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 tubingensis 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, when fermenting under the optimal conditions of a limonin addition amount of 4.0 mg / L, an inoculum size of 10% (v / v), an initial pH value of 5.0, a stirring speed of 250 rpm, and a temperature of 30 °C, the maximum 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 a 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] The present invention belongs to the technical field of enzyme preparations, and specifically relates to a preparation method and application of an enzyme preparation for enzymatically hydrolyzing bitter substances in citrus juice. Background Art

[0002] The bitter substances in citrus mainly include two categories. One is flavonoid substances, including naringin, neohesperidin, aurapten, hesperetin, nobiletin, quercetin, etc. Among them, naringin has the highest content and is the main flavanone glycoside bitter substance. The other is highly oxidized triterpenoid compounds - limonoid analogs, among which limonin is the main bitter substance. The main bitter substances in citrus fruits, naringin and limonin, have the highest content in pomelos, followed by grapefruit, mandarins or hybrid mandarins, tangerines, navel oranges, and common sweet oranges. The "delayed bitterness" or "post-bitter taste" of these natural bitter substances present in citrus fruits during the processing process severely restricts the development of its processing industry and is one of the huge challenges currently faced by the citrus processing industry. Therefore, industrially, it is necessary to remove the bitterness of citrus fruits and improve the quality of their processed products.

[0003] Limonin, the main substance responsible for the bitterness and "post-bitter taste" in citrus fruits, greatly affects the taste of citrus juice. Limonin, also known as nomilin, is a triterpenoid compound with a bitterness threshold of 1.0 mg / kg in aqueous solution and 3.4 mg / L in fruit juice, about 20 times that of naringin. Limonin has a low content in fresh fruits or freshly squeezed juices and mostly exists as non-bitter limonin A-ring lactone in the cytoplasm. However, prolonged storage or heat treatment, etc. will intensify its bitterness. This "post-bitter taste" phenomenon is due to the conversion of limonin A-ring lactone into bitter limonin under the catalysis of limonin D-ring lactone hydrolase under acidic conditions (pH < 6.5). Under conditions such as freezing, mechanical damage, or heat treatment of the pulp tissue, its acidity will increase, thereby promoting the formation of limonin during processing or storage.

[0004]

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

[0006]

[0007] The hydrolysis of naringin is mainly completed in two steps. Naringin (4'-5,7'-trihydroxyflavanone-7-rhamnoglucoside) is hydrolyzed by rhamnosidase to rhamnose and prunin, and the bitterness of prunin is about one-third of that of naringin; prunin is then hydrolyzed by β-D-glucosidase to non-bitter naringenin and glucose.

[0008] From the above, it can be seen that the enzymatic hydrolysis principles of limonin and naringin are completely different.

[0009] The methods currently reported for debittering juice include β -Cyclodextrin method, supercritical CO2 method, membrane separation method, addition of bitterness inhibitors, bioenzyme method, etc. Compared with other debittering methods, the bioenzyme method not only has the characteristics of good effect, no pollution, simple process, mild reaction conditions, etc., but also its enzymatic hydrolysis products can enhance the flavor of juice to a certain extent, retain the nutritional components of juice, and are more suitable for industrial production. The main reason why the bioenzyme method has not been widely used in the citrus processing industry is the lack of highly active enzyme preparation products. Therefore, it is very important to prepare enzyme preparations with stable performance and high activity. The naringinase powder currently sold on the market has only naringin as its substrate, has almost no effect on limonin and nomilin bitter substances, and has an activity of only 0.1-0.4 U / mL. Pure naringinase could be imported from abroad before, but in recent years, several major foreign enzyme preparation companies have no stock, and need to be customized more than half a year in advance, and the price is expensive, which is very limited.

[0010] Chinese patent CN104404016B discloses a method for producing naringinase, which uses a traditional liquid fermentation method. The activity of the obtained naringinase is not high, and the thermal stability is only 45°C. Chinese patent CN101914451A discloses a method for producing α-L-rhamnosidase from Alternaria alternata. The α-L-rhamnosidase produced by Alternaria alternata is an intracellular enzyme that needs to be separated and extracted from the cells. The subsequent separation and purification are difficult, which directly leads to high use costs. Chinese patent CN105441410A discloses a method for producing rhamnosidase from Chaetomium species, and the obtained enzyme activity is 500 U / mL, but the substrate specificity is not studied. These research results reflect that the research on α-L-rhamnosidase or naringinase has attracted much attention in recent years, but the research on enzyme preparations with composite debittering enzyme characteristics is rarely involved. Summary of the invention

[0011] The invention provides a preparation method of an enzyme preparation for enzymatically hydrolyzing bitter substances in citrus juice and its application, and a method for preparing an enzyme preparation capable of simultaneously enzymatically hydrolyzing bitter substances in citrus juice, namely limonin and naringin.

[0012] In order to achieve the above object, the present invention adopts the following technical solutions:

[0013] A tubingensis Aspergillus, the name of the tubingensis Aspergillus is Aspergillus tubingensis UA13, deposited in the China Center for Type Culture Collection on June 27, 2022, with a deposit number of CCTCC NO: M2022989, and the deposit address is Wuhan University, Wuhan, Hubei.

[0014] Preferably, Aspergillus tubingensis is applied to the preparation of limonin debittering enzyme by fermentation method.

[0015] More preferably, in the application, limonin or lemon peel powder containing limonin is added to the fermentation medium of Aspergillus tubingensis, and the addition amount of limonin is 4.0 - 6.0 mg / L.

[0016] Preparation of lemon peel powder: Remove the white albedo layer from fresh lemon peel, slice it, dry it, and then grind it to 100 meshes for standby.

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

[0018] Even more preferably, in the fermentation medium:

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

[0020] The application of the Aspergillus tubingensis in the preparation of a complex enzyme preparation whose main bitter substances to be enzymolyzed are limonin, and the application includes:

[0021] Solid plate medium: MgSO4·7H2O is 0.80 - 1.20 g / L, KH2PO4 is 0.80 - 1.20 g / L, (NH4)2SO4 is 1.30 - 1.70 g / L, KCl is 0.30 - 0.80 g / L, KNO3 is 1.20 - 1.70 g / L, CaCl2 is 0.05 - 0.15 g / L, yeast extract is 1.50 - 2.50 g / L, limonin is 0.50 - 1.50 mg / L, agar powder is 8.00 - 12.00 g / L, and the natural pH;

[0022] Seed culture medium: Limonin 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;

[0023] In the fermentation medium: 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 powder 1.50 - 2.50 g / L, peptone 1.50 - 2.50 g / L, limonin 4.00 - 5.00 mg / L;

[0024] Solid plate medium, seed culture medium and fermentation medium are used for successive subculture.

[0025] The application of Aspergillus tubingensis in the preparation of a complex enzyme preparation in which the enzyme-hydrolyzed bitter substances are mainly naringin, and the application includes:

[0026] Add naringin powder 8.00 - 12.00 g / L or naringin 4.00 - 6.00 g / L to the solid plate medium, add naringin powder 8.00 - 12.00 g / L to the components of the seed culture medium, and add naringin powder 8.00 - 12.00 g / L or naringin 4.00 - 6.00 g / L to the fermentation medium; the other components of the medium are respectively:

[0027] Solid plate 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;

[0028] 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;

[0029] In the fermentation medium: 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 powder 1.50 - 2.50 g / L, peptone 1.50 - 2.50 g / L.

[0030] Solid plate medium, seed medium and fermentation medium are used for successive scale-up cultivation.

[0031] Preparation of pomelo peel powder: Remove the white albedo layer from fresh pomelo peels, slice them, dry them, and then grind them into powder with a mesh size of 100 for later use.

[0032] Cultivation conditions: Liquid fermentation is carried out at an initial pH value of 5.0 - 6.0, a cultivation temperature of 30 - 35 °C, an aeration rate of 1.3 - 2.5 min / L, and a stirring speed of 250 - 300 rpm for 70 - 77 h to obtain a complex enzyme fermentation broth with the maximum enzyme activity.

[0033] The application of the Aspergillus tubingensis in the preparation of a complex enzyme preparation whose main bitter substances to be enzymolyzed are limonoids, and the said application includes:

[0034] Add 8.00 - 12.00 g / L of lemon peel powder or 4.00 - 6.00 g / L of limonin to the solid plate medium, add 8.00 - 12.00 g / L of lemon peel powder to the components of the seed medium, and add 8.00 - 12.00 g / L of lemon peel powder or 4.00 - 6.00 g / L of limonin to the fermentation medium;

[0035] The other components of the medium are respectively:

[0036] Solid plate 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;

[0037] 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;

[0038] In the fermentation medium: 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 powder 1.50 - 2.50 g / L, peptone 1.50 - 2.50 g / L.

[0039] Solid plate medium, seed culture medium and fermentation medium are used for successive scale-up cultivation.

[0040] Preparation of lemon peel powder: Fresh lemon peels are sliced and dried, then ground into powder of 100 meshes for standby;

[0041] Cultivation conditions: Liquid fermentation is carried out at an initial pH value of 5.0 - 6.0, cultivation temperature of 31 - 34 °C, aeration rate of 1.3 - 1.8 min / L, and stirring speed of 250 - 280 rpm for 70 - 76 h to obtain a complex enzyme fermentation broth with the maximum enzyme activity.

[0042] The application of Aspergillus tubingensis in the preparation of a complex enzyme preparation for enzymatically hydrolyzing bitter substances mainly including naringin and limonin, and the application includes:

[0043] Adding 8.00 - 12.00 g / L of pomelo peel powder or 4.00 - 6.00 g / L of naringin to the solid plate medium, adding 8.00 - 12.00 g / L of lemon peel powder to the components of the seed culture medium, and adding 8.00 - 12.00 g / L of lemon peel powder to the fermentation medium;

[0044] Other components of the culture medium are respectively:

[0045] Solid plate 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;

[0046] 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;

[0047] In the fermentation medium: 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 powder 1.50 - 2.50 g / L, peptone 1.50 - 2.50 g / L.

[0048] Solid plate medium, seed culture medium and fermentation medium are used for successive scale-up culture.

[0049] Cultivation conditions: Liquid fermentation is carried out at an initial pH value of 5.0 - 6.0, the cultivation temperature is 30 - 35 °C, the aeration rate is 1.3 - 1.8 min / L, and the stirring speed is 250 - 300 rpm for 65 - 75 h to obtain a complex enzyme fermentation broth with the maximum enzyme activity.

[0050] Advantages of the present invention:

[0051] 1. The enzyme produced by strain UA13 has a strong affinity for limonin. At the same time, the research results show that the limonin debittering enzyme can still efficiently hydrolyze naringin, that is, the strain UA13 of the present invention can simultaneously enzymatically hydrolyze limonin, naringin and hesperidin. Then this enzyme has very good application prospects in the citrus juice processing industry.

[0052] 2. The limonin debittering enzyme has good heat resistance and acid resistance, which is convenient for the wide use of enzyme preparations and can be applied to different types of citrus fruits, different ripening periods of citrus fruits or different processing conditions.

[0053] 3. The enzyme produced by strain UA13 has enzymatic hydrolysis ability for both limonoids and naringin as substrates, and the enzymatic hydrolysis ability varies slightly according to different substrates.

[0054] 4. During the process of strain cultivation to produce enzymes, according to the different requirements of the substrates, corresponding inducers can be used to induce the production of enzymes, so as to obtain specific enzyme substances. Description of the drawings

[0055] Figure 1Fermentation curve of strain UA13 in a 1 L fermenter under unoptimized fermentation conditions;

[0056] Figure 2 Effect of stirring speed on enzyme production by strain UA13;

[0057] Figure 3 Effect of fermentation temperature on enzyme production by strain UA13;

[0058] Figure 4 Effect of initial pH value of fermentation broth on enzyme production by strain UA13;

[0059] Figure 5 Lineweaver - Burk curve of limonin debittering enzyme catalyzing different substrates. Specific embodiments

[0060] The present invention will be further described below in conjunction with specific embodiments. A more detailed description of the present invention is given, but it is not limited to these embodiments.

[0061] Example 1

[0062] An Aspergillus tubingensis, named Aspergillus tubingensis UA13, was deposited at the China Center for Type Culture Collection on June 27, 2022, with the deposit number CCTCC NO: M2022989 and the deposit address being Wuhan University, Wuhan, Hubei.

[0063] Example 2

[0064] Using the strain of Example 1, limonin debittering enzyme was prepared. The fermentation conditions were explored as follows:

[0065] Solid 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, limonin 1.00 mg / L, agar powder 10.00 g / L, natural pH.

[0066] Seed medium: limonin 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%.

[0067] In the fermentation medium: 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 powder 2.00 g / L, peptone 2.00 g / L, limonin 4.00 mg / L.

[0068] 1 Experimental method

[0069] 1.1 Fermentation culture

[0070] Sporulation suspension: Inoculate the slant strain Aspergillus tubingensis UA13 stored in the laboratory onto a solid plate medium and culture at 30 °C for 3 - 4 d until the spores are mature. Wash down the spores with 0.85% sterile physiological saline and disperse them to obtain a bacterial suspension. Examine and count the sporulation suspension under a microscope, and adjust the concentration to about 10 7 cells / mL.

[0071] Seed culture: Inoculate the sporulation suspension with adjusted concentration into the seed medium at an inoculation amount of 10% (v / v), and culture in a constant temperature shaker at 30 °C and 180 r / min.

[0072] Strain fermentation: Use a sterile pipette tip to aspirate 5 mL (inoculation amount 10%, v / v) of the adjusted sporulation suspension and inoculate it into the sterilized fermentation medium with a liquid volume of 50 mL / 250 mL (5 glass beads). Culture in a constant temperature shaker at 30 °C and 180 r / min for 72 h.

[0073] 1.2 Enzyme activity assay

[0074] (1) Preparation of crude enzyme solution: Take the fermentation broth cultured in a shaking flask for 72 h and place it in a centrifuge tube, centrifuge at 5000 r / min for 20 min.

[0075] (2) Determination of limonin content: Respectively take 0.3, 0.6, 0.9, 1.2, 1.5, 1.8 mL of 200 μg / mL limonin standard solution and place them in 25 mL stoppered test tubes, make up to 2 mL with absolute ethanol, then add 5 mL of chromogenic solution to each, shake well and let stand for color development for 30 min, and measure the absorbance at a wavelength of 500 nm. Taking the limonin content as the abscissa and the absorbance as the ordinate, the standard curve equation is A = 0.0038C + 0.0379, R 2 = 0.9997.

[0076] Preparation of the chromogenic solution (prepare and use immediately): Accurately weigh 125 mg of p-diaminobenzaldehyde and dissolve it in 100 mL of a sulfuric acid-anhydrous ethanol mixture (V:V = 65:35, use after cooling), then add 0.5 mL of 0.9% FeCl3 and mix well.

[0077] (3) Determination of the activity of limonin debittering enzyme: Add 0.5 mL of 200 μg / mL substrate limonin to a stoppered test tube, then add 0.1 mL of enzyme solution with a certain concentration, and make up to 2 mL with anhydrous ethanol. Place it in a constant temperature shaker at 60 °C (150 rpm) for enzymatic hydrolysis for 30 min, take out the enzymatic hydrolysis solution and immediately place it in a boiling water bath at 100 °C to inactivate the enzyme for 5 min; add 5 mL of the chromogenic solution to the enzymatic hydrolysis solution, mix well and let it stand for color development for 30 min. Take out and inject it into a 1 cm cuvette, and measure the absorbance at 500 nm with a spectrophotometer.

[0078] All samples are measured in parallel three times, and the average value is taken for analysis (the same below).

[0079] Definition of enzyme activity: Under the conditions of 60 °C and pH 4.0, the amount of enzyme required to consume 1 μg of limonin per minute is defined as one enzyme activity unit (U).

[0080]

[0081] Definition of relative enzyme activity: 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 (%).

[0082] 1.3 Optimization of enzyme production conditions

[0083] 1.3.1 Optimization of shake flask fermentation

[0084] Using the activity of limonin debittering enzyme as the evaluation index, a single factor experiment was used to explore the optimal enzyme production conditions of the strain. Under the initial culture conditions, inoculate the seed liquid with a certain seed age, and change the inoculation amount (4% - 16%, v / v), fermentation temperature (25 - 40 °C), and initial pH value of the fermentation broth (4.0 - 9.0) of the fermentation medium to determine the optimal fermentation environment of the strain.

[0085] 1.3.2 Optimization experiment in a 1 L fermenter

[0086] Synchronous continuous fermentation in a 1-L four-tank system: The seed liquid was synchronously cultured and then inoculated into 600 mL of sterilized fermentation broth (inoculation volume 10%, v / v). Scale-up cultivation was carried out under the initial conditions of an aeration rate of 0.4 L / min, a stirring speed of 300 rpm, and an initial pH value of 5.0. Based on the optimized results in shake flasks, the stirring speed (200 - 350 rpm), fermentation temperature (25 - 40 °C), and initial fermentation pH value (4.0 - 7.0) were optimized respectively, and the optimal enzyme-producing conditions were determined by observing the growth of the strain and enzyme production during fermentation.

[0087] The analysis of the improvement multiple of enzyme activity was carried out based on the enzyme activity optimized in shake flask fermentation for comparative study.

[0088] 2 Results

[0089] 2.1 Optimization of enzyme-producing conditions

[0090] 2.1.1 Effect of strain inoculation volume on enzyme production

[0091] The concentration of the seed liquid and the physiological state of the strain directly affect the fermentation effect. The cultivation time of the seed medium to a certain extent determines the strength of the seed vitality. Especially for filamentous fungi, the morphology of the mycelium and nutrient mass transfer will be affected. Based on previous studies, the seed medium cultured for 48 h with different inoculation volumes was inoculated into shake flasks for fermentation, and the enzyme production activities of the strains are shown in Table 1.

[0092] Table 1 Effect of different inoculation volumes on enzyme production of strain UA13

[0093]

[0094] As can be seen from Table 1 above, the inoculation volume has little effect on the enzyme production activity of the strain. When the inoculation volume is in the range of 4% - 10% (v / v), the enzyme production ability of the strain remains at a relatively high level. Considering both enzyme production activity and fermentation time, this study determined an inoculation volume of 10% (v / v) as the basis for subsequent research (the same below).

[0095] 2.1.2 Effect of fermentation temperature on enzyme production

[0096] The fermentation temperature was changed to explore the optimal cultivation temperature for the strain to produce limonin-debittering enzyme, and the results are shown in Table 2.

[0097] Table 2 Effect of fermentation temperature on enzyme production of strain UA13

[0098]

[0099] As can be seen from Table 2 above, when the strain was fermented and cultured in the range of 25 - 40 °C, the enzyme production activity of the strain first increased and then decreased with the increase of the culture temperature, and the enzyme activity reached the maximum at 35 °C. Therefore, in this study, the fermentation temperature of 35 °C was used for subsequent experiments.

[0100] 2.1.3 Effect of initial pH value on enzyme production

[0101] The initial pH value of the medium can affect the structure and function of the enzyme, the structure of the cell, the charge status of the cell membrane, etc. The metabolic products produced by microorganisms during growth can also cause changes in the pH value of the medium. The pH range for the growth and enzyme production of molds is generally between 3.0 and 8.5.

[0102] Table 3 Effect of initial fermentation pH value on enzyme production of strain UA13

[0103]

[0104] The suitable pH value ranges for the growth metabolism and enzyme production of different strains are different. As can be seen from Table 3 above, when the initial fermentation pH value is in the range of 5.0 - 6.0, the enzyme production ability of the strain is stronger, and when the initial pH is 5.0, the enzyme activity reaches the highest. Therefore, in this study, the initial pH of 5.0 was used for subsequent experiments.

[0105] On the basis of the single-factor experiment, using 4.0 mg / L of limonin as the carbon source and inducer, inoculating 10% (v / v) of the seed medium fermented for 48 h, and fermenting for 72 h under the conditions of the initial medium pH of 5.0 and the fermentation temperature of 35 °C, the highest activity of limonin debittering enzyme can be achieved by strain UA13.

[0106] 2.2 Fermenter optimization experiment

[0107] Synchronous continuous fermentation in a 1 L fermenter four-tank system. After synchronous cultivation of the seed liquid, it was inoculated into 600 mL of sterilized fermentation broth (inoculation amount 10%, v / v). Scale-up cultivation was carried out under the initial conditions of an aeration rate of 0.4 L / min, a stirring speed of 300 rpm, and an initial pH value of 5.0. The change in dissolved oxygen during fermentation can reflect the growth state of the strain. As Figure 1 shown, in the first 24 h of fermentation, the bacteria were in the lag phase, the dissolved oxygen decreased sharply, and the oxygen in the medium was fully utilized. Subsequently, the dissolved oxygen began to gradually increase, the biosynthesis of the enzyme began, the bacteria entered the exponential growth phase, and the enzyme activity began to increase continuously. The enzyme activity reached the highest of 25.48 U / mL after 72 h of fermentation. The pH value of the medium is dynamically changing. Its value rapidly decreases from the start of fermentation, then gradually increases, and finally fluctuates within the range of 4.3 - 4.8.

[0108] 2.2.1 Effect of stirring speed on enzyme production

[0109] During shake flask cultivation, the rotation of the shaker and the addition of an appropriate amount of glass beads were used to promote the fusion of various substances, avoiding the agglomeration of mycelia and affecting enzyme production by fermentation. When scaling up from the shake flask to the fermenter, the control of stirring parameters largely determines the mixing of substances in the tank and the efficiency of enzyme production by fermentation. If the stirring speed is too fast, the excessive shear force will affect the generation and composition of cells, having an adverse effect on the enzyme production by the mycelia fermentation; if the stirring speed is too low, the mycelia will entangle into clusters, which is not conducive to fermentation production.

[0110] As Figure 2 shown, when the stirring speed was 200 rpm, due to the low stirring speed, the growth of mycelia was slow, the lag phase was long, and the logarithmic growth phase was entered at 36 h. The enzyme production capacity of the mycelia reached the maximum of 34.04 U / mL at 48 h and then continued to decline. From the shake flask experiment, it was known that the enzyme activity of the strain usually reached the highest at 72 h. The reason for the difference might be that the rotation speed of the fermenter was too low, and the mycelia did not continue to break and reproduce subsequently, but a large number of them wound into balls on the baffle in the tank, affecting enzyme production; when the stirring speed was 350 rpm, the dissolved oxygen in the fermenter increased, but the excessive shear force would accelerate the death of the mycelia, and the enzyme activity reached the highest of 33.78 U / mL at 60 h of fermentation; the fermentation change trends at 250 rpm and 300 rpm were not very different: at a rotation speed of 250 rpm, the enzyme activity of the strain reached the highest of 42.61 U / mL at 48 h, an increase of 67.23%. At a rotation speed of 300 rpm, the enzyme activity of the strain reached the highest of 43.23 U / mL at 60 h, an increase of 69.66%. Considering energy conservation in actual fermentation and the small difference in enzyme activity at the two rotation speeds, it was more appropriate to choose 250 rpm as the optimal rotation speed.

[0111] 2.2.2 Effect of fermentation temperature on enzyme production

[0112] As Figure 3 shown, the fermentation curve trends of the strain at different temperatures were basically the same: when the fermentation temperature was controlled at 25 °C and 40 °C, the enzyme activities both reached the highest at 48 h, which were 28.80 U / mL and 30.46 U / mL respectively, and the enzyme activities were relatively low, consistent with the shake flask fermentation situation; at 30 °C and 35 °C, the enzyme production activities of the strain reached 40.10 U / mL and 35.61 U / mL respectively at 36 h, and secondary growth occurred in both at 60 h of fermentation. To sum up, the strain had good enzyme production capacity at 30 °C - 35 °C. At a cultivation temperature of 30 °C, the enzyme activity of the strain reached the highest of 40.10 U / mL at 36 h in advance, an increase of 57.38% compared with that before optimization, and the fermentation cycle was shortened. Therefore, a cultivation temperature of 30 °C was selected for the subsequent fermenter optimization experiment.

[0113] 2.2.3 Effect of initial pH value of fermentation broth on enzyme production

[0114] As Figure 4 shown, under the condition of initial pH 5.0, the dissolved oxygen decreased rapidly in the first 12 h of fermentation. From 12 h to 48 h, the dissolved oxygen in the fermenter approached zero, basically maintaining below 10%, indicating that the strain had strong metabolism and was more suitable for growth under the condition of initial pH 5.0, which was consistent with the results of the shake flask experiment. At 48 h, the debittering enzyme activity of limonin had reached 48.52 U / mL, which was 90.42% higher than that before optimization; under the condition of initial pH 6.0 of the fermentation broth, the strain maintained good enzyme production ability from 48 h to 72 h, and the highest enzyme activity could reach 40.16 U / mL; when the initial pH of the fermentation broth was 4.0, the enzyme reached the highest activity of 38.82 U / mL at 96 h. To sum up, the optimal initial pH value of the fermentation broth was 5.0, and Aspergillus tubingensis UA13 had strong growth and enzyme production ability in the range of initial pH 4.0 - 6.0. This strain was more suitable for survival in a slightly acidic environment, which was consistent with the results of the shake flask experiment.

[0115] The optimization experiment of the strain was carried out in a 1 L fermenter. Under the optimal conditions of 4.0 mg / L of limonin addition amount, 10% (v / v) of inoculation amount, initial pH value of 5.0, stirring speed of 250 rpm, and 30 °C in the 1 L fermenter medium, the highest debittering enzyme activity of limonin could reach 48.52 U / mL, which was 90.42% higher than that before optimization.

[0116] Among them, the culture medium was as follows:

[0117] 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, limonin 1.00 mg / L, agar powder 10.00 g / L, natural pH.

[0118] Seed culture medium: limonin 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, inoculation amount 10%.

[0119] In the fermentation medium: 0.50 g / L of MgSO4·7H2O, 1.50 g / L of KH2PO4, 1.50 g / L of K2HPO4, 4.00 g / L of (NH4)2SO4, 0.10 g / L of ZnSO4·7H2O, 0.10 g / L of CaCl2, 1.00 g / L of yeast extract, 2.00 g / L of soybean powder, 2.00 g / L of peptone, and 4.00 mg / L of limonin.

[0120] Example 3

[0121] The limonin debittering enzyme was prepared under the optimal conditions of Example 2.

[0122] The conditions were as follows: In a 1 L fermentor medium, with a limonin addition amount of 4.00 mg / L, an inoculation amount of 10% (v / v), an initial pH value of 5.0, a stirring speed of 250 rpm, and fermentation under the optimal conditions of 30°C, the maximum activity of the limonin debittering enzyme could reach 48.52 U / mL.

[0123] Among them, the medium was as follows:

[0124] Solid medium: 1.00 g / L of MgSO4·7H2O, 1.00 g / L of KH2PO4, 1.50 g / L of (NH4)2SO4, 0.50 g / L of KCl, 1.50 g / L of KNO3, 0.10 g / L of CaCl2, 2.00 g / L of yeast extract, 1.00 mg / L of limonin, 10.00 g / L of agar powder, natural pH.

[0125] Seed medium: 4.00 mg / L of limonin, 5.00 g / L of MgSO4·7H2O, 5.00 g / L of K2HPO4, 5.00 g / L of KCl, 0.10 g / L of FeSO4·5H2O, natural pH, inoculation amount 10%.

[0126] In the fermentation medium: 0.50 g / L of MgSO4·7H2O, 1.50 g / L of KH2PO4, 1.50 g / L of K2HPO4, 4.00 g / L of (NH4)2SO4, 0.10 g / L of ZnSO4·7H2O, 0.10 g / L of CaCl2, 1.00 g / L of yeast extract, 2.00 g / L of soybean powder, 2.00 g / L of peptone, and 4.00 mg / L of limonin.

[0127] The supernatant after centrifugation of the fermentation broth is the crude enzyme solution. The changes in enzyme activity of the crude enzyme solution were investigated at different enzymatic hydrolysis temperatures (30 - 70 °C) and pH values (2.0 - 8.0). Taking the highest enzyme activity measured in the same group of experiments as 100%, the relative enzyme activity under other conditions was calculated.

[0128] Substrate affinity of the enzyme: The Michaelis constant Km values of the enzyme catalyzing different substrates (limonin, naringin, hesperidin) were determined to explore the substrate affinity of the enzyme, and Km was obtained by the double reciprocal method (Linewear - Burk).

[0129] 1. Research on the properties of limonin debittering enzyme

[0130] 1.1 Research on the optimal enzymatic hydrolysis temperature of limonin debittering enzyme

[0131] Temperature is an important factor affecting the substrate conversion efficiency. When the temperature is too low, many enzyme molecules are not activated and the enzymatic hydrolysis effect is extremely low. When the temperature is too high and exceeds the enzyme thermal stability range, the enzyme protein is inactivated. An appropriate enzymatic hydrolysis temperature can maximize the substrate conversion efficiency.

[0132] Table 4 Effect of enzymatic hydrolysis temperature on the activity of limonin debittering enzyme

[0133]

[0134] As can be seen from Table 4, when the enzymatic hydrolysis temperature is in the range of 30 - 70 °C, the activity of limonin debittering enzyme first increases and then decreases with the increase of temperature, and the enzyme activity reaches the highest at 60 °C; when the enzymatic hydrolysis temperature is 70 °C, the activity of limonin debittering enzyme is still 59.5%. Therefore, the limonin debittering enzyme in this study has good heat resistance, and the subsequent experiments were carried out at an enzymatic hydrolysis temperature of 60 °C.

[0135] 1.2 Research on the optimal enzymatic hydrolysis pH value of limonin debittering enzyme

[0136] Generally, the optimal pH value of fungal naringinase is in the range of 3.0 - 6.0. An inappropriate pH value will affect the conformation of the enzyme protein, thereby reducing the enzyme activity.

[0137] Table 5 Effect of different enzymatic hydrolysis pH values on the activity of limonin debittering enzyme

[0138]

[0139] As can be seen from Table 5, when the strain UA13 is in the enzymatic hydrolysis environment with an enzymatic hydrolysis pH of 2.0 - 8.0, the enzyme activity shows a trend of first increasing and then decreasing. The enzyme activity reaches the highest at an enzymatic hydrolysis pH of 5.0; the relative enzyme activity is 90.58% at pH 3.0; the relative enzyme activity is 81.11% at pH 7.0. Therefore, this enzyme has good acid resistance, and the optimal enzymatic hydrolysis pH is 5.0.

[0140] 2. Substrate Affinity Study

[0141] The reaction rates of limonin debittering enzyme at different substrate concentrations were measured, and the corresponding kinetic parameters were obtained using the Linewear-Burk curve. This study found that the enzyme produced by the strain induced by limonin catalyzed the reactions of three different substrates (limonin, hesperidin, and naringin) in accordance with the Michaelis-Menten kinetic law ( Figure 5 ).

[0142] From the experiment, when the substrate was limonin, K m = 0.622 mmol / L, V max = 0.017 mmol / (L·min); when the substrate was hesperidin, K m = 0.967 mmol / L, V max = 0.010 mmol / (L·min); when the substrate was naringin, K m = 0.999 mmol / L, V max = 0.018 mmol / (L·min). This enzyme has the ability to hydrolyze limonin, hesperidin, and naringin. The hydrolysis rates of limonin, hesperidin, and naringin within 30 min reached 48%, 38%, and 33% respectively. The dynamic law of this enzyme hydrolysis conforms to the enzyme-catalyzed reaction kinetics. From the K m value, it can be known that the order of the enzyme's affinity for substrates is: limonin > hesperidin > naringin. Therefore, the enzyme has a better affinity for limonin than naringin, which may be due to the fact that limonin serves as both an inducer and a carbon source during the growth of the strain.

[0143] The strain was fermented and cultured with naringin as the inducer, that is, the limonin in the medium in this example was changed to naringin, to explore the size of the enzyme's affinity for the substrate. It was found that the enzyme induced by naringin had a higher affinity for naringin (K m = 0.480 mmol / L), a poorer substrate affinity for limonin (K m = 91.920 mmol / L), and the hydrolysis rates of limonin, hesperidin, and naringin within 30 min reached 37%, 47%, and 52% respectively. Therefore, it can be inferred that different inducers have a great impact on the growth and enzyme production of the strain. The strain after long-term induction and optimization has a better substrate affinity for the corresponding inducer and a stronger ability to enzymatically hydrolyze the corresponding bitter substances.

[0144] Example 4

[0145] Based on the research results of Example 3, a preparation method for three composite enzymes was developed.

[0146] The enzymes were all prepared using Aspergillus tubingensis UA13, which was deposited at the China Center for Type Culture Collection on June 27, 2022, with the deposit number CCTCC NO: M 2022989, and the deposit address being Wuhan University, Wuhan, Hubei.

[0147] 1. Preparation method of a complex enzyme preparation in which the bitter substance to be enzymatically hydrolyzed is mainly naringin, the method comprising:

[0148] Adding 10.00 g / L of pomelo peel powder to the solid plate medium, 10.00 g / L of pomelo peel powder to the seed medium components, and 10.00 g / L of pomelo peel powder to the fermentation medium; the other components of the medium are respectively:

[0149] The other components of the medium are respectively:

[0150] Solid medium: 1.00 g / L of MgSO4·7H2O, 1.00 g / L of KH2PO4, 1.50 g / L of (NH4)2SO4, 0.50 g / L of KCl, 1.50 g / L of KNO3, 0.10 g / L of CaCl2, 2.00 g / L of yeast extract, 10.00 g / L of agar powder, natural pH.

[0151] Seed medium: 5.00 g / L of MgSO4·7H2O, 5.00 g / L of K2HPO4, 5.00 g / L of KCl, 0.10 g / L of FeSO4·5H2O, natural pH, inoculation amount 10%.

[0152] In the fermentation medium: 0.50 g / L of MgSO4·7H2O, 1.50 g / L of KH2PO4, 1.50 g / L of K2HPO4, 4.00 g / L of (NH4)2SO4, 0.10 g / L of ZnSO4·7H2O, 0.10 g / L of CaCl2, 1.00 g / L of yeast extract, 2.00 g / L of soybean powder, 2.00 g / L of peptone.

[0153] Preparation of pomelo peel powder: Remove the white pith layer from fresh pomelo peel, slice and dry, then grind to 100 mesh for standby;

[0154] Cultivation conditions: Conduct liquid fermentation at an initial pH value of 6.0, cultivation temperature of 35 °C, aeration rate of 1.6 min / L, stirring speed of 300 rpm for 75 h, and a complex enzyme fermentation broth with the maximum enzyme activity of 49.70 U / mL can be obtained.

[0155] The hydrolysis rates of the prepared complex enzyme on limonin, hesperidin, and naringin reach 32%, 50%, and 57% respectively within 30 min.

[0156] 2. The method for preparing a complex enzyme preparation using Aspergillus tubingensis for enzymatically hydrolyzing bitter substances mainly composed of limonoids. Limonoids include limonin, nomilin, deacetylnomilin, obacunone, and limonic acid, all of which are triterpenoids with a furan ring.

[0157] The method includes:

[0158] Add 10.00 g / L of lemon peel powder to the solid plate medium, 10.00 g / L of lemon peel powder to the seed medium components, and 10.00 g / L of lemon peel powder to the fermentation medium;

[0159] The other components of the medium are as follows:

[0160] Solid medium: 1.00 g / L of MgSO4·7H2O, 1.00 g / L of KH2PO4, 1.50 g / L of (NH4)2SO4, 0.50 g / L of KCl, 1.50 g / L of KNO3, 0.10 g / L of CaCl2, 2.00 g / L of yeast extract, 10.00 g / L of agar powder, natural pH.

[0161] Seed medium: 5.00 g / L of MgSO4·7H2O, 5.00 g / L of K2HPO4, 5.00 g / L of KCl, 0.10 g / L of FeSO4·5H2O, natural pH, inoculation amount 10%.

[0162] In the fermentation medium: 0.50 g / L of MgSO4·7H2O, 1.50 g / L of KH2PO4, 1.50 g / L of K2HPO4, 4.00 g / L of (NH4)2SO4, 0.10 g / L of ZnSO4·7H2O, 0.10 g / L of CaCl2, 1.00 g / L of yeast extract, 2.00 g / L of soybean powder, 2.00 g / L of peptone.

[0163] Preparation of lemon peel powder: Cut fresh lemon peel into slices, dry it, and then grind it to 100 mesh for standby;

[0164] Cultivation conditions: Perform liquid fermentation at an initial pH value of 5.0, cultivation temperature of 33 °C, aeration rate of 1.6 min / L, and stirring speed of 250 rpm for 70 h to obtain a complex enzyme fermentation broth with a maximum enzyme activity of 40.16 U / mL.

[0165] The hydrolysis rates of the prepared composite enzyme for limonin, hesperidin, and naringin reach 60%, 49%, and 41% respectively within 30 min.

[0166] 3. The preparation method of Aspergillus tubingensis in the preparation of a composite enzyme preparation whose main bitter substances for enzymatic hydrolysis are naringin and limonin, the method comprising:

[0167] Add 10.00 g / L of pomelo peel powder to the solid plate medium, add 10.00 g / L of lemon peel powder to the seed medium components, and add 10.00 g / L of lemon peel powder to the fermentation medium;

[0168] The other components of the medium are respectively:

[0169] Solid medium: 1.00 g / L of MgSO4·7H2O, 1.00 g / L of KH2PO4, 1.50 g / L of (NH4)2SO4, 0.50 g / L of KCl, 1.50 g / L of KNO3, 0.10 g / L of CaCl2, 2.00 g / L of yeast extract, 10.00 g / L of agar powder, natural pH.

[0170] Seed medium: 5.00 g / L of MgSO4·7H2O, 5.00 g / L of K2HPO4, 5.00 g / L of KCl, 0.10 g / L of FeSO4·5H2O, natural pH, inoculation amount 10%.

[0171] In the fermentation medium: 0.50 g / L of MgSO4·7H2O, 1.50 g / L of KH2PO4, 1.50 g / L of K2HPO4, 4.00 g / L of (NH4)2SO4, 0.10 g / L of ZnSO4·7H2O, 0.10 g / L of CaCl2, 1.00 g / L of yeast extract, 2.00 g / L of soybean powder, 2.00 g / L of peptone.

[0172] Cultivation conditions: Carry out liquid fermentation at an initial pH value of 5.0, cultivation temperature of 34 °C, aeration rate of 2.0 min / L, stirring speed of 250 rpm, and ferment for 72 h to obtain a composite enzyme fermentation broth with a maximum enzyme activity of 40.10 U / mL.

[0173] The hydrolysis rates of the prepared composite enzyme for limonin, hesperidin, and naringin reach 49%, 41%, and 52% respectively within 30 min.

[0174] Centrifuge the fermentation broth in a refrigerated centrifuge (15000 rpm) for 30 min, collect the supernatant, concentrate the enzyme solution through a membrane separation device, and directly prepare it into a liquid enzyme preparation, or place it in a freeze dryer to prepare a freeze-dried powder of the enzyme preparation.

[0175] The above embodiments are only the preferred technical solutions of the present invention and should not be regarded as limitations on the present invention. In the present application, the embodiments and the features in the embodiments can be arbitrarily combined with each other without conflict. The protection scope of the present invention shall be the technical solutions recorded in the claims, including the equivalent replacement solutions of the technical features in the technical solutions recorded in the claims. That is, the equivalent replacement improvements within this scope are also within the protection scope of the present invention.

Claims

1. An Aspergillus tubingensis UA13, characterized in that: The Aspergillus tubingensis was deposited at the China Center for Type Culture Collection on June 27, 2022, with the deposit number CCTCC NO: M 2022989, and the deposit address being Wuhan University, Wuhan, Hubei.

2. Use of Aspergillus tubingensis as described in claim 1, characterized in that: The Aspergillus tubingensis is applied to the preparation of limonin debittering enzyme by fermentation method; The specific application is as follows: Limonin or lemon peel powder containing limonin is added to the fermentation medium of Aspergillus tubingensis, and the addition amount of limonin is 4.0 - 6.0 mg / L.

3. Use of Aspergillus tubingensis according to claim 2, characterized in that: The preparation of the lemon peel powder: The fresh lemon peel is removed of the white albedo layer, sliced, dried, and then ground into powder for standby.

4. Use of Aspergillus tubingensis according to claim 2, characterized in that: In the fermentation medium: 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 powder 1.50 - 2.50 g / L, peptone 1.50 - 2.50 g / L, limonin 4.00 - 5.00 mg / L, natural pH.

5. Use of Aspergillus tubingensis according to claim 2, characterized in that: In the fermentation medium: 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 powder 2.00 g / L, peptone 2.00 g / L, limonin 4.00 mg / L.

6. Use of Aspergillus tubingensis according to claim 1 in the preparation of a complex enzyme preparation for enzymatically hydrolyzing the bitter substance limonin, characterized in that, The said application includes: Solid plate 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, limonin 0.50 - 1.50 mg / L, agar powder 8.00 - 12.00 g / L, natural pH; Seed medium: limonin 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; In the fermentation medium: 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 powder 1.50 - 2.50 g / L, peptone 1.50 - 2.50 g / L, limonin 4.00 - 5.00 mg / L; Solid plate medium, seed medium and fermentation medium are used for successive scale-up cultivation.

7. Use of Aspergillus tubingensis as claimed in claim 1 in the preparation of a complex enzyme preparation for enzymatically hydrolyzing the bitter substance naringin, characterized in that, The applications include: Adding 8.00 - 12.00 g / L of pomelo peel powder or 4.00 - 6.00 g / L of naringin to the solid plate medium, 8.00 - 12.00 g / L of pomelo peel powder to the seed medium components, and 8.00 - 12.00 g / L of pomelo peel powder or 4.00 - 6.00 g / L of naringin to the fermentation medium; the other components of the medium are respectively: Solid plate 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; Seed 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; In the fermentation medium: 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 powder 1.50 - 2.50 g / L, peptone 1.50 - 2.50 g / L; Solid plate medium, seed medium and fermentation medium are used for successive scale-up cultivation; Preparation of pomelo peel powder: Remove the white pith layer from fresh pomelo peel, slice it, dry it, and then grind it into powder for standby; Cultivation conditions: Liquid fermentation is carried out at an initial pH value of 5.0 - 6.0, a cultivation temperature of 30 - 35 °C, an aeration rate of 1.3 - 2.5 min / L, and a stirring speed of 250 - 300 rpm for 70 - 77 h to obtain a complex enzyme fermentation broth with the maximum enzyme activity.

8. Use of Aspergillus tubingensis according to claim 1 in the preparation of a complex enzyme preparation for enzymatically hydrolyzing bitter substances limoninoids, characterized in that, The said applications include: Adding 8.00 - 12.00 g / L of lemon peel powder or 4.00 - 6.00 g / L of limonin to the solid plate medium, adding 8.00 - 12.00 g / L of lemon peel powder to the components of the seed medium, and adding 8.00 - 12.00 g / L of lemon peel powder or 4.00 - 6.00 g / L of limonin to the fermentation medium; The other components of the medium are respectively: Solid plate 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; Seed 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; In the fermentation medium: 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 powder 1.50 - 2.50 g / L, peptone 1.50 - 2.50 g / L; The solid plate medium, the seed medium and the fermentation medium are used for successive scale-up cultivation; Preparation of lemon peel powder: Fresh lemon peels are sliced and dried, and then ground into powder for standby; Cultivation conditions: Liquid fermentation is carried out at an initial pH value of 5.0 - 6.0, a cultivation temperature of 31 - 34 °C, an aeration rate of 1.3 - 1.8 min / L, and a stirring speed of 250 - 280 rpm for 70 - 76 h to obtain a complex enzyme fermentation broth with the maximum enzyme activity.

9. Use of Aspergillus tubingensis as claimed in claim 1 in the preparation of a complex enzyme preparation for enzymatically hydrolyzing bitter substances into naringin and limonin, characterized in that, The said applications include: Adding 8.00 - 12.00 g / L of pomelo peel powder or 4.00 - 6.00 g / L of naringin to the solid plate medium, adding 8.00 - 12.00 g / L of lemon peel powder to the components of the seed medium, and adding 8.00 - 12.00 g / L of lemon peel powder to the fermentation medium; The other components of the medium are respectively: Solid plate 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; Seed 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; In the fermentation medium: 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 powder 1.50 - 2.50 g / L, peptone 1.50 - 2.50 g / L; The solid plate medium, seed medium and fermentation medium are used for successive scale-up culture; Cultivation conditions: Liquid fermentation is carried out at an initial pH value of 5.0 - 6.0, the cultivation temperature is 30 - 35 °C, the aeration rate is 1.3 - 1.8 min / L, and the stirring speed is 250 - 300 rpm for 65 - 75 h to obtain a complex enzyme fermentation broth with the maximum enzyme activity.

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