Preparation method and application of an enzyme preparation for enzymatically hydrolyzing bitter substances in citrus juice

Abstract

The invention discloses a preparation method and application of an enzyme preparation for enzymatically hydrolyzing bitter substances in citrus juice. Using Aspergillus tabingensis UA13 isolated in the early stage of the laboratory and mutagenized by atmospheric and room temperature plasma (ARTP) as the starting strain, limonin in lemon peel powder is used as an inducer to induce enzyme production, and the fermentation enzyme production process of the strain and the substrate affinity of the enzyme are studied. The results show that: in a 1 L fermentor medium, under the optimal conditions of limonin addition amount of 4.0 mg / L, inoculation amount of 10% (v / v), initial pH value of 5.0, stirring speed of 250 rpm, and fermentation at 30 °C, the highest limonin debittering enzyme activity can reach 48.52 U / mL. The results of the study on the substrate affinity of the enzyme for limonin show that the Km value of the limonin debittering enzyme for limonin is 0.622 mmol / L, indicating strong affinity. At the same time, the enzyme can still efficiently hydrolyze other bitter substances such as naringin and hesperidin, has good heat resistance and acid resistance, and has very good application effects in the citrus juice processing industry.

Description

Technical Field

[0001] This invention belongs to the field of enzyme preparation technology, specifically relating to a method for preparing an enzyme preparation for enzymatically hydrolyzing bitter substances in citrus juice and its application. Background Technology

[0002] The bitter substances in citrus fruits mainly fall into two categories. One category consists of flavonoids, including naringin, neohesperidin, citrus glycosides, tangeretin, nobiletin, and quercetin, with naringin being the most abundant and the most prominent flavanone glycoside bitter substance. The other category comprises highly oxidized triterpenoid compounds—limonene analogues, with limonene being the primary bittering agent. The main bitter substances in citrus fruits, naringin and limonene, are most abundant in grapefruit, followed by grapefruit, citrus or hybrid citrus fruits, mandarins, navel oranges, and common sweet oranges. The "delayed bitterness" or "post-bitterness" resulting from these naturally occurring bitter substances in citrus fruits during processing severely restricts the development of the citrus processing industry and is one of the major challenges currently facing the industry. Therefore, industrially, it is essential to debitter citrus fruits to improve the quality of processed products.

[0003] Limonin is the main substance responsible for the bitterness and "after-bitterness" of citrus fruits, significantly affecting the taste of citrus juice. Also known as limonin, it is a triterpenoid compound with a bitter threshold of 1.0 mg / kg in aqueous solution and 3.4 mg / L in juice, approximately 20 times that of naringin. Limonin is present in low concentrations in fresh fruit or juice, primarily as the non-bitter limonin A-cyclic lactone in the cytoplasm. However, prolonged storage or heat treatment intensifies its bitterness. This "after-bitterness" phenomenon occurs because, under acidic conditions (pH < 6.5), limonin A-cyclic lactone is converted to the bitter limonin by limonin D-cyclic lactone hydrolase. Freezing, mechanical damage, or heat treatment of fruit pulp increases acidity, thus promoting limonin formation during processing or storage.

[0004]

[0005] The enzymatic hydrolysis mechanism of naringin is as follows:

[0006]

[0007] The hydrolysis of naringin is mainly completed in two steps. Naringin (4'-5,7'-trihydroxydihydroflavon-7-rhamnosylglucoside) is hydrolyzed by rhamnosidase into rhamnose and purine. The bitterness of purine is about one-third that of naringin. Purine is then hydrolyzed by β-D-glucosidase into non-bitter naringenin and glucose.

[0008] As can be seen from the above, the enzymatic hydrolysis mechanisms of limonene and naringin are completely different.

[0009] Currently reported methods for debittering fruit juice include the β-cyclodextrin method, supercritical CO2 method, membrane separation method, addition of bitterness inhibitors, and enzymatic methods. Compared with other debittering methods, enzymatic methods not only have the advantages of good effect, no pollution, simple process, and mild reaction conditions, but also their enzymatic hydrolysis products can enhance the flavor of the juice to a certain extent and retain the nutritional components of the juice, making them more suitable for industrial production. The main reason why enzymatic methods have not been widely used in the citrus processing industry is the lack of highly active enzyme preparations. Therefore, it is crucial to prepare enzyme preparations with stable performance and high activity. Currently, the naringinase powder sold on the market only has naringin as its substrate, and has almost no effect on limonene and nomilin bitter substances, with an activity of only 0.1-0.4 U / mL. Pure naringinase used to be able to be imported from abroad, but in recent years, major foreign enzyme preparation companies have not had it in stock, requiring orders to be placed more than six months in advance, and the price is expensive, which is very limiting.

[0010] Chinese patent CN104404016B discloses a method for producing naringinase using a traditional liquid fermentation method. However, the resulting naringinase exhibits low activity and a thermal stability of only 45℃. Chinese patent CN101914451A discloses a method for producing α-L-rhamnosidase from *Alternaria*. The α-L-rhamnosidase produced by *Alternaria* is an intracellular enzyme, requiring extraction from cells, which is difficult and directly leads to high usage costs. Chinese patent CN105441410A discloses a method for producing rhamnosidase from *Chaetoceros*, yielding an enzyme activity of 500 U / mL, but it does not investigate substrate specificity. These research findings demonstrate the significant attention given to α-L-rhamnosidase or naringinase research in recent years; however, research on enzyme preparations with complex debittering enzyme properties is scarce. Summary of the Invention

[0011] This invention proposes a method for preparing an enzyme preparation for enzymatically hydrolyzing bitter substances in citrus juice and its application, which is an enzyme preparation capable of simultaneously enzymatically hydrolyzing bitter substances in citrus juice—limonin and naringin.

[0012] To achieve the above objectives, the present invention adopts the following technical solution:

[0013] A species of Aspergillus tubingensis, named Aspergillus tubingensis UA13, was deposited on June 27, 2022, at the China Center for Type Culture Collection (CCTCCNO: M20222989), located at Wuhan University, Wuhan, Hubei Province.

[0014] Preferably, Aspergillus tabineum is used in the fermentation process to prepare limonene debittering enzyme.

[0015] More preferably, the application involves adding limonene or lemon peel powder containing limonene to the fermentation medium of Aspergillus tabbinus, with the amount of limonene added being 4.0-6.0 mg / L.

[0016] Preparation of lemon peel powder: Remove the white pith from fresh lemon peel, slice it, dry it, and then grind it into powder to 100 mesh for later use.

[0017] More preferably, the culture medium comprises a fermentation medium containing: MgSO4·7H2O 0.40-0.60 g / L, KH2PO4 1.20-1.80 g / L, K2HPO4 1.20-1.80 g / L, (NH4)2SO4 3.50-4.50 g / L, ZnSO4·7H2O 0.05-0.15 g / L, CaCl2 0.05-0.15 g / L, yeast extract 0.80-1.20 g / L, soybean flour 1.50-2.50 g / L, peptone 1.50-2.50 g / L, limonene 4.00-5.00 mg / L, and natural pH.

[0018] More preferably, in the fermentation medium:

[0019] MgSO4·7H2O 0.50g / L, KH2PO4 1.50g / L, K2HPO4 1.50g / L, (NH4)2SO4 4.00g / L, ZnSO4·7H2O 0.10g / L, CaCl2 0.10g / L, yeast extract 1.00g / L, soybean flour 2.00g / L, peptone 2.00g / L, limonene 4.00mg / L.

[0020] The application of Aspergillus tabineum in the preparation of a complex enzyme preparation for enzymatic hydrolysis of bitter substances, mainly limonene.

[0021] The applications include:

[0022] Solid plate culture medium: MgSO4·7H2O 0.80-1.20 g / L, KH2PO4 0.80-1.20 g / L, (NH4)2SO4 1.30-1.70 g / L, KCl 0.30-0.80 g / L, KNO3 1.20-1.70 g / L, CaCl2 0.05-0.15 g / L, yeast extract 1.50-2.50 g / L, limonene 0.50-1.50 mg / L, agar powder 8.00-12.00 g / L, natural pH;

[0023] Seed culture medium: limonene 3.50-4.50 mg / L, MgSO4·7H2O 4.00-6.00 g / L, K2HPO4 4.00-7.00 g / L, KCl 4.00-6.00 g / L, FeSO4·5H2O 0.05-0.15 g / L, natural pH;

[0024] The fermentation medium contains: MgSO4·7H2O 0.40-0.60 g / L, KH2PO4 1.20-1.80 g / L, K2HPO4 1.20-1.80 g / L, (NH4)2SO4 3.50-4.50 g / L, ZnSO4·7H2O 0.05-0.15 g / L, CaCl2 0.05-0.15 g / L, yeast extract 0.80-1.20 g / L, soybean flour 1.50-2.50 g / L, peptone 1.50-2.50 g / L, and limonene 4.00-5.00 mg / L.

[0025] Solid plate culture medium, seed culture medium, and fermentation culture medium were used to expand the culture in sequence.

[0026] The application of *Aspergillus tabineus* in the preparation of a complex enzyme preparation for enzymatic hydrolysis of bitter substances, mainly naringin, includes:

[0027] Add 8.00-12.00 g / L of pomelo peel powder or 4.00-6.00 g / L of naringin to the solid plate culture medium; add 8.00-12.00 g / L of pomelo peel powder to the seed culture medium; add 8.00-12.00 g / L of pomelo peel powder or 4.00-6.00 g / L of naringin to the fermentation culture medium; the other components of the culture medium are as follows:

[0028] Solid plate culture medium: MgSO4·7H2O 0.80-1.20 g / L, KH2PO4 0.80-1.20 g / L, (NH4)2SO4 1.30-1.70 g / L, KCl 0.30-0.80 g / L, KNO3 1.20-1.70 g / L, CaCl2 0.05-0.15 g / L, yeast extract 1.50-2.50 g / L, agar powder 8.00-12.00 g / L, natural pH;

[0029] Seed culture medium: MgSO4·7H2O 4.00-6.00 g / L, K2HPO4 4.00-7.00 g / L, KCl 4.00-6.00 g / L, FeSO4·5H2O 0.05-0.15 g / L, natural pH;

[0030] The fermentation medium contains: MgSO4·7H2O 0.40-0.60 g / L, KH2PO4 1.20-1.80 g / L, K2HPO4 1.20-1.80 g / L, (NH4)2SO4 3.50-4.50 g / L, ZnSO4·7H2O 0.05-0.15 g / L, CaCl2 0.05-0.15 g / L, yeast extract 0.80-1.20 g / L, soybean flour 1.50-2.50 g / L, and peptone 1.50-2.50 g / L.

[0031] Solid plate culture medium, seed culture medium, and fermentation culture medium were used to expand the culture in sequence.

[0032] Preparation of grapefruit peel powder: Remove the white pith layer from fresh grapefruit peel, slice it, dry it, and then grind it into powder to 100 mesh for later use.

[0033] Culture conditions: Liquid fermentation is carried out at an initial pH of 5.0-6.0, a culture temperature of 30-35℃, an aeration rate of 1.3-2.5 min / L, and a stirring speed of 250-300 rpm for 70-77 h to obtain the compound enzyme fermentation broth with maximum enzyme activity.

[0034] The application of *Aspergillus tabineus* in the preparation of complex enzyme preparations for enzymatic hydrolysis of bitter substances, mainly limonene, includes the following applications:

[0035] Add 8.00-12.00 g / L of lemon peel powder or 4.00-6.00 g / L of limonene to the solid plate culture medium, add 8.00-12.00 g / L of lemon peel powder to the seed culture medium component, and add 8.00-12.00 g / L of lemon peel powder or 4.00-6.00 g / L of limonene to the fermentation culture medium;

[0036] The other components of the culture medium are as follows:

[0037] Solid plate culture medium: MgSO4·7H2O 0.80-1.20 g / L, KH2PO4 0.80-1.20 g / L, (NH4)2SO4 1.30-1.70 g / L, KCl 0.30-0.80 g / L, KNO3 1.20-1.70 g / L, CaCl2 0.05-0.15 g / L, yeast extract 1.50-2.50 g / L, agar powder 8.00-12.00 g / L, natural pH;

[0038] Seed culture medium: MgSO4·7H2O 4.00-6.00 g / L, K2HPO4 4.00-7.00 g / L, KCl 4.00-6.00 g / L, FeSO4·5H2O 0.05-0.15 g / L, natural pH;

[0039] The fermentation medium contains: MgSO4·7H2O 0.40-0.60 g / L, KH2PO4 1.20-1.80 g / L, K2HPO4 1.20-1.80 g / L, (NH4)2SO4 3.50-4.50 g / L, ZnSO4·7H2O 0.05-0.15 g / L, CaCl2 0.05-0.15 g / L, yeast extract 0.80-1.20 g / L, soybean flour 1.50-2.50 g / L, and peptone 1.50-2.50 g / L.

[0040] Solid plate culture medium, seed culture medium, and fermentation culture medium were used to expand the culture in sequence.

[0041] Preparation of lemon peel powder: Fresh lemon peel is sliced, dried, and then ground into powder to 100 mesh for later use;

[0042] Culture conditions: Liquid fermentation is carried out at an initial pH of 5.0-6.0, a culture temperature of 31-34℃, an aeration rate of 1.3-1.8 min / L, and a stirring speed of 250-280 rpm for 70-76 h to obtain the compound enzyme fermentation broth with maximum enzyme activity.

[0043] The application of *Aspergillus tabineus* in the preparation of a complex enzyme preparation for enzymatic hydrolysis of bitter substances, mainly naringin and limonene, includes:

[0044] Add 8.00-12.00 g / L of grapefruit peel powder or 4.00-6.00 g / L of naringin to the solid plate culture medium; add 8.00-12.00 g / L of lemon peel powder to the seed culture medium component; add 8.00-12.00 g / L of lemon peel powder to the fermentation culture medium.

[0045] The other components of the culture medium are as follows:

[0046] Solid plate culture medium: MgSO4·7H2O 0.80-1.20 g / L, KH2PO4 0.80-1.20 g / L, (NH4)2SO4 1.30-1.70 g / L, KCl 0.30-0.80 g / L, KNO3 1.20-1.70 g / L, CaCl2 0.05-0.15 g / L, yeast extract 1.50-2.50 g / L, agar powder 8.00-12.00 g / L, natural pH;

[0047] Seed culture medium: MgSO4·7H2O 4.00-6.00 g / L, K2HPO4 4.00-7.00 g / L, KCl 4.00-6.00 g / L, FeSO4·5H2O 0.05-0.15 g / L, natural pH;

[0048] The fermentation medium contains: MgSO4·7H2O 0.40-0.60 g / L, KH2PO4 1.20-1.80 g / L, K2HPO4 1.20-1.80 g / L, (NH4)2SO4 3.50-4.50 g / L, ZnSO4·7H2O 0.05-0.15 g / L, CaCl2 0.05-0.15 g / L, yeast extract 0.80-1.20 g / L, soybean flour 1.50-2.50 g / L, and peptone 1.50-2.50 g / L.

[0049] Solid plate culture medium, seed culture medium, and fermentation culture medium were used to expand the culture in sequence.

[0050] Culture conditions: Liquid fermentation is carried out at an initial pH of 5.0-6.0, a culture temperature of 30-35℃, an aeration rate of 1.3-1.8 min / L, and a stirring speed of 250-300 rpm for 65-75 h to obtain the compound enzyme fermentation broth with maximum enzyme activity.

[0051] Beneficial effects of this invention:

[0052] 1. The enzyme produced by strain UA13 has a strong affinity for limonin. Furthermore, research results show that the limonin debittering enzyme can still efficiently hydrolyze naringin, meaning that strain UA13 of this invention can simultaneously enzymatically hydrolyze limonin, naringin, and hesperidin. Therefore, this enzyme has excellent application prospects in the citrus juice processing industry.

[0053] 2. Limonene debittering enzyme has good heat resistance and acid resistance, which facilitates the widespread use of enzyme preparations. It can be used for different types of citrus fruits, citrus fruits at different maturity stages, or different processing conditions.

[0054] 3. The enzymes produced by strain UA13 have enzymatic hydrolysis capabilities regardless of whether the substrates are limonenes or naringin. The enzymatic hydrolysis capabilities vary slightly depending on the substrate.

[0055] 4. During the enzyme production process of bacterial strain culture, enzyme production can be induced by corresponding inducers according to the different requirements of the substrate, thereby obtaining specific enzyme substances. Attached Figure Description

[0056] Figure 1 Fermentation curve of strain UA13 in a 1L fermenter under unoptimized fermentation conditions;

[0057] Figure 2 The effect of stirring speed on enzyme production by strain UA13;

[0058] Figure 3 The effect of fermentation temperature on enzyme production by strain UA13;

[0059] Figure 4 The effect of initial pH of fermentation broth on enzyme production by strain UA13;

[0060] Figure 5 Linewear-Burk curves of different substrates catalyzed by limonene debittering enzyme. Detailed implementation method:

[0061] The present invention will be further described below with reference to specific embodiments. The present invention will be described in further detail, but is not limited to these embodiments.

[0062] Example 1

[0063] A species of Aspergillus tubingensis, named Aspergillus tubingensis UA13, was deposited on June 27, 2022, at the China Center for Type Culture Collection (CCTCCNO: M20222989), located at Wuhan University, Wuhan, Hubei Province.

[0064] Example 2

[0065] Using the strain from Example 1, limonene debittering enzyme was prepared. The fermentation conditions were adjusted, and the specific process is as follows:

[0066] Solid culture medium: MgSO4·7H2O 1.00 g / L, KH2PO4 1.00 g / L, (NH4)2SO4 1.50 g / L, KCl 0.50 g / L, KNO3 1.50 g / L, CaCl2 0.10 g / L, yeast extract 2.00 g / L, limonene 1.00 mg / L, agar powder 10.00 g / L, natural pH.

[0067] Seed culture medium: limonene 4.00 mg / L, MgSO4·7H2O 5.00 g / L, K2HPO4 5.00 g / L, KCl 5.00 g / L, FeSO4·5H2O 0.10 g / L, natural pH, inoculum size 10%.

[0068] The fermentation medium contained the following components: MgSO4·7H2O 0.50 g / L, KH2PO4 1.50 g / L, K2HPO4 1.50 g / L, (NH4)2SO4 4.00 g / L, ZnSO4·7H2O 0.10 g / L, CaCl2 0.10 g / L, yeast extract 1.00 g / L, soybean flour 2.00 g / L, peptone 2.00 g / L, and limonene 4.00 mg / L.

[0069] 1 Experimental Methods

[0070] 1.1 Fermentation Culture

[0071] Spore suspension: The laboratory-stored slant strain *Aspergillus tabingensis* UA13 was inoculated onto solid agar plates and incubated at 30°C for 3–4 days until spores matured. The spores were washed off with 0.85% sterile physiological saline and dispersed to obtain a bacterial suspension. The spore suspension was examined and counted under a microscope, and the concentration was adjusted to 10⁻⁶. 7 Approximately one per mL is sufficient.

[0072] Seed culture: Inoculate the seed culture medium with the adjusted spore suspension at an inoculation rate of 10% (v / v) and culture it in a constant temperature shaker at 30℃ and 180r / min.

[0073] Fermentation: Using a sterile pipette tip, 5 mL of the adjusted spore suspension (10% inoculum, v / v) was inoculated into a sterilized 50 mL / 250 mL fermentation medium (containing 5 glass beads). The medium was then incubated at 30°C and 180 rpm for 72 h in a constant-temperature shaker.

[0074] 1.2 Enzyme activity assay

[0075] (1) Preparation of crude enzyme solution: Take the fermentation broth from the shake flask fermentation culture for 72 h into a centrifuge tube and centrifuge at 5000 r / min for 20 min.

[0076] (2) Determination of limonene content: 0.3, 0.6, 0.9, 1.2, 1.5, and 1.8 mL of 200 μg / mL limonene standard solution were placed in 25 mL stoppered test tubes, respectively. Anhydrous ethanol was added to bring the volume to 2 mL, and then 5 mL of colorimetric reagent was added to each tube. After thorough mixing, the tubes were allowed to stand for 30 min for color development, and the absorbance was measured at 500 nm. The standard curve equation, with limonene content as the x-axis and absorbance as the y-axis, was A = 0.0038C + 0.0379, R... 2 =0.9997.

[0077] Preparation of colorimetric solution (prepare immediately before use): Accurately weigh 125 mg of p-diaminobenzaldehyde and dissolve it in 100 mL of sulfuric acid-anhydrous ethanol mixture (V:V = 65:35, use after cooling), then add 0.5 mL of 0.9% FeCl and mix well.

[0078] (3) Limonene debittering enzyme activity assay: Add 0.5 mL of 200 μg / mL limonene substrate to a stoppered test tube, then add 0.1 mL of enzyme solution of a certain concentration, and make up to 2 mL with anhydrous ethanol. Place the solution in a constant temperature shaker (150 rpm) at 60℃ for 30 min to inactivate the enzyme. Immediately place the digest in a boiling water bath at 100℃ for 5 min to inactivate the enzyme. Add 5 mL of colorimetric solution to the digest, mix well, and let stand for 30 min to develop color. Pour the solution into a 1 cm cuvette and measure the absorbance at 500 nm using a spectrophotometer.

[0079] All samples were measured in triplicate, and the average value was used for analysis (the same applies below).

[0080] Enzyme activity is defined as the amount of enzyme required to consume 1 μg of limonene per minute at 60°C and pH 4.0.

[0081]

[0082] Relative enzyme activity is defined as follows: the highest enzyme activity in the same group of experiments is defined as 100%, and the ratio of the enzyme activity value under other conditions to the highest value × 100 is defined as the relative enzyme activity (%).

[0083] 1.3 Optimization of enzyme production conditions

[0084] 1.3.1 Shake Flask Fermentation Optimization

[0085] Using limonene debittering enzyme activity as the evaluation index, single-factor experiments were conducted to explore the optimal enzyme production conditions of the strain. Under initial culture conditions, seed culture of a certain age was inoculated, and the inoculum amount (4%–16%, v / v), fermentation temperature (25–40℃), and initial pH of the fermentation broth (4.0–9.0) of the fermentation broth were varied to determine the optimal fermentation environment for the strain.

[0086] 1.3.2 Optimization test of 1L fermenter

[0087] Simultaneous continuous fermentation was carried out in a four-tank 1L fermenter. After simultaneous cultivation of the seed culture, it was inoculated into 600 mL of sterilized fermentation broth (10% inoculum, v / v). Scale-up cultivation was conducted under initial conditions of 0.4 L / min aeration, 300 rpm stirring speed, and an initial pH of 5.0. Based on the shake-flask optimization results, the stirring speed (200–350 rpm), fermentation temperature (25–40℃), and initial pH (4.0–7.0) were further optimized. The optimal enzyme production conditions were determined by observing the strain growth and enzyme production during fermentation.

[0088] The analysis of the fold increase in enzyme activity was based on a comparative study using enzyme activities optimized by shake-flask fermentation.

[0089] 2 Results

[0090] 2.1 Optimization of enzyme production conditions

[0091] 2.1.1 Effect of strain inoculum size on enzyme production

[0092] The concentration of the seed culture and the physiological state of the microorganisms directly affect the fermentation effect. The incubation time of the seed culture medium determines the strength of the seed viability to a certain extent, especially for filamentous fungi, where cell morphology and nutrient mass transfer are affected. Based on previous studies, seed culture medium inoculated with different inoculation amounts for 48 hours was fermented in shake flasks, and the enzyme production activities of the strains are shown in Table 1.

[0093] Table 1. Effects of different inoculum sizes on enzyme production by strain UA13

[0094]

[0095]

[0096] As shown in Table 1 above, the inoculum size has a relatively small impact on the enzyme activity produced by the strain during fermentation. When the inoculum size is within the range of 4%–10% (v / v), the enzyme production capacity of the strain remains at a relatively high level. Considering both enzyme activity and fermentation time, this study determined an inoculum size of 10% (v / v) as the basis for subsequent studies (the same applies below).

[0097] 2.1.2 Effect of fermentation temperature on enzyme production

[0098] The optimal culture temperature for the production of limonene debittering enzyme by changing the fermentation temperature was investigated, and the results are shown in Table 2.

[0099] Table 2 Effect of fermentation temperature on enzyme production by strain UA13

[0100] Temperature / °C Relative enzyme activity (%) 20 59.56 25 68.29 30 83.94 35 100.00 40 74.33

[0101] As shown in Table 2 above, when the strain was fermented within the range of 25–40℃, the enzyme activity of the strain first increased and then decreased with increasing culture temperature, reaching its maximum at 35℃. Therefore, this study used a fermentation temperature of 35℃ for subsequent experiments.

[0102] 2.1.3 Effect of initial pH on enzyme production

[0103] The initial pH of a culture medium can affect the structure and function of enzymes, cell structure, and cell membrane charge. Metabolic products produced by microorganisms during growth also cause changes in the pH of the culture medium. The pH range for fungal growth and enzyme production is approximately 3.0–8.5.

[0104] Table 3 Initial fermentation p Effect of H value on enzyme production by strain UA13

[0105] initial pH value Relative enzyme activity (%) 4 55.34 5 100.00 5.5 78.02 6 76.31 7 73.96 8 69.98 9 28.39

[0106] Different bacterial strains have different optimal pH ranges for growth, metabolism, and enzyme production. As shown in Table 3 above, the strains exhibit stronger enzyme production capacity when the initial fermentation pH is in the range of 5.0–6.0, with enzyme activity reaching its peak at an initial pH of 5.0. Therefore, this study used an initial pH of 5.0 for subsequent experiments.

[0107] Based on the single-factor experiment, using 4.0 mg / L limonene as the carbon source and inducer, and inoculating with 10% (v / v) of seed culture medium fermented for 48 h, and fermenting for 72 h at an initial culture medium pH of 5.0 and a fermentation temperature of 35℃, strain UA13 produced limonene debittering enzyme with the highest activity.

[0108] 2.2 Fermenter Optimization Experiment

[0109] Simultaneous continuous fermentation was performed in a four-tank 1L fermenter. After simultaneous cultivation of the seed culture, it was inoculated into 600mL of sterilized fermentation broth (10% inoculum, v / v). Scale-up cultivation was carried out under initial conditions of 0.4L / min aeration, 300rpm stirring speed, and an initial pH of 5.0. Changes in dissolved oxygen during fermentation reflected the growth status of the strain. Figure 1 As shown, during the first 24 hours of fermentation, the cells were in the lag phase, with a sharp drop in dissolved oxygen, indicating that the oxygen in the culture medium was being fully utilized. Subsequently, dissolved oxygen gradually increased, enzyme biosynthesis began, and the cells entered the exponential growth phase. Enzyme activity then began to increase continuously, reaching a peak of 25.48 U / mL after 72 hours of fermentation. The pH of the culture medium was dynamic, decreasing rapidly at the start of fermentation, then gradually increasing, and finally fluctuating within the range of 4.3–4.8.

[0110] 2.2.1 Effect of stirring speed on enzyme production

[0111] During shake flask cultivation, the rotation of the shaker and the addition of an appropriate amount of glass beads promote the mixing of various substances and prevent mycelial clumping, which would affect enzyme production during fermentation. When scaling up from shake flasks to fermenters, the control of stirring parameters largely determines the efficiency of material mixing and enzyme production within the fermenter. If the stirring speed is too fast, excessive shear force will affect cell formation and composition, adversely affecting enzyme production during fermentation; if the stirring speed is too low, the mycelium will clump together, which is detrimental to fermentation production.

[0112] like Figure 2As shown, at a stirring speed of 200 rpm, due to the low stirring speed, mycelial growth was slow and the lag phase was long, entering the logarithmic growth phase at 36 h. The enzyme production capacity of the cells reached its maximum of 34.04 U / mL at 48 h, and then continued to decline. However, the shake flask experiment showed that the strain usually reached its highest enzyme activity at 72 h. The difference may be due to the low stirring speed of the fermenter, which prevented the mycelium from continuing to break down and reproduce, but instead caused it to entangle into balls on the baffles inside the tank, affecting enzyme production. At a stirring speed of 350 rpm, dissolved oxygen in the fermenter increased, but the excessive shear force accelerated the mycelial death, and the enzyme activity reached its maximum of 33.78 U / mL at 60 h of fermentation. The fermentation trends at stirring speeds of 250 rpm and 300 rpm were not significantly different: at a stirring speed of 250 rpm, the enzyme activity of the strain reached its maximum of 42.61 U / mL at 48 h, an increase of 67.23%. At a fermentation speed of 300 rpm, the enzyme activity of the strain reached a maximum of 43.23 U / mL after 60 hours, an increase of 69.66%. Considering energy conservation in actual fermentation and the small difference in enzyme activity between the two speeds, 250 rpm was chosen as the optimal speed.

[0113] 2.2.2 Effect of fermentation temperature on enzyme production

[0114] like Figure 3 As shown, the fermentation curves of the strain at different temperatures exhibited a generally consistent trend: when the fermentation temperature was controlled at 25℃ and 40℃, the enzyme activity reached its maximum at 48h, at 28.80 U / mL and 30.46 U / mL respectively, which was relatively low, consistent with the shake-flask fermentation. At 30℃ and 35℃, the enzyme activity of the strain reached 40.10 U / mL and 35.61 U / mL respectively at 36h, and both showed secondary growth at 60h. In summary, the strain has good enzyme production capacity at 30℃–35℃. At a culture temperature of 30℃, the strain reached its maximum enzyme activity of 40.10 U / mL earlier at 36h, an increase of 57.38% compared to before optimization, thus shortening the fermentation cycle. Therefore, a culture temperature of 30℃ was selected for subsequent fermenter optimization experiments.

[0115] 2.2.3 Effect of initial pH of fermentation broth on enzyme production

[0116] like Figure 4As shown, under initial pH 5.0 conditions, dissolved oxygen rapidly decreased in the first 12 hours of fermentation. From 12 to 48 hours, the dissolved oxygen in the fermenter approached zero, generally remaining below 10%, indicating that the strain exhibited vigorous metabolism and was more suitable for growth under initial pH 5.0 conditions, consistent with the shake flask experiment results. At 48 hours, the activity of limonene debittering enzyme reached 48.52 U / mL, an increase of 90.42% compared to before optimization. Under initial fermentation pH 6.0 conditions, the strain maintained good enzyme production capacity from 48 to 72 hours, with the highest enzyme activity reaching 40.16 U / mL. At an initial pH of 4.0, the enzyme reached its highest activity of 38.82 U / mL at 96 hours. In summary, the optimal initial pH value for the fermentation broth is 5.0, and Aspergillus tabineii UA13 exhibits strong growth and enzyme production capacity within the initial pH range of 4.0–6.0. ​​This strain is more suitable for survival in a slightly acidic environment, consistent with the shake flask experiment results.

[0117] The strain was optimized in a 1L fermenter. Under the optimal conditions of limonene addition of 4.0 mg / L, inoculum size of 10% (v / v), initial pH of 5.0, stirring speed of 250 rpm, and 30℃ in a 1L fermenter medium, the limonene debittering enzyme activity reached a maximum of 48.52 U / mL, which is 90.42% higher than before optimization.

[0118] The culture medium is:

[0119] Solid culture medium: MgSO4·7H2O 1.00 g / L, KH2PO4 1.00 g / L, (NH4)2SO4 1.50 g / L, KCl 0.50 g / L, KNO3 1.50 g / L, CaCl2 0.10 g / L, yeast extract 2.00 g / L, limonene 1.00 mg / L, agar powder 10.00 g / L, natural pH.

[0120] Seed culture medium: limonene 4.00 mg / L, MgSO4·7H2O 5.00 g / L, K2HPO4 5.00 g / L, KCl 5.00 g / L, FeSO4·5H2O 0.10 g / L, natural pH, inoculum size 10%.

[0121] The fermentation medium contained the following components: MgSO4·7H2O 0.50 g / L, KH2PO4 1.50 g / L, K2HPO4 1.50 g / L, (NH4)2SO4 4.00 g / L, ZnSO4·7H2O 0.10 g / L, CaCl2 0.10 g / L, yeast extract 1.00 g / L, soybean flour 2.00 g / L, peptone 2.00 g / L, and limonene 4.00 mg / L.

[0122] Example 3

[0123] Limonene debittering enzyme was prepared using the optimal conditions of Example 2.

[0124] The optimal conditions for fermentation were: 4.00 mg / L limonene added, 10% (v / v) inoculum, initial pH 5.0, stirring speed 250 rpm, and 30℃. The activity of limonene debittering enzyme reached a maximum of 48.52 U / mL.

[0125] The culture medium is:

[0126] Solid culture medium: MgSO4·7H2O 1.00 g / L, KH2PO4 1.00 g / L, (NH4)2SO4 1.50 g / L, KCl 0.50 g / L, KNO3 1.50 g / L, CaCl2 0.10 g / L, yeast extract 2.00 g / L, limonene 1.00 mg / L, agar powder 10.00 g / L, natural pH.

[0127] Seed culture medium: limonene 4.00 mg / L, MgSO4·7H2O 5.00 g / L, K2HPO4 5.00 g / L, KCl 5.00 g / L, FeSO4·5H2O 0.10 g / L, natural pH, inoculum size 10%.

[0128] The fermentation medium contained the following components: MgSO4·7H2O 0.50 g / L, KH2PO4 1.50 g / L, K2HPO4 1.50 g / L, (NH4)2SO4 4.00 g / L, ZnSO4·7H2O 0.10 g / L, CaCl2 0.10 g / L, yeast extract 1.00 g / L, soybean flour 2.00 g / L, peptone 2.00 g / L, and limonene 4.00 mg / L.

[0129] The supernatant after centrifugation of the fermentation broth is the crude enzyme solution. The changes in enzyme activity of the crude enzyme solution under different enzymatic hydrolysis temperatures (30–70℃) and pH values ​​(2.0–8.0) were investigated. The highest enzyme activity measured in the same group of experiments was taken as 100%, and the relative enzyme activities under other conditions were calculated.

[0130] Enzyme substrate affinity: The Michaelis constant Km of enzymes catalyzing different substrates (limonene, naringin, hesperidin) was determined to explore the enzyme affinity for the substrates. Km was obtained using the double reciprocal method (Linewear-Burk).

[0131] 1. Study on the properties of limonene debittering enzyme

[0132] 1.1 Study on the optimal enzymatic hydrolysis temperature of limonene debittering enzyme

[0133] Temperature is a crucial factor affecting substrate conversion efficiency. At excessively low temperatures, many enzyme molecules remain unactivated, resulting in extremely low enzymatic hydrolysis efficiency. Conversely, temperatures exceeding the enzyme's thermal stability range lead to enzyme inactivation. Optimal enzymatic hydrolysis temperatures maximize substrate conversion efficiency.

[0134] Table 4. Effect of enzymatic hydrolysis temperature on the activity of limonin debittering enzyme

[0135]

[0136]

[0137] Table 4 shows that within the enzymatic hydrolysis temperature range of 30–70℃, the activity of limonene debittering enzyme first increases and then decreases with increasing temperature, reaching its highest level at 60℃. Even at 70℃, the activity of limonene debittering enzyme remains at 59.5%. Therefore, the limonene debittering enzyme in this study exhibits good heat resistance, and subsequent experiments were conducted at an enzymatic hydrolysis temperature of 60℃.

[0138] 1.2 Study on the optimal pH value for limonene debittering enzyme

[0139] Generally, the optimal pH range for fungal naringin production is between 3.0 and 6.0. Inappropriate pH values ​​can affect the conformation of the enzyme protein, thereby reducing enzyme activity.

[0140] Table 5 Different enzymatic hydrolysates p Effect of H value on the activity of limonin debittering enzyme

[0141] Enzymatic hydrolysis pH value Relative enzyme activity (%) 2 67.51 3 90.58 4 98.36 5 100.00 6 94.66 7 81.11 8 54.52

[0142] Table 5 shows that the enzyme activity of strain UA13 initially increased and then decreased under enzymatic hydrolysis conditions ranging from pH 2.0 to 8.0. The enzyme activity reached its highest level at pH 5.0; the relative enzyme activity was 90.58% at pH 3.0; and 81.11% at pH 7.0. Therefore, this enzyme exhibits good acid resistance, and its optimal hydrolysis pH is 5.0.

[0143] 2. Substrate affinity studies

[0144] The reaction rates of limonin debittering enzyme at different substrate concentrations were determined, and the corresponding kinetic parameters were obtained using Linewear-Burk curves. This study found that the fermentation of three different substrates (limonin, hesperidin, and naringin) by the limonin-induced strain followed Michaelis-Menten kinetics. Figure 5 ).

[0145] Experiments show that when the substrate is limonene, K m =0.622mmol / L, Vmax =0.017mmol / (L·min); when the substrate is hesperidin, K m =0.967mmol / L, V max =0.010 mmol / (L·min); when the substrate is naringin, K m =0.999mmol / L, V max =0.018 mmol / (L·min). This enzyme has the ability to hydrolyze limonene, hesperidin, and naringin, with hydrolysis rates of 48%, 38%, and 33% for limonene, hesperidin, and naringin, respectively, within 30 min. The dynamic law of this enzyme's hydrolysis conforms to the kinetics of enzyme-catalyzed reactions, determined by K... m The values ​​indicate that the enzyme's affinity for the substrate is in the order: limonin > hesperidin > naringin. Therefore, this enzyme has a greater affinity for limonin than for naringin, which may be due to limonin acting as both an inducer and a carbon source during the strain's growth.

[0146] The strain was fermented using naringin as an inducer, that is, limonene in the culture medium of this example was replaced with naringin to investigate the affinity of the enzyme for the substrate. It was found that the enzyme induced by naringin had a higher affinity for naringin (K). m =0.480mmol / L), with poor substrate affinity for limonene (K m =91.920 mmol / L), and the hydrolysis rates of limonene, hesperidin, and naringin reached 37%, 47%, and 52% respectively within 30 min. Therefore, it can be inferred that different inducers have a great influence on the growth and enzyme production of the strain. The strains that have been induced and optimized for a long time will have better substrate affinity for the corresponding inducers and stronger ability to enzymatically hydrolyze the corresponding bitter substances.

[0147] Example 4

[0148] Based on the research results of Example 3, three methods for preparing composite enzymes were developed.

[0149] The enzymes were prepared using Aspergillus tubingensis UA13, which was deposited on June 27, 2022, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20222989, located at Wuhan University, Wuhan, Hubei Province.

[0150] 1. A method for preparing a complex enzyme preparation that enzymatically hydrolyzes bitter substances, primarily naringin, the method comprising:

[0151] 10.00 g / L of grapefruit peel powder was added to the solid plate culture medium, 10.00 g / L of grapefruit peel powder was added to the seed culture medium, and 10.00 g / L of grapefruit peel powder was added to the fermentation medium; the other components of the culture medium were as follows:

[0152] The other components of the culture medium are as follows:

[0153] Solid culture medium: MgSO4·7H2O 1.00 g / L, KH2PO4 1.00 g / L, (NH4)2SO4 1.50 g / L, KCl 0.50 g / L, KNO3 1.50 g / L, CaCl2 0.10 g / L, yeast extract 2.00 g / L, agar powder 10.00 g / L, natural pH.

[0154] Seed culture medium: MgSO4·7H2O 5.00g / L, K2HPO4 5.00g / L, KCl 5.00g / L, FeSO4·5H2O 0.10g / L, natural pH, inoculum size 10%.

[0155] The fermentation medium contained the following components: MgSO4·7H2O 0.50 g / L, KH2PO4 1.50 g / L, K2HPO4 1.50 g / L, (NH4)2SO4 4.00 g / L, ZnSO4·7H2O 0.10 g / L, CaCl2 0.10 g / L, yeast extract 1.00 g / L, soybean flour 2.00 g / L, and peptone 2.00 g / L.

[0156] Preparation of grapefruit peel powder: Remove the white pith layer from fresh grapefruit peel, slice it, dry it, and then grind it into powder to 100 mesh for later use;

[0157] Culture conditions: Liquid fermentation was carried out at an initial pH of 6.0, a culture temperature of 35℃, an aeration rate of 1.6 min / L, and a stirring speed of 300 rpm for 75 h to obtain a compound enzyme fermentation broth with maximum enzyme activity of 49.70 U / mL.

[0158] The prepared compound enzyme achieved hydrolysis rates of 32%, 50%, and 57% for limonin, hesperidin, and naringin, respectively, within 30 minutes.

[0159] 2. The method for preparing a complex enzyme preparation of Aspergillus tabineus for enzymatic hydrolysis of bitter substances, mainly limonene compounds. Limonene compounds include limonene, nomiline, deacetylated nomiline, berberine, and mirinic acid, all of which are triterpenoid compounds with furan rings.

[0160] The method includes:

[0161] Add 10.00 g / L of lemon peel powder to the solid plate culture medium, add 10.00 g / L of lemon peel powder to the seed culture medium component, and add 10.00 g / L of lemon peel powder to the fermentation culture medium;

[0162] The other components of the culture medium are as follows:

[0163] Solid culture medium: MgSO4·7H2O 1.00 g / L, KH2PO4 1.00 g / L, (NH4)2SO4 1.50 g / L, KCl 0.50 g / L, KNO3 1.50 g / L, CaCl2 0.10 g / L, yeast extract 2.00 g / L, agar powder 10.00 g / L, natural pH.

[0164] Seed culture medium: MgSO4·7H2O 5.00g / L, K2HPO4 5.00g / L, KCl 5.00g / L, FeSO4·5H2O 0.10g / L, natural pH, inoculum size 10%.

[0165] The fermentation medium contained the following components: MgSO4·7H2O 0.50 g / L, KH2PO4 1.50 g / L, K2HPO4 1.50 g / L, (NH4)2SO4 4.00 g / L, ZnSO4·7H2O 0.10 g / L, CaCl2 0.10 g / L, yeast extract 1.00 g / L, soybean flour 2.00 g / L, and peptone 2.00 g / L.

[0166] Preparation of lemon peel powder: Fresh lemon peel is sliced, dried, and then ground into powder to 100 mesh for later use;

[0167] Culture conditions: Liquid fermentation was carried out at an initial pH of 5.0, a culture temperature of 33℃, an aeration rate of 1.6 min / L, and a stirring speed of 250 rpm for 70 h to obtain a compound enzyme fermentation broth with maximum enzyme activity of 40.16 U / mL.

[0168] The prepared compound enzyme achieved hydrolysis rates of 60%, 49%, and 41% for limonin, hesperidin, and naringin, respectively, within 30 minutes.

[0169] 3. The preparation method of Aspergillus tabine in the preparation of a complex enzyme preparation for enzymatic hydrolysis of bitter substances mainly consisting of naringin and limonene, the method comprising:

[0170] Add 10.00 g / L of grapefruit peel powder to the solid plate culture medium, add 10.00 g / L of lemon peel powder to the seed culture medium component, and add 10.00 g / L of lemon peel powder to the fermentation culture medium;

[0171] The other components of the culture medium are as follows:

[0172] Solid culture medium: MgSO4·7H2O 1.00 g / L, KH2PO4 1.00 g / L, (NH4)2SO4 1.50 g / L, KCl 0.50 g / L, KNO3 1.50 g / L, CaCl2 0.10 g / L, yeast extract 2.00 g / L, agar powder 10.00 g / L, natural pH.

[0173] Seed culture medium: MgSO4·7H2O 5.00g / L, K2HPO4 5.00g / L, KCl 5.00g / L, FeSO4·5H2O 0.10g / L, natural pH, inoculum size 10%.

[0174] The fermentation medium contained the following components: MgSO4·7H2O 0.50 g / L, KH2PO4 1.50 g / L, K2HPO4 1.50 g / L, (NH4)2SO4 4.00 g / L, ZnSO4·7H2O 0.10 g / L, CaCl2 0.10 g / L, yeast extract 1.00 g / L, soybean flour 2.00 g / L, and peptone 2.00 g / L.

[0175] Culture conditions: Liquid fermentation was carried out at an initial pH of 5.0, a culture temperature of 34℃, an aeration rate of 2.0 min / L, a stirring speed of 250 rpm, and fermentation was carried out for 72 h to obtain a compound enzyme fermentation broth with maximum enzyme activity of 40.10 U / mL.

[0176] The prepared compound enzyme achieved hydrolysis rates of 49%, 41%, and 52% for limonin, hesperidin, and naringin, respectively, within 30 minutes.

[0177] The fermentation broth was centrifuged in a refrigerated centrifuge (15,000 rpm) for 30 minutes. The supernatant was collected and the enzyme solution was concentrated using a membrane separation device to directly prepare a liquid enzyme preparation, or it was prepared into a freeze-dried enzyme powder using a freeze dryer.

[0178] The above embodiments are merely preferred technical solutions of the present invention and should not be considered as limitations on the present invention. The embodiments and features described in these embodiments can be arbitrarily combined without conflict. The scope of protection of the present invention should be limited to the technical solutions described in the claims, including equivalent substitutions of the technical features described in the claims. That is, equivalent substitutions and improvements within this scope are also within the scope of protection of the present invention.

Claims

1. A type of Aspergillus tabineus, characterized in that: The Aspergillus tubingensis mentioned is named Aspergillus tubingensis UA13. It was deposited at the China Center for Type Culture Collection on June 27, 2022, with accession number CCTCC NO: M 20222989, and the deposit address is Wuhan University, Wuhan, Hubei Province.

2. An application of Aspergillus tabineum as described in claim 1, characterized in that: Aspergillus tabineum is used in the fermentation process to prepare limonin debittering enzyme.

3. The application of Aspergillus tabineum according to claim 2, characterized in that: The application involves adding limonene or lemon peel powder containing limonene to the fermentation medium of Aspergillus tabbinensis, with the amount of limonene added being 4.0-6.0 mg / L; Preparation of lemon peel powder: Remove the white pith from fresh lemon peel, slice it, dry it, and then grind it into powder for later use.

4. The application of Aspergillus tabineum according to claim 3, characterized in that: The culture medium includes a fermentation medium containing: MgSO4·7H2O 0.40-0.60 g / L, KH2PO4 1.20-1.80 g / L, and K2HPO4. 4 1.20-1.80 g / L, (NH4)2SO4 3.50-4.50 g / L, ZnSO4·7H2O 0.05-0.15 g / L, CaCl2 0.05-0.15 g / L, yeast extract 0.80-1.20 g / L, soybean flour 1.50-2.50 g / L, peptone 1.50-2.50 g / L, limonene 4.00-5.00 mg / L, natural pH.

5. The limonin according to claim 2 for inducing high production of limonin debittering enzyme in Aspergillus tabineus, characterized in that: In the fermentation medium: MgSO4·7H2O 0.50 g / L, KH2PO4 1.50 g / L, K2HPO 4 1.50 g / L, (NH4)2SO4 4.00 g / L, ZnSO4·7H2O 0.10 g / L, CaCl2 0.10 g / L, yeast extract 1.00 g / L, soybean flour 2.00 g / L, peptone 2.00 g / L, limonene 4.00 mg / L.

6. The application of Aspergillus tabineum according to claim 1 in the preparation of a complex enzyme preparation for enzymatic hydrolysis of bitter substances, mainly limonene, is characterized in that... The applications include: Solid agar plate culture medium: MgSO4·7H2O 0.80-1.20 g / L, KH2PO4 4 0.80-1.20 g / L, (NH4)2SO 4 1.30-1.70 g / L, KCl 0.30-0.80 g / L, KNO3 1.20-1.70 g / L, CaCl2 0.05-0.15 g / L, yeast extract 1.50-2.50 g / L, limonene 0.50-1.50 mg / L, agar powder 8.00-12.00 g / L, natural pH; Seed culture medium: limonene 3.50-4.50 mg / L, MgSO4·7H2O 4.00-6.00 g / L, K2HPO4 4.00-7.00 g / L, KCl 4.00-6.00 g / L, FeSO4·5H2O 0.05-0.15 g / L, natural pH; Fermentation medium: MgSO4·7H2O 0.40-0.60 g / L, KH2PO4 1.20-1.80 g / L, K2HPO4 4 1.20-1.80 g / L, (NH4)2SO4 3.50-4.50 g / L, ZnSO4·7H2O 0.05-0.15 g / L, CaCl2 0.05-0.15 g / L, yeast extract 0.80-1.20 g / L, soybean flour 1.50-2.50 g / L, peptone 1.50-2.50 g / L, limonene 4.00-5.00 mg / L; Solid plate culture medium, seed culture medium, and fermentation culture medium were used to expand the culture in sequence.

7. The application of Aspergillus tabineum according to claim 1 in the preparation of a complex enzyme preparation for enzymatic hydrolysis of bitter substances, mainly naringin, is characterized in that, The applications include: Add 8.00-12.00 g / L of pomelo peel powder or 4.00-6.00 g / L of naringin to the solid plate culture medium; add 8.00-12.00 g / L of pomelo peel powder to the seed culture medium; add 8.00-12.00 g / L of pomelo peel powder or 4.00-6.00 g / L of naringin to the fermentation culture medium; the other components of the culture medium are as follows: Solid agar plate culture medium: MgSO4·7H2O 0.80-1.20 g / L, KH2PO4 4 0.80-1.20 g / L, (NH4)2SO 4 1.30-1.70 g / L, KCl 0.30-0.80 g / L, KNO3 1.20-1.70 g / L, CaCl2 0.05-0.15 g / L, yeast extract 1.50-2.50 g / L, agar powder 8.00-12.00 g / L, natural pH; Seed culture medium: MgSO4·7H2O 4.00-6.00 g / L, K2HPO4 4.00-7.00 g / L, KCl 4.00-6.00 g / L, FeSO4·5H2O 0.05-0.15 g / L, natural pH; Fermentation medium: MgSO4·7H2O 0.40-0.60 g / L, KH2PO4 1.20-1.80 g / L, K2HPO4 4 1.20-1.80 g / L, (NH4)2SO4 3.50-4.50 g / L, ZnSO4·7H2O 0.05-0.15 g / L, CaCl2 0.05-0.15 g / L, yeast extract 0.80-1.20 g / L, soybean flour 1.50-2.50 g / L, peptone 1.50-2.50 g / L; Solid plate culture medium, seed culture medium, and fermentation culture medium were used for scale-up cultivation in sequence. Preparation of grapefruit peel powder: Remove the white pith layer from fresh grapefruit peel, slice it, dry it, and then grind it into powder for later use; Culture conditions: Liquid fermentation is carried out at an initial pH of 5.0-6.0, a culture temperature of 30-35℃, an aeration rate of 1.3-2.5 min / L, and a stirring speed of 250-300 rpm for 70-77 h to obtain the compound enzyme fermentation broth with maximum enzyme activity.

8. The application of Aspergillus tabineum according to claim 1 in the preparation of a complex enzyme preparation for enzymatic hydrolysis of bitter substances, mainly limonene, is characterized in that, The applications include: Add 8.00-12.00 g / L of lemon peel powder or 4.00-6.00 g / L of limonene to solid plate culture medium, add 8.00-12.00 g / L of lemon peel powder to seed culture medium components, and add 8.00-12.00 g / L of lemon peel powder or 4.00-6.00 g / L of limonene to fermentation culture medium; The other components of the culture medium are as follows: Solid agar plate culture medium: MgSO4·7H2O 0.80-1.20 g / L, KH2PO4 4 0.80-1.20 g / L, (NH4)2SO 4 1.30-1.70 g / L, KCl 0.30-0.80 g / L, KNO3 1.20-1.70 g / L, CaCl2 0.05-0.15 g / L, yeast extract 1.50-2.50 g / L, agar powder 8.00-12.00 g / L, natural pH; Seed culture medium: MgSO4·7H2O 4.00-6.00 g / L, K2HPO4 4.00-7.00 g / L, KCl 4.00-6.00 g / L, FeSO4·5H2O 0.05-0.15 g / L, natural pH; Fermentation medium: MgSO4·7H2O 0.40-0.60 g / L, KH2PO4 1.20-1.80 g / L, K2HPO4 4 1.20-1.80 g / L, (NH4)2SO4 3.50-4.50 g / L, ZnSO4·7H2O 0.05-0.15 g / L, CaCl2 0.05-0.15 g / L, yeast extract 0.80-1.20 g / L, soybean flour 1.50-2.50 g / L, peptone 1.50-2.50 g / L; Solid plate culture medium, seed culture medium, and fermentation culture medium were used for scale-up cultivation in sequence. Preparation of lemon peel powder: Fresh lemon peel is sliced, dried, and then ground into powder for later use; Culture conditions: Liquid fermentation is carried out at an initial pH of 5.0-6.0, a culture temperature of 31-34℃, an aeration rate of 1.3-1.8 min / L, and a stirring speed of 250-280 rpm for 70-76 h to obtain the compound enzyme fermentation broth with maximum enzyme activity.

9. The application of Aspergillus tabineum according to claim 1 in the preparation of a complex enzyme preparation for enzymatic hydrolysis of bitter substances mainly consisting of naringin and limonene, characterized in that, The applications include: Add 8.00-12.00 g / L of grapefruit peel powder or 4.00-6.00 g / L of naringin to the solid plate culture medium; add 8.00-12.00 g / L of lemon peel powder to the seed culture medium component; add 8.00-12.00 g / L of lemon peel powder to the fermentation culture medium. The other components of the culture medium are as follows: Solid agar plate culture medium: MgSO4·7H2O 0.80-1.20 g / L, KH2PO4 4 0.80-1.20 g / L, (NH4)2SO 4 1.30-1.70 g / L, KCl 0.30-0.80 g / L, KNO3 1.20-1.70 g / L, CaCl2 0.05-0.15 g / L, yeast extract 1.50-2.50 g / L, agar powder 8.00-12.00 g / L, natural pH; Seed culture medium: MgSO4·7H2O 4.00-6.00 g / L, K2HPO4 4.00-7.00 g / L, KCl 4.00-6.00 g / L, FeSO4·5H2O 0.05-0.15 g / L, natural pH; Fermentation medium: MgSO4·7H2O 0.40-0.60 g / L, KH2PO4 1.20-1.80 g / L, K2HPO4 4 1.20-1.80 g / L, (NH4)2SO4 3.50-4.50 g / L, ZnSO4·7H2O 0.05-0.15 g / L, CaCl2 0.05-0.15 g / L, yeast extract 0.80-1.20 g / L, soybean flour 1.50-2.50 g / L, peptone 1.50-2.50 g / L; Solid plate culture medium, seed culture medium, and fermentation culture medium were used for scale-up cultivation in sequence. Culture conditions: Liquid fermentation is carried out at an initial pH of 5.0-6.0, a culture temperature of 30-35 ℃, an aeration rate of 1.3-1.8 min / L, and a stirring speed of 250-300 rpm for 65-75 h to obtain the compound enzyme fermentation broth with maximum enzyme activity.

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