation, microfiltration sterilization, and ultrafiltration, and the enzyme activity was diluted to 42900 ± 30 U / ml. The enzyme solution could be stored at 4 °C for more than 3 months after the addition of 30% (v / v) glycerol, 0.2% (m / v) potassium sorbate, and 0.1% (m / v) methyl paraben.

[0175] (2) Hesperidin glycosylation reaction

[0176] Hesperidin (5 g, purchased from Sichuan Qingyi Pure) and maltodextrin (20 g) were mixed, and sodium hydroxide was added to adjust the solution to alkaline (pH 13), at which time the hesperidin was better dissolved. The pH of the substrate solution was then adjusted to 9.5, and the volume was made up to 90 ml. Liquid enzyme preparation prepared from B. forestii HW253 in Example 3 was added (10 g), and the reaction was carried out at 60 °C for 30 hours. Finally, the solution was sterilized at 90 °C for 30 minutes. Hesperetin glycoside reactants (including 12% unreacted hesperidin and 88% polyglucosyl hesperidin) were obtained.

[0177] (3) Hydrolysis reaction of polyglucosyl hesperidin and hesperidin

[0178] The enzyme solution of step (1) was mixed with the above hesperetin glycoside reactants (mass ratio 1:100) at pH 5.0 and 55 °C with vigorous stirring for 3 hours. After the hydrolysis was complete, the pH was adjusted to 3.0, and the temperature was raised to 90 °C for 30 minutes to inactivate the enzyme. At this time, the hydrolyzate was still clear, and high-performance liquid chromatography was used to detect the hydrolyzate. As shown in FIG. 6, the hydrolyzate contained α-GH and hesperidin. Figure 10

[0179] A. niger CICC 2279 is a commonly used strain for producing glucoamylase, and the glucose amylase secreted by the strain can convert polyglucosyl hesperidin into α-GH, but does not produce α-L-rhamnosidase and β-D-glucosidase, and cannot hydrolyze hesperidin into hesperetin. Therefore, not all A. niger strains have the ability to produce α-L-rhamnosidase and β-D-glucosidase.​

[0180] Example 2: Production of a-GH using the enzyme solution obtained by fermentation of Aspergillus niger CICC 2279 and hesperidinase

[0181] (1) Preparation of glucoamylase

[0182] According to Example 1, the enzyme solution was prepared using Aspergillus niger CICC 2279.

[0183] (2) Hesperidin glycosylation reaction

[0184] 5 g of hesperidin (purchased from Sichuan Qingyi Pure) and 20 g of maltodextrin were weighed and mixed, and sodium hydroxide was added to adjust the solution to alkaline (pH 13), at which time the hesperidin was better dissolved. Then the pH of the substrate solution was adjusted to 9.5, and the volume was made up to 90 ml. 10 g of liquid enzyme preparation prepared from B. forestii HW253 in Example 3 was added, and the reaction was carried out at 60°C for 30 hours. Finally, sterilization was carried out at 90°C for 30 minutes. The hesperidin glycoside reactant (including 12% unreacted hesperidin and 88% polyglucosyl hesperidin) was obtained.

[0185] (3) Hydrolysis reaction of polyglucosyl hesperidin and hesperidin

[0186] According to the mass fraction, 1 part of the enzyme solution prepared from Aspergillus niger CICC 2279 and 1 part of hesperidinase (containing a-L-rhamnose glycosidase and b-D-glucosidase, purchased from Zhengzhou Alpha Chemical Co., Ltd.) were mixed with 98 parts of the above-mentioned hesperidin glycoside reactant, and the pH was adjusted. Under the condition of 55°C, it was fully stirred for 3 hours. After the hydrolysis was completed, the pH was adjusted to 3.0, and the temperature was raised to 90°C, and the enzyme was inactivated for 30 minutes. At this time, the hydrolysis solution was clear. The hydrolysis solution was detected by high performance liquid chromatography, as shown in Figure 11 . It can be seen that the hydrolysis solution contains a-GH and hesperetin.

[0187] As can be seen, under normal circumstances, the crude enzyme solution (containing glucoamylase) produced by Aspergillus niger and hesperidinase (including a-L-rhamnose glycosidase and b-D-glucosidase) can finally hydrolyze polyglucosyl hesperidin and hesperidin into a-GH and hesperetin, but hesperetin does not precipitate.

[0188] Example 3: Production of a-GH using glucoamylase and hesperidinase

[0189] (1) Hesperidin glycosylation reaction

[0190] Take 5 g of hesperidin (purchased from Sichuan Qingyi Pure) and 20 g of maltodextrin, add sodium hydroxide to adjust the solution to alkaline (pH 13), at which time the hesperidin will be better dissolved, then adjust the pH of the substrate solution to 9.5, and then add 10 g of liquid enzyme preparation prepared by forest bacillus HW253 in Example 3, and react at 60℃ for 30 hours, and finally sterilize at 90℃ for 30 minutes. Hesperitin glycoside reactant (including 12% unreacted hesperidin and 88% polyglucosyl hesperidin) can be obtained.

[0191] (2) Hydrolysis reaction of polyglucosyl hesperidin and hesperidin

[0192] According to the mass fraction, take 1 part of commercially available glucoamylase (purchased from Shandong Longkete Enzyme Preparation Co., Ltd., activity is 150000 U / ml), 1 part of hesperidinase (Zhengzhou Alpha Chemical Co., Ltd.) and 500 parts of the above hesperitin glycoside reactant, and mix them under the condition of pH 5.0 and 55℃, and fully stir for 3 hours. After hydrolysis is completed, adjust the pH to 3.0, and heat to 90℃, and keep for 30 minutes to inactivate the enzyme. At this time, the hydrolysis solution is still clear. The hydrolysis solution is detected by high performance liquid chromatography, such as Figure 12 It can be seen that the hydrolysis solution contains α-GH and hesperitin.

[0193] As can be seen, under normal circumstances, using a complex enzyme system (including glucoamylase, α-L-rhamnosidase, β-D-glucosidase) can also finally hydrolyze polyglucosyl hesperidin and hesperidin into α-GH and hesperitin, but hesperitin will not automatically precipitate.

Claims

1. A type of Aspergillus niger, characterized in that, The preservation number of the Aspergillus niger is CGMCC No. 42150.

2. A fermentation broth, characterized in that, The fermentation broth is obtained by fermentation with Aspergillus niger as described in claim 1.

3. The fermentation broth as described in claim 2, characterized in that, The fermentation meets one or more of the following conditions: (1) The fermentation medium comprises the following components by weight percentage: 1.0%-5.0% corn starch, 0.1%-2.0% yeast extract, 0.1%-2.0% peptone, 0.1%-2.0% ammonium sulfate, 0.1%-1.0% potassium dihydrogen phosphate, 0.01%-0.10% calcium chloride and 0.001%-0.10% magnesium sulfate; for example, it includes: 1.0% corn starch, 0.5% yeast extract, 2.0% peptone, 0.5% ammonium sulfate, 0.2% potassium dihydrogen phosphate, 0.05% calcium chloride and 0.01% magnesium sulfate; (2) The inoculum amount is 1%-10%, which is the volume ratio of the seed liquid containing the Aspergillus niger to the fermentation medium; (3) The fermentation temperature is 25-30℃; (4) Fermentation time is 24-72 hours; and (5) Fermentation is carried out under aerated conditions.

4. An enzyme preparation, characterized in that, The enzyme preparation is obtained by extraction from the fermentation broth as described in claim 2 or 3.

5. The enzyme preparation according to claim 4, characterized in that, The extraction includes the following steps: the fermentation broth is centrifuged and filtered to obtain the supernatant; The centrifugation speed is preferably 4000-10000 rpm; The centrifugation time is preferably 5 min to 30 min, for example 10 min; The filtration is preferably carried out using filter cloth, filter paper, or filter membrane, and the pore size of the filter membrane is, for example, 0.22 μm. Preferably, the filtration process further includes a concentration step; the concentration is preferably carried out using a membrane, and the membrane has a pore size of, for example, 3 kDa. Preferably, the concentration process further includes a step of mixing the resulting concentrate with excipients, such as glycerin, potassium sorbate, and methylparaben.

6. The use of Aspergillus niger as described in claim 1, the fermentation broth as described in claim 2 or 3, or the enzyme preparation as described in claim 4 or 5 in the preparation of α-monoglucosylhesperidin.

7. The application as described in claim 6, characterized in that, The preparation of α-monoglucosylhesperidin includes the following steps: Under the action of the fermentation broth or the enzyme preparation, the hesperidin glycosylation product is hydrolyzed, followed by solid-liquid separation, and the supernatant is taken to obtain the product; the hesperidin glycosylation product includes polyglucosylhesperidin and unreacted hesperidin. Preferably, the hesperidin glycosylation product is prepared by a method comprising the following steps: The product is prepared by glycosylation of a reaction substrate containing hesperidin and a glycosyl donor under the action of glycosyltransferase.

8. The application as described in claim 7, characterized in that, The preparation of α-monoglucosylhesperidin satisfies one or more of the following conditions: (1) The hesperidin glycosylation product comprises 86%-88% polyglucosyl hesperidin and 12%-14% unreacted hesperidin; the percentages are the mass percentages of each component in the hesperidin glycosylation product. (2) Based on the comprehensive enzyme activity of the fermentation broth or enzyme preparation being 30,000 U / mL, the mass ratio of the hesperidin glycosylation product to "the fermentation broth or enzyme preparation" is (80-120):1, for example, 100:1; (3) The pH of the hydrolysis reaction is 4.0-6.0, preferably 4.5-5.5, for example 5.0; (4) The temperature of the hydrolysis reaction is 40-60℃, preferably 50-55℃; (5) The hydrolysis reaction takes 1-3 hours; and (6) The hydrolysis reaction is followed by an inactivation step; the inactivation is preferably carried out by heating at pH 3.0 and 85-100°C for 10-30 min, for example, heating at pH 3.0 and 90°C for 30 min.

9. The application as described in claim 7, characterized in that, The preparation of α-monoglucosylhesperidin satisfies one or two of the following conditions: (1) The solid-liquid separation is performed by centrifugation or filtration; The centrifugation speed is preferably 4000-10000 rpm; The centrifugation time is preferably 5-30 min, for example 10 min; and (2) The solid-liquid separation is followed by a purification step; the purification is preferably ultrafiltration purification or resin purification; The ultrafiltration purification is preferably performed using an ultrafiltration membrane, the pore size of which is, for example, 150-200 Da. Preferably, the ultrafiltration purification is performed using pure water for washing, and the pure water is added, for example, in equal volumes. The ultrafiltration purification process can be further dried, for example, by spray drying. The resin purification is preferably performed using HP-10 resin. The resin purification preferably includes the following steps: loading the sample, washing with water, and then eluting with ethanol; The resin can be further concentrated and / or dried after purification; the concentration is, for example, vacuum concentration, and the drying is, for example, spray drying.

10. The application as described in claim 7, characterized in that, The preparation of the hesperidin glycosylation product satisfies one or more of the following conditions: (1) The glycosyl donor includes one or more of maltodextrin, β-cyclodextrin and starch, such as maltodextrin; (2) The mass ratio of the hesperidin to the glycosyl donor is 1:(2-5), for example 1:4; (3) The pH of the reaction substrate is 7.5-10.5, for example 8.0; (4) The glycosyltransferase is a cyclodextrin glycosyltransferase, for example, β-cyclomaltodextrin glycosyltransferase; (5) The glycosyltransferase is used in the form of the glycosyltransferase itself, fermentation broth containing the glycosyltransferase, or glycosyltransferase enzyme solution; (6) The temperature of the glycosylation reaction is 45-75℃, for example 55-60℃; (7) The glycosylation reaction takes 1-40 hours, for example, 30 hours; and (8) The glycosylation reaction is followed by an inactivation step; the inactivation is preferably carried out by heating at 85-100°C for 10-30 min, for example, heating at 90°C for 30 min.

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