Aspergillus aculeatus for efficiently producing ester-type catechin degraded by tannase and application thereof

Abstract

This invention belongs to the field of food enzyme engineering technology, specifically relating to a strain of *Aspergillus echinococcosis* that produces highly efficient ester-degrading catechin tannin enzymes and its applications. The strain is classified as *Aspergillus echinococcosis* (…). Aspergillus aculeatus LS06, with accession number CGMCC No. 42170. The *Aspergillus echinococcosis* strain provided by this invention serves as a tannin-producing strain, using inexpensive agricultural waste bran as raw material, resulting in low production costs. Simultaneously, the produced tannins exhibit high enzyme activity and high catalytic efficiency for ester-type catechins, demonstrating promising application prospects in tea beverage processing.

Description

Technical Field

[0001] This invention belongs to the field of food enzyme engineering technology, specifically relating to a highly efficient Aspergillus echinococcosis that produces tannins to degrade ester-type catechins and its applications. Background Technology

[0002] Tea, with its unique flavor and significant antioxidant, anti-cancer, and neuroprotective effects, is highly favored worldwide. Tea polyphenols are crucial secondary metabolites in tea, directly influencing its color, aroma, and taste, and serving as a vital material basis for its various health benefits. Ester-type catechins have been proven to be major contributors to the bitterness and astringency of tea, and during storage, they easily form complexes with proteins and other components, resulting in precipitation, commonly known as "cold-induced cloudiness." This phenomenon worsens with prolonged storage, significantly reducing the aesthetic appeal of tea beverages and consequently decreasing consumer purchasing desire.

[0003] The full name of tanninase is tannic acid acyl hydrolase (EC3.1.1.20). It hydrolyzes the ester bonds in ester-type catechins, thereby reducing their binding sites, decreasing intermolecular interactions, and inhibiting precipitation. Simultaneously, the product gallic acid can competitively bind with phenolic compounds to form water-soluble substances with caffeine, reducing the turbidity of tea beverages. In addition, it also improves flavor, reduces bitterness, and enhances antioxidant capacity, thus improving the quality and nutritional value of tea beverages.

[0004] Existing technologies disclose several preparation techniques and application scenarios for tanninases. Publications CN120158397A, CN116121080A, CN116240125A, and CN119837209A disclose applications of different tanninase sources in baijiu (Chinese liquor), pomegranate peel, tobacco leaves, and fruit juice, respectively. Patent CN117384879A screened a lactic acid bacterium producing acid-resistant tanninases from acidic tea, but its hydrolytic ability against ester-type catechins is weak. Although there is a wealth of research on tanninases, there are few reports on screening tanninase-producing strains capable of efficiently degrading ester-type catechins in tea infusion.

[0005] Publication number CN116121080A discloses a strain of Aspergillus echinosus and its application in the production of tanninase and gallic acid, mainly focusing on the production of tanninase and gallic acid for the green degradation of industrial and agricultural waste. This patent does not involve the relevant research on the degradation of ester-type catechins by tanninase and substrate specificity in tea beverage systems, and the tanninase produced by the patent strain has significantly different enzymatic properties from the tanninase described in this invention. Summary of the Invention

[0006] To address the problem of low efficiency in degrading ester-type catechins in existing technologies, this invention provides Aspergillus echinococcosis and its application in tannin enzyme production.

[0007] The present invention is implemented using the following technical solutions:

[0008] In a first aspect, the present invention provides *Aspergillus echinosporum* (… Aspergillus aculeatus LS06, this strain was identified as Aspergillus aculeatus Named Aspergillus aculeatus LS06. It is deposited at the China General Microbiological Culture Collection Center, CGMCC No. 42170, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The deposit date is August 18, 2025. The viability of this biological material (strain) was tested by the collection center on August 18, 2025, and the result was that it was viable.

[0009] Secondly, this invention provides the application of Aspergillus echinosporum LS06 in the production of tanninase.

[0010] Thirdly, the present invention provides a method for producing tanninase, wherein the Aspergillus echinosporum LS06 is activated, filtered and inoculated into a solid fermentation medium for fermentation, wherein the solid fermentation medium comprises bran and salt solution to obtain tanninase solution.

[0011] Further, the mass ratio of wheat bran to salt solution is 1:0.5-1.5, and the salt solution comprises: 25-35 g / L tannic acid, 2-4 g / L NaNO3, 0.5-1.5 g / L KH2PO4, 0.005-0.015 g / L MgSO4·7H2O, with a pH of 4.5-5.5. Further, the initial pH of the solid-state fermentation medium is 4.5-5.5.

[0012] Furthermore, the concentration of the Aspergillus echinococcosis spore suspension in the solid-state fermentation medium is 1×10⁻⁶. 7 -1×10 9 1 spore / mL, inoculum size 15%-25% v / w.

[0013] Furthermore, the fermentation conditions are cultured at 28-32℃ for 60-84 h.

[0014] Fourthly, the present invention provides a tanninase prepared by the production method described above.

[0015] Fifthly, the present invention provides the application of tanninase in the degradation of ester-type catechins.

[0016] Furthermore, the ester-type catechins include epigallocatechin gallate, epigallocatechin gallate, gallocatechin gallate, and catechin gallate.

[0017] In a sixth aspect, the present invention provides a method for degrading ester-type catechins by adding the fermentation product obtained by fermenting Aspergillus echinosus LS06 to a tea extract.

[0018] The present invention has the following beneficial effects:

[0019] (1) The Aspergillus echinosus strain provided by the present invention is used as a raw material for producing tannins, and the production cost is low.

[0020] (2) The tanninase produced by Aspergillus echinosus provided by the present invention has high enzyme activity, and therefore has high catalytic efficiency for ester-type catechins, and has good application prospects in tea beverage processing.

[0021] (3) This invention predicts the binding sites and catalytic mechanisms of tanninase and ester-type catechins through molecular docking, which can further guide the targeted modification of tanninase, the optimization of enzyme reaction conditions, and its subsequent application in the transformation of tea components.

[0022] (4) This invention discloses a tannin-producing Aspergillus echinosus, which can efficiently degrade ester-type catechins in tea infusion, and molecular docking has revealed that it has a high affinity for ester-type catechins. Attached Figure Description

[0023] Figure 1 This is a diagram showing the isolation and purification of Aspergillus echinococcosis in Example 1;

[0024] Figure 2 This is a diagram showing the effect of Aspergillus echinococcosis on tannin selection medium in Example 1;

[0025] Figure 3 In Example 1 Aspergillus aculeatus Microscopic morphological characteristics of LS06;

[0026] Figure 4 In Example 2 Aspergillus aculeatus Electrophoresis image of the PCR amplification sequence of LS06;

[0027] Figure 5 In Example 2 Aspergillus aculeatus Phylogenetic tree of LS06;

[0028] Figure 6 In Example 5 Aspergillus aculeatus Comparison of the degradation effects of tanninase produced by LS06 and commercially available enzymes on ester-type catechins;

[0029] Figure 7In Example 6 Aspergillus aculeatus Enzymatic properties of tanninase produced by LS06 (A is the optimal temperature of tanninase; B is the temperature stability of tanninase; C is the optimal pH of tanninase; D is the pH stability of tanninase);

[0030] Figure 8 is a schematic diagram of the molecular docking pattern between Aspergillus echinococcosis tanninase and EGCG in Example 7;

[0031] Figure 9 is a schematic diagram of the molecular docking pattern between the commercial tanninase (from Aspergillus oryzae) and EGCG in Example 7;

[0032] Figure 10 is a schematic diagram of the molecular docking pattern between Aspergillus echinococcosis tanninase and ECG in Example 7;

[0033] Figure 11 is a schematic diagram of the molecular docking pattern between the commercial tanninase (from Aspergillus oryzae) of Example 7 and ECG;

[0034] Figure 12 is a schematic diagram of the molecular docking pattern between Aspergillus echinococcosis tanninase and GCG in Example 7;

[0035] Figure 13 is a schematic diagram of the molecular docking pattern between the commercial tanninase (aspergillus oryzae source) and GCG in Example 7;

[0036] Figure 14 is a schematic diagram of the molecular docking pattern between Aspergillus echinococcosis tanninase and CG in Example 7;

[0037] Figure 15 is a schematic diagram of the molecular docking pattern between the commercial tanninase (aspergillus oryzae source) and CG in Example 7. Detailed Implementation

[0038] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features can be combined with each other. Unless otherwise specified, the methods used in the embodiments of the present invention are conventional methods, and the reagents used are commercially available.

[0039] Example 1: Screening of tannin-producing fungi

[0040] Fungal isolation and culture: 5 g of fermented sour tea rich in active bacteria was mixed with 45 mL of physiological saline and shaken thoroughly on a shaker to obtain a dilution of 10. -1 The bacterial culture was prepared, and then 0.5 mL of the bacterial culture was added to 4.5 mL of physiological saline for serial dilution to 10⁻⁶. -40.1 mL of bacterial suspension from each dilution gradient was spread onto potato dextrose agar (PDA) plates for screening and incubated at 30°C for 3 days. Microscopic examination was performed. Figure 1 ), and obtained 62 fungal strains.

[0041] Preliminary screening of tannin-producing fungi using the color change plate method: Fungi from the aforementioned PDA plates are inoculated onto tannin selection medium, which uses tannin as the sole carbon source and contains bromophenol blue indicator. If a strain can grow on this medium, it is considered capable of degrading tannic acid through tannin production. Since the product gallic acid alters the pH of the medium, causing bromophenol blue to change from blue-purple to yellow, the size of the color change zone can be used to preliminarily determine the strain's enzyme production ability. If a strain cannot produce tannin enzymes, it indicates that the strain cannot use tannin as a carbon source for growth and will not produce a color change zone on the tannin selection medium. Figure 2 (Left side); while strains that can grow on tannin selection medium and show obvious color change ( Figure 2 (Right side). Using the color-changing zone as a screening indicator, 12 tannin-producing strains were selected.

[0042] Solid-state fermentation for enzyme production rescreening: Tannin screening medium can only screen strains that can utilize tannins as their sole carbon source, and the enzyme production capacity of a strain does not necessarily correspond perfectly to the size of the color change zone. Therefore, solid-state fermentation is needed again for screening and verification to determine the true enzyme production capacity of each strain. The tannin-producing fungi rescreened are transferred to PDA slant medium. 10 mL of sterile physiological saline is used to wash the spores from the PDA slant medium into a sterile Erlenmeyer flask (scraped clean with an inoculation loop), then shaken thoroughly. 1 mL of the suspension is serially diluted and examined under a microscope for counting. The spore suspension is then diluted to 1*10⁻⁶. 8 Before transferring the inoculum to the fermentation flask, the sample was filtered through sterile gauze and then transferred to solid-state fermentation medium at an inoculum rate of 20% (v / w). The medium was then incubated at 30°C for 72 h. After fermentation, 500% (v / w) citrate buffer (0.1 M, pH 5.0) was added to the medium, and the mixture was shaken at 200 rpm / min for 60 min at 30°C. After removing solid residue by vacuum filtration, the filtrate was centrifuged at 10000 g for 10 min at 4°C. The supernatant was collected for enzyme activity assay, yielding one strain with the strongest tannin-producing ability. The microscopic results are shown below. Figure 3 As shown.

[0043] PDA medium (g / L): Potato 300, glucose 20, agar 15, chloramphenicol 0.1.

[0044] Tannin screening medium (g / L): tannic acid 10, agar 30, NaNO3 3, KH2PO4 1, MgSO4·7H2O 0.05, KCl 0.5, FeSO4·7H2O 0.01, bromophenol blue 0.04; pH=5.0.

[0045] Solid-state fermentation medium: wheat bran and salt solution (tannic acid 30 g / L, NaNO3 3 g / L, KH2PO4 1 g / L, MgSO4·7H2O 0.01 g / L) are mixed in a 1:1 ratio, pH=5.0.

[0046] Citrate buffer (0.1 M, pH 5.0): Prepare 0.1 M solutions of citric acid and sodium citrate by mixing them at a ratio of 8.2:11.8 (v / v).

[0047] Example 2: Molecular identification of tannin-producing Aspergillus echinococcosis

[0048] The strains obtained in the previous screening step were sent to Shanghai Paisennong Biotechnology Co., Ltd. for ITS sequence sequencing. PCR amplification was performed using upstream primer ITS1 (SEQ ID NO.1, 5-TCCGTAGGTGAACCTGCGG-3) and downstream primer ITS4 (SEQ ID NO.2, 5-TCCTCCGCTTATTGATATGC-3). The PCR results were analyzed by agarose gel electrophoresis. Figure 4 As shown. PCR products were recovered and purified, and DNA sequencing was performed using an ABI 3730-XL sequencer. The assembled sequence file (see SEQ ID NO.3) was compared with data in the NCBI nucleic acid database using the NCBIL program, and a phylogenetic tree was constructed. Figure 5 ), identified as Aspergillus aculeatus Named Aspergillus aculeatus LS06. It is deposited at the China General Microbiological Culture Collection Center, CGMCC No. 42170, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing.

[0049] Example 3: Solid-state fermentation of Aspergillus echinospora LS06 to produce tannin enzyme solution

[0050] Activation of glycerol tubes: Aspergillus echinococcosis stored in an ultra-low temperature freezer Aspergillus aculeatus Using a sterile inoculation loop, LS06 was dipped into a glycerol tube and streaked onto a PDA slant culture medium. The medium was incubated at 30°C for 72 h until spores were fully developed. The spores were then washed from the PDA slant culture medium into a sterile Erlenmeyer flask with an appropriate amount of sterile physiological saline (after scraping the spores clean with an inoculation loop). After filtration through sterile gauze, the resulting spore suspension (approximately 1.0 × 10⁻⁶ spores) was obtained. 8(spores / mL) is used for the fermentation production of tannins.

[0051] Preparation of solid-state fermentation medium: Weigh out the following ingredients in the following proportions: 0.3% NaNO3, 0.1% KH2PO4, 0.05% MgSO4·7H2O, 0.005% KCl, and 0.001% FeSO4·7H2O. Dissolve the solutions in an appropriate amount of water and sterilize with wheat bran at 121℃ for 20 min. Weigh out 3% tannic acid, dissolve it in a small amount of water, filter it through a sterile membrane to remove bacteria, and mix it with the above salt solution. Adjust the pH to 5.0, and then mix it with wheat bran at a 1:1 ratio (v / w). Stir thoroughly to obtain the solid-state fermentation medium.

[0052] Fermentation preparation of enzyme solution: The spore suspension prepared above was inoculated into a solid fermentation medium at a ratio of 20% (v / w) and cultured at 30℃ for 72 h. After fermentation, 500% (v / w) citrate buffer (0.1 M, pH 5.0) was added, and the mixture was extracted by shaking at 200 rpm / min for 60 min at 30℃. After removing solid residue by vacuum filtration, the filtrate was centrifuged at 10000xg for 10 min at 4℃. The supernatant was collected, which is the tannin enzyme solution.

[0053] Example 4: Determination of tanninase activity

[0054] Tanninase activity assay: Take two clean 10 mL centrifuge tubes, add 0.25 mL of enzyme solution and heat-inactivated enzyme solution as a control, respectively, and incubate at 30℃ for 5 min. Then add substrate solution (0.01 M methyl gallate dissolved in pH 5.5, 50 mM citrate-sodium citrate buffer) to both tubes and react precisely for 5 min. Then add 0.3 mL of methanol ragulanin (0.667%, w / v), shake to mix, and incubate at 30℃ for 5 min. Then add 0.2 mL of KOH solution (0.5 M), and incubate for another 5 min. Finally, add 4 mL of distilled water to dilute, and after standing for 10 min, measure the absorbance of each solution at 520 nm.

[0055] Under the above measurement conditions, the amount of enzyme required to release 1 µmol of gallic acid per minute is defined as 1 enzyme activity unit (U).

[0056] Therefore, Aspergillus echinospora was found. Aspergillus aculeatus The tanninase activity in the enzyme solution prepared by LS06 according to the method in Example 3 was 330 U / L.

[0057] Example 5: Determination of the high-efficiency degradation ability of tanninases derived from Aspergillus echinospora LS06 on ester-type catechins

[0058] The tanninase activity assay method defined in Example 4 was used, with commercially available tanninase produced by Beijing Weinuoen Engineering Biotechnology Co., Ltd. as the control group (CK), and the above-mentioned Aspergillus echinosporum was used. Aspergillus aculeatus The tanninase from LS06 fermentation was used in the experimental group (LS06). The same dose of tanninase (0.2 U / mL) was added to the tea extract (material-to-liquid ratio 1:110), and the reaction was carried out for different times (0-80 min) under optimal conditions. Figure 6 It was found that the levels of the four ester-type catechins—epigallocatechin gallate (EGCG), epicatechin gallate (ECG), gallocatechin gallate (GCG), and catechin gallate (CG)—decreased to varying degrees after different reaction times, while the levels of the corresponding non-ester-type catechins—epigallocatechin (EGC), epicatechin (EC), gallocatechin (GC), and gallic acid (GA)—increased. Specifically, in the original tea infusion, the contents of EGCG, ECG, GCG, and CG were 144.35 mg / L, 188.88 mg / L, 6.64 mg / L, and 3.23 mg / L, respectively; while the contents of the non-ester-type catechins EGC, EC, GC, and GA were 133.13 mg / L, 27.83 mg / L, 9.05 mg / L, and 13.10 mg / L, respectively. After LS06 was applied to the tea infusion for 80 min, the contents of EGCG, ECG, GCG, and CG were 19.51 mg / L, 3.35 mg / L, 4.62 mg / L, and 1.05 mg / L, respectively, with hydrolysis degrees of 86.48%, 82.87%, 30.51%, and 67.35%, respectively. The contents of non-esterified catechins EGC, EC, GC, and GA were 293.55 mg / L, 33.77 mg / L, 11.01 mg / L, and 74.50 mg / L, respectively, with the formation of each substance increasing by 2.21 times, 1.21 times, 1.22 times, and 5.69 times, respectively. After 80 min of treatment with CK, the contents of EGCG, ECG, GCG, and CG in tea infusion were 55.53 mg / L, 3.52 mg / L, 5.15 mg / L, and 2.13 mg / L, respectively, with hydrolysis degrees of 61.53%, 81.37%, 22.51%, and 33.94%. The contents of non-esterified catechins EGC, EC, GC, and GA were 249.20 mg / L, 33.50 mg / L, 10.13 mg / L, and 57.53 mg / L, respectively, with the formation of each substance increasing by 1.87 times, 1.20 times, 1.12 times, and 4.39 times. Comparative analysis revealed that LS06 exhibits a stronger ability to degrade esterified catechins.

[0059] Example 6: Enzymatic Properties Analysis of Tannins Derived from Aspergillus echinospora LS06

[0060] The enzyme solution was placed under different temperatures and pH conditions for enzyme activity determination, with the highest enzyme activity being 100%, to study the enzymatic properties of this tanninase. Figure 7 The results showed that the optimal temperature for this tanninase was 35°C. Between 30-50°C, 70% of the enzyme activity was maintained. Above 50°C, the relative enzyme activity decreased to approximately 25%. Treatment at 45°C for 2 hours maintained 80% of the enzyme activity, while above 55°C, the enzyme activity was almost completely lost. The optimal pH was 6.0. Within the pH range of 2.0-10.0, 60% of the enzyme activity was maintained. Treatment at pH 3.0-7.0 for 12 hours resulted in almost no loss of enzyme activity, but activity decreased significantly above pH 8. These findings indicate that the tanninase is suitable for low-temperature reactions and exhibits good stability within the pH range of 3.0-7.0. Analysis revealed that the enzymatic properties of the tanninase from LS06 differed significantly from those of the tanninase in publication number CN116121080A, indicating that these two tanninases from different Aspergillus strains may have significant differences in their catalytic properties.

[0061] Example 7: Analysis of the molecular binding ability of tanninases derived from Aspergillus echinospora LS06 to ester-type catechins

[0062] The three-dimensional structure of tanninases from Aspergillus echinococcosis was predicted using SWISS-MODEL (https: / / beta.swissmodel.expasy.org / ), and the accuracy was greater than 80%, indicating that the modeling results are reliable.

[0063] Molecular docking was performed using Autodock 4 software and visualized using Pymol software. Figure 8-Figure 15 The results showed that the binding energies of tanninases derived from *Aspergillus echinense* with EGCG, ECG, GCG, and CG were -7.45 kJ / mol, -12.34 kJ / mol, -12.97 kJ / mol, and -8.49 kJ / mol, respectively, while the binding energies of commercially available tanninases with EGCG, ECG, GCG, and CG were -2.59 kJ / mol, -6.19 kJ / mol, and -8.41 kJ / mol, respectively. Lower binding energies indicate more stable intermolecular binding and stronger affinity. Therefore, the tanninases derived from *Aspergillus echinense* exhibited tighter binding with EGCG, ECG, and GCG, demonstrating a higher affinity for ester-type catechins. Furthermore, the tanninases derived from *Aspergillus echinense* showed more abundant hydrogen bond formation with ester-type catechins, further indicating that they possess a stronger binding capacity with ester-type catechins.

Claims

1. Aspergillus echinosporum ( Aspergillus aculeatus )LS06, with accession number CGMCC No.42170.

2. The application of Aspergillus echinococcosis LS06 as described in claim 1 in the production of tanninase.

3. A method for producing tanninase, characterized in that, After activating the Aspergillus echinococcosis LS06 according to claim 1, it is filtered and inoculated into a solid-state fermentation medium for fermentation. The solid-state fermentation medium comprises wheat bran and a salt solution to obtain a tannin enzyme solution. The mass ratio of wheat bran to salt solution is 1:0.5-1.

5. The salt solution comprises: tannic acid 25-35 g / L, NaNO3 2-4 g / L, KH2PO4 0.5-1.5 g / L, MgSO4·7H2O 0.005-0.015 g / L, and pH 4.5-5.

5.

4. The production method as described in claim 3, characterized in that, The concentration of the spore suspension of said Aspergillus aculeatus in the solid-state fermentation medium is 1 x 10 7 -1 x 10 9 spores / mL, with an inoculum of 15-25% v / w.

5. The production method as described in claim 3, characterized in that, The fermentation conditions are as follows: cultured at 28-32 ℃ for 60-84 h.

6. Tanninase prepared by any of the production methods shown in claims 3-5.

7. The application of the tanninase as described in claim 6 in the degradation of ester-type catechins.

8. The application as described in claim 7, characterized in that, The ester-type catechins include epigallocatechin gallate, epigallocatechin gallate, gallocatechin gallate, and catechin gallate.

9. A method for degrading ester-type catechins, characterized in that, The fermentation product obtained by fermenting Aspergillus echinosporum LS06 as described in claim 1 is added to the tea extract.

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