Aspergillus aculeatus and its application in the production of tannase and gallic acid

Abstract

The present invention discloses an Aspergillus aculeatus and its application in the production of tannase and gallic acid, belonging to the field of microorganisms and tannin degradation technology. The microorganism is classified and named Aspergillus aculeatus WYT2101, which has been deposited in the Guangdong Provincial Microbial Culture Collection Center with a deposit number of GDMCC No. 62691, a deposit date of August 10, 2022, and a deposit address of 5th Floor, Building 59, Institute of Microbiology, Guangdong Academy of Sciences. The Aspergillus aculeatus can use its tannase gene to prepare tannase with high enzyme activity (the tannase activity of the fermentation supernatant can reach 44.45U / mL) and good thermal stability; it can also use pomegranate peel to efficiently produce tannase (2.14U / mL) and gallic acid (the output reaches 1.7mg / mL), and has the potential to be applied to industrial mass production of tannase and gallic acid.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and tannin degradation, and particularly relates to Aspergillus aculeatus and application thereof in the production of tannase and gallic acid. Background Art

[0002] Tannins are the second most abundant phenolic compound in nature, primarily found in the roots, stems, and fruits of plants. Tannins have a distinctive astringent taste and can strongly complex with various minerals, as well as macromolecules such as proteins, pectins, and alkaloids, hindering their biodegradation. Tannase (EC 3.1.1.20), also known as tannin acyl hydrolase, effectively hydrolyzes the ester and carboxyphenol bonds in hydrolyzable tannins (including ellagitannins (ETs), gallic acid (GTs), and other gallic acid esters), producing polyphenolic compounds such as gallic acid. Tannase research and application have been extensively applied in feed processing, food processing, brewing, cosmetics production, and pharmaceutical manufacturing.

[0003] 3,4,5-Trihydroxybenzoic acid, commonly known as gallic acid, is the primary component of the tannic acid molecule found in plants and fruits. Gallic acid is an important fine chemical with pharmacological properties such as antibacterial, antiallergic, antioxidant, antimutagenic, anti-inflammatory, neuroprotective, and anticancer activities. It is widely used in the pharmaceutical, chemical, and food industries. Global demand for gallic acid is approximately 8,000 tons per year. Traditional chemical methods for producing gallic acid have disadvantages such as low purity, high cost, low yield, and severe environmental pollution. Microbial tannase can be used to hydrolyze tannic acid into gallic acid. The enzymatic production of gallic acid using tannase has a short reaction time, high conversion rate, and is pollution-free.

[0004] However, most existing tannase-producing strains suffer from low tannase activity, poor thermal stability, and difficulty in recycling, limiting their large-scale application. Therefore, developing microbial strains capable of high tannase production and tannic acid degradation has become an urgent challenge. Summary of the Invention

[0005] In view of the above-mentioned problems existing in the prior art, the technical problem to be solved by the present invention is to provide an Aspergillus aculeatus and its application in the production of tannase and gallic acid. The Aspergillus aculeatus can utilize its tannase gene to prepare tannase with high enzyme activity and good thermal stability, and has the potential to be applied to the industrial mass production of tannase and gallic acid.

[0006] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0007] A type of Aspergillus aculeatus, classified and named Aspergillus aculeatus WYT2101, has been deposited in the Guangdong Provincial Microbial Culture Collection Center with the accession number: GDMCC No. 62691, the deposit date: August 10, 2022, and the deposit address is 5th Floor, Building 59, Institute of Microbiology, Guangdong Academy of Sciences.

[0008] Aspergillus aculeatus was obtained by screening from soil samples under tea trees in Fujian.

[0009] Biological characteristics of Aspergillus aculeatus: This strain is cultured in PDA medium. In the early stage of growth, white hyphae grow. As the culture time increases, dark brown round or oval colonies are formed, and the surface protrusions are velvety. The hyphae have transverse septa and many branches, the top capsule is flask-shaped, and the conidia are spherical or oval.

[0010] The ITS identification results of the Aspergillus aculeatus strain are shown in SEQ ID NO: 1. The measured ITS sequence was submitted to the National Center of Biotechnology Information (NCBI) of the United States. The basic local alignment search tool (BLAST) was used to compare the sequence with the known sequence, and a phylogenetic tree was constructed using the ITS sequence and the downloaded sequences of related strains. The results showed that strain WYT2101 and Aspergillus aculeatus were clustered in the same branch. Combined with the colony morphology and cell morphology observation of the strain and the comparison results, the strain was identified as Aspergillus aculeatus.

[0011] The application of the above-mentioned Aspergillus aculeatus in the production of tannase.

[0012] The application of the above-mentioned Aspergillus aculeatus in the production of gallic acid.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] The Aspergillus aculeatus provided by the present invention can efficiently produce tannase. After fermentation and culture, the tannase with high enzyme activity (the tannase activity of the fermentation supernatant can reach 44.45 U / mL) and good thermal stability can be obtained. The conditions of the enzymatic hydrolysis reaction have a wide pH adaptability, so that the degradation of tannins can be efficiently catalyzed in a short time without easily causing material denaturation, and the tannase has good application value.

[0015] The Aspergillus aculeatus provided by the present invention has a fast growth rate and is simple to control fermentation, so that the production cost of tannase is low, which is conducive to the industrialized production of tannase and tannin degradation. It is green, safe, and pollution-free, and can effectively improve economic benefits.

[0016] The Aspergillus aculeatus provided by the present invention can utilize industrial and agricultural waste, including but not limited to pomegranate peel, tea leaves, rapeseed cake, etc., to efficiently produce tannase, so that the production cost of tannase is low, and the product is green, safe, and pollution-free, thereby effectively improving economic benefits.

[0017] The Aspergillus aculeatus provided by the present invention can efficiently produce tannase (2.14 U / mL) and gallic acid (yield reaches 1.7 mg / mL) by using pomegranate peel. The method is simple, has few steps, and is pollution-free, which is conducive to the industrial production of tannase and gallic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Figures show the colony of Aspergillus aculeatus and the results of microscopic observation (A: colony morphology of Aspergillus aculeatus; B: microscopic observation of the acrocyst and hyphae; C: microscopic observation of spores);

[0019] Figure 2 This is the ITS phylogenetic tree of Aspergillus aculeatus;

[0020] Figure 3 This is the enzymatic characteristic diagram of tannase produced by Aspergillus aculeatus (A: effect of temperature on tannase activity; B: effect of pH on tannase activity; C: temperature stability of tannase; D: pH stability of tannase). DETAILED DESCRIPTION

[0021] The present invention will be further described below with reference to specific embodiments.

[0022] The Aspergillus aculeatus provided by the present invention is isolated from a soil sample under a tea tree in Fujian Province. The isolation and screening method of Aspergillus aculeatus can adopt a conventional method for isolating and screening new strains in the art, and can be selected according to actual needs.

[0023] For example, in a preferred embodiment of the present invention, the separation and screening method comprises:

[0024] S1. Collect soil samples from under tea trees in Fujian;

[0025] S2. Gallnut powder is selected as the sole carbon source of the enrichment medium, and the soil sample is added to the enrichment medium for enrichment culture.

[0026] S3. Select bromophenol blue medium supplemented with tannic acid as the primary screening medium, apply the enriched culture solution to the primary screening medium after gradient dilution, and perform primary screening. Since tannase-producing strains hydrolyze tannic acid in the medium to produce gallic acid, the bromophenol blue indicator changes from blue-purple to yellow. The strains that form color change circles in the primary screening medium are screened to obtain the primary screening strains.

[0027] S4. Inoculate the primary screened strain into the secondary screened culture medium for shake flask fermentation. After fermentation, determine the activity of tannase using the methanol rhodanine method, select the target strain, and obtain the Aspergillus aculeatus.

[0028] Preferably, the enrichment medium may include 3-15 g of gallnut powder and 30-150 mL of water; the primary screening medium may include 20-40 g / L of sucrose, 10-60 g / L of tannic acid, 1-5 g / L of sodium nitrate; 0.5-2.0 g / L of dipotassium hydrogen phosphate, 0.1-1.0 g / L of magnesium sulfate, 0.03-0.05 g / L of bromophenol blue, and 15-40 g / L of agar; the secondary screening medium may include 20-40 g / L of sugar (which may be at least one of sucrose, glucose, xylose, fructose, and lactose), 10-60 g / L of tannic acid, 1-5 g / L of sodium nitrate; 0.5-2.0 g / L of dipotassium hydrogen phosphate, and 0.1-1.0 g / L of magnesium sulfate, and the initial pH value may be 3-7. The conditions for the rescreening seed culture at least meet the following requirements: temperature of 28-34°C, rotation speed of 120-200 rpm, and time of 12-48 h; the conditions for the rescreening fermentation culture at least meet the following requirements: inoculation amount of 0.5-10 volume %, temperature of 25-35°C, rotation speed of 120-200 rpm, and time of 24-96 h.

[0029] The Aspergillus aculeatus provided by the present invention can produce a large number of viable Aspergillus aculeatus cells after cultivation. The present invention has no particular limitation on the cultivation method, as long as the Aspergillus aculeatus can be proliferated in large quantities by the cultivation method. For example, the Aspergillus aculeatus can be inoculated into a seed culture medium and cultured at 30°C±5°C and 120-200 rpm for 20-50 hours to prepare a seed liquid; the seed liquid is inoculated into a fermentation medium containing tannic acid, with an inoculum amount accounting for 0.5%-20% of the volume of the fermentation medium; after inoculation, the culture is cultured at 30°C±5°C and 120-200 rpm for 24-72 hours to obtain a fermentation liquid. The seed culture medium can be potato broth medium or modified Czapek liquid medium.

[0030] The fermentation medium containing tannic acid may include 5-20 g / L of sugar (which may be at least one of sucrose, glucose, xylose, fructose and lactose), 10-60 g / L of tannic acid, 1-5 g / L of tea seed cake powder; 0.5-2.0 g / L of dipotassium hydrogen phosphate, 0.2-0.8 g / L of magnesium sulfate, and the initial pH value may be 3-7.

[0031] The present invention can further separate the Aspergillus aculeatus cells in the above-mentioned culture solution. There is no particular limitation on the separation method, as long as the cells can be separated from the culture solution. For example, it can be achieved by centrifugation and / or filtration. The conditions for the centrifugation and filtration can be conventional conditions in the art.

[0032] The present invention provides a bacterial agent containing the above-mentioned Aspergillus aculeatus. In the present invention, there is no particular limitation on the concentration of Aspergillus aculeatus in the bacterial agent, and the concentration can be selected according to specific circumstances.

[0033] Furthermore, in the above application, the bacterial agent contains at least one of live cells, dead cells, or fermentation products of Aspergillus aculeatus. In the present invention, the term "fermentation product" refers to metabolites (including intracellular metabolites and / or extracellular metabolites) produced by Aspergillus aculeatus during the fermentation or cultivation process.

[0034] According to the present invention, there is no particular limitation on the dosage form of the bacterial agent. Depending on the intended use, the agent can be prepared into different dosage forms and contain corresponding excipients and other ingredients. For example, the bacterial agent can be a liquid agent (e.g., a fermentation broth and / or a fermentation supernatant obtained by centrifugation after cell disruption, or an extract of a solid fermentation product) and / or a solid agent (e.g., freeze-dried bacterial cells or a tannase preparation). The addition of the excipients to the dosage form can be determined using conventional techniques well known to those skilled in the art.

[0035] The present invention provides the use of the Aspergillus aculeatus in preparing tannase.

[0036] According to the present invention, the Aspergillus aculeatus can efficiently produce tannase through fermentation culture, that is, tannase is a fermentation product of the Aspergillus aculeatus, and the process of preparing tannase can adopt conventional cell disruption, tannase extraction and purification methods.

[0037] The present invention provides a method for preparing tannase and gallic acid by using the Aspergillus aculeatus.

[0038] In the present invention, Aspergillus aculeatus can be inoculated into a seed culture medium and cultured at 30°C ± 5°C and 120-200 rpm for 20-50 hours to prepare a seed solution; the seed solution is inoculated into a fermentation medium containing a tannin-rich raw material, with the inoculation amount accounting for 0.5% to 20% of the volume of the fermentation medium. After inoculation, the culture is carried out at 30°C ± 5°C and 120-200 rpm for 24-72 hours to obtain a fermentation liquid. The seed culture medium can be potato broth medium or modified Czapek liquid medium.

[0039] The fermentation medium for tannin-rich raw materials can include 50-200 g / L of tannin-rich raw materials (which can be at least one of tannin-rich agricultural and forestry processing residues such as pomegranate peel, tea leaves, rapeseed cake, etc.), 10-20 g / L of nitrogen source (which can be inorganic nitrogen sources such as sodium nitrate, ammonium chloride, ammonium sulfate, or organic nitrogen sources such as urea, yeast extract, peptone, bran, soybean meal, tea bran, etc.); 0.5-2.0 g / L of dipotassium hydrogen phosphate, 0.2-0.8 g / L of magnesium sulfate, and the initial pH value can be 3-7.

[0040] According to the present invention, the fermentation satisfies at least the following conditions: pH is 3-7, specifically 3, 4, 5, 6, 7, or any value between the above two values; temperature is 25-35°C, specifically 25°C, 30°C, 35°C, or any value between the above two values; rotation speed is 120-200rpm, specifically 120rpm, 140rpm, 160rpm, 180rpm, 200rpm, or any value between the above two values; time is 24-72h, specifically 24h, 36h, 48h, 60h, 72h, or any value between the above two values.

[0041] The specific embodiments of the present invention will be further explained below through examples, but it is not intended to limit the scope of protection of the present invention to the scope described in the examples.

[0042] In the following examples, the enrichment medium for Aspergillus aculeatus: 3 g of tannic acid, 30 mL of water, sterilized at 121° C. for 20 min.

[0043] The components of the screening medium are: sucrose 30 g / L, tannic acid 20 g / L, dipotassium hydrogen phosphate 1 g / L, magnesium sulfate 0.5 g / L, bromophenol blue 0.04 g / L, and agar 30 g / L.

[0044] The components of potato agar (PDA) medium are: 200 g / L potato, 20 g / L sucrose, and 20 g / L agar.

[0045] The components of the seed culture medium are: 5 g / L potato extract powder, 10 g / L peptone, 15 g / L glucose, and 5 g / L sodium chloride.

[0046] The components of the rescreening culture medium are: sucrose 20 g / L, tannic acid 1 g / L, sodium nitrate 3 g / L, dipotassium hydrogen phosphate 1 g / L, magnesium sulfate 0.5 g / L, and natural pH.

[0047] Example 1

[0048] 1. Preliminary screening of bacterial strains

[0049] (1) Sampling: Soil samples were collected from the soil under tea trees in Fujian;

[0050] (2) Enrichment culture: 1 g of soil sample was added to a 250 mL Erlenmeyer flask containing 30 mL of enrichment medium and cultured in a shaking incubator at 30 °C and 180 rpm for 48 h.

[0051] (3) Separation and screening: dilute the enriched culture medium to an appropriate multiple (10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 ), 0.2 mL of each concentration was evenly spread on the screening medium, cultured at 30°C for 48 hours, and the growth was observed every 6 hours. Since tannase-producing strains hydrolyze tannic acid in the culture medium to produce gallic acid, the bromophenol blue indicator changes from blue-purple to yellow. By measuring the hydrolysis zone diameter (D) and colony diameter (d), the D / d value was used as the basis for initial screening to screen strains with higher enzyme production capacity and purify them five times. Tannase-producing bacteria were selected and plated on a slant medium (PDA) and stored in a refrigerator at 4°C. All obtained strains were stored in glycerol cryovials (containing 20% sterile glycerol) and stored in a -20°C refrigerator.

[0052] 2. Rescreening of strains

[0053] The strains with high Dp / Dc ratios identified in the initial screening were activated and inoculated into seed culture medium. The culture was then incubated at 30°C and 180 rpm for 24 hours to obtain a seed solution. The seed solution of the strains was inoculated into a secondary screening culture medium at a 6% inoculum rate and incubated at 30°C and 180 rpm for 24 hours. The fermentation broth was then centrifuged at 12,000 rpm for 10 minutes to obtain a crude enzyme solution. Tannase activity was measured for each strain, ultimately identifying the highly active Aspergillus aculeatus WYT2101 strain.

[0054] Tannase activity determination: Three clean test tubes, labeled blank, control, and test tubes, were added with 0.25 mL of propyl gallate solution. To the blank tube, 0.25 mL of 0.1 mol / L sodium citrate buffer was added, and to the test tube, 0.25 mL of enzyme solution was added. The enzymatic hydrolysis conditions were 30°C for 5 min. To each of the three test tubes, 0.5 mL of rhodanine methanol solution (0.667%) was added, and the reaction was incubated at 30°C for 5 min. To each of the three test tubes, 3 mL of 0.5 mol / L KOH solution was added, and to the control tube, 0.25 mL of enzyme solution was added, and the reaction was incubated at 30°C for 10 min. The mixture was thoroughly mixed, and the absorbance at a wavelength of 520 nm was measured using the liquid in the blank tube as a control. Tannase activity was calculated based on the change in absorbance.

[0055] Enzyme activity definition: 1 unit (U) is the amount of enzyme required to produce 1 μmol / L gallic acid per minute at pH 5.0 and 30°C.

[0056] 3. Inoculate the target strain into PDA culture medium for cultivation. In the early stage of growth, white hyphae grow out. As the culture time increases, round or oval colonies are formed. The colonies are dark brown and the surface protrusions are fluffy. The hyphae have transverse septa, many branches, flask-shaped top capsules, and spherical or oval conidia (such as Figure 1 The internal transcribed spacer (ITS) sequence analysis was commissioned to Beijing Haichuang Keye Biotechnology Co., Ltd. The ITS sequence (shown in SEQ ID NO: 1) and the phylogenetic tree (shown in Figure 2 The analysis results showed that strain WYT2101 and Aspergillus aculeatus were clustered in the same branch; combined with the colony morphology, cell morphology observation and comparison results of the strain, strain WYT2101 was identified as Aspergillus aculeatus and sent to Guangdong Provincial Microbiological Culture Collection Center (GDMCC, address: 5th Floor, Building 59, Institute of Microbiology, Guangdong Academy of Sciences, No. 100 Xianlie Middle Road, Guangzhou, China) for preservation on August 10, 2022, with the preservation number GDMCC No. 62691.

[0057] Example 2

[0058] Characteristics of tannase produced by Aspergillus aculeatus (results as shown Figure 3 shown)

[0059] 1. Optimum Reaction Temperature: Maintain a substrate addition volume of 0.25 mL and mix thoroughly with 0.25 mL of tannase enzyme solution to a reaction volume of 0.5 mL. Incubate the reaction at 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, and 80°C for 5 minutes. Determine tannase activity, with the highest activity defined as 100%. The inactivated enzyme solution serves as the blank control. Results indicate that tannase activity peaks at 30°C and retains nearly 80% activity at 80°C.

[0060] Optimal pH for the Enzymatic Reaction: The enzyme solution was diluted to the appropriate multiple with buffer solutions of pH 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0. 0.25 mL was mixed with an equal volume of substrate and the tannase activity was measured at 30°C. The highest enzyme activity was set as 100%, and the enzyme solutions inactivated at different pH conditions served as blank controls. The results showed that the optimal pH for the tannase-catalyzed reaction was 5.0.

[0061] 3. Thermal stability and pH stability of tannase

[0062] The enzyme solution was placed in a water bath at 30.0℃, 40.0℃, 50.0℃, 60.0℃ and 70.0℃ for 4 hours, quickly cooled, and then the enzyme activity was measured at 30.0℃ and pH = 5.0. The results showed that the tannase had strong thermal stability. When placed in a water bath at a temperature of 30-70℃ for 4 hours, the relative activity of the tannase remained above 80%.

[0063] Tannase was placed at pH 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0 for 4 hours at the optimal temperature, and the enzyme activity was measured at 30.0°C and pH 5.0. The results showed that after 4 hours of treatment at pH 3.0-6.0, the residual tannase activity remained above 80%. After 4 hours of treatment at pH 7.0, the enzyme activity remained above 70%. After 4 hours of treatment at pH 8, the tannase activity dropped to above 34.56%. The tannase activity decreased rapidly with extended treatment time, indicating that the tannase has excellent stability within the pH range of 3.0-7.0 and has potential for development and utilization.

[0064] Example 3

[0065] Application of Aspergillus aculeatus in the production of tannase

[0066] After activation, Aspergillus aculeatus was inoculated into a seed culture medium and cultured with shaking at 30°C and 180 rpm for 24 hours to obtain a seed solution. The seed solution was inoculated into a tannase fermentation medium at a 2% inoculum rate and cultured with shaking at 28°C and 120 rpm for 48 hours. Tannase was obtained by centrifugation or filtration, and the tannase activity reached 44.45 U / mL.

[0067] The components of the tannase fermentation medium are: tannic acid 50 g / L, glucose 10 g / L, tea seed cake powder 2 g / L, K2HPO4 1 g / L, MgSO4 0.5 g / L, and the initial pH value of the medium is adjusted to 3.0.

[0068] Example 4

[0069] Application of Aspergillus aculeatus in the production of tannase and gallic acid from pomegranate peel

[0070] After activation, Aspergillus aculeatus was inoculated into a seed culture medium and cultured with shaking at 30°C and 180 rpm for 24 hours to obtain a seed solution. The seed solution was inoculated into a 250 mL Erlenmeyer flask containing 50 mL of pomegranate peel fermentation medium at a 0.5% inoculum concentration. The culture was shaken at 30°C and 140 rpm for 48 hours. Tannase was obtained by centrifugation or filtration, and the tannase activity reached 2.2 U / mL, and the gallic acid yield reached 1.7 mg / mL.

[0071] The components of the pomegranate peel fermentation medium are: pomegranate peel 200 g / L, soybean meal 20 g / L, K2HPO4 1 g / L, and MgSO4 0.5 g / L.

Claims

1. An Aspergillus aculeatus, characterized in that Its classification name is Aspergillus aculeatus ( Aspergillus aculeatus )WYT2101 has been deposited in Guangdong Provincial Microbiological Culture Collection Center with the accession number: GDMCC No.62691, the deposit date: August 10, 2022, and the deposit address is 5th Floor, Building 59, Institute of Microbiology, Guangdong Academy of Sciences.

2. The use of Aspergillus aculeatus according to claim 1 in producing tannase, characterized in that The specific method is as follows: after activating Aspergillus aculeatus, inoculate it into a seed culture medium, shake culture it at 30°C and 180 rpm for 24 h to obtain a seed solution; inoculate the seed solution into a tannase fermentation medium at a 2% inoculum rate, shake culture it at 28°C and 120 rpm for 48 h, and obtain tannase by centrifugation or filtration; The components of the seed culture medium are: potato extract powder 5 g / L, peptone 10 g / L, glucose 15 g / L, sodium chloride 5 g / L; The components of tannase fermentation medium are: tannic acid 50 g / L, glucose 10 g / L, tea seed cake powder 2 g / L, K2HPO4 1 g / L, MgSO4 0.5 g / L, and the initial pH value of the medium is adjusted to 3.

0.

3. The use of Aspergillus aculeatus according to claim 1 in producing gallic acid, characterized in that The specific method is as follows: after activating Aspergillus aculeatus, inoculate it into the seed culture medium, culture it at 30℃ and 180 rpm for 24 hours to obtain the seed solution; Inoculate 0.5% of the seed solution into a 250 mL Erlenmeyer flask containing 50 mL of pomegranate peel fermentation medium, shake culture at 30°C and 140 rpm for 48 h, and then centrifuge or filter. The components of the seed culture medium are: potato extract powder 5 g / L, peptone 10 g / L, glucose 15 g / L, sodium chloride 5 g / L; The components of pomegranate peel fermentation medium are: pomegranate peel 200 g / L, soybean meal 20 g / L, K2HPO4 1 g / L, MgSO4 0.5 g / L.

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