Tea saponin-degrading bacterial strain and application thereof

Abstract

The application discloses a theasapogenin-degrading strain and application thereof, and belongs to the technical field of microbial fermentation engineering. The strain is Aspergillus niger AN-8, is preserved in the China General Microbiological Culture Collection Center, and the preservation time is June 5, 2025, and the preservation number is CGMCC NO.41992. The application provides an Aspergillus niger strain which can simultaneously and efficiently degrade theasapogenin, improve the antibacterial activity of theasapogenin and improve the hemolytic performance, greatly improves the degradation rate of theasapogenin, and the degradation rate of theasapogenin reaches 70% after fermentation for 144 hours, the degradation rate of theasapogenin reaches 100% after fermentation for 216 hours, the antibacterial performance is obviously improved after fermentation, the hemolytic performance is obviously reduced, and the biological activity and use safety of theasapogenin are greatly improved.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation engineering technology, and more specifically to a tea saponin degrading strain and its application. Background Technology

[0002] my country has the widest distribution and the most varieties of Camellia oleifera (Camellia oleifera Abel) in the world, boasting abundant resources. Camellia seed meal is rich in nutrients, with crude protein accounting for 10%–20% and sugars for 30%–40%, making it a high-quality unconventional feed ingredient. In traditional oil processing, Camellia seed meal is often used for low-value-added products such as fertilizers, pond cleaning agents, or even discarded directly.

[0003] Camellia seed meal contains 15% to 20% tea saponin. A small amount of tea saponin has anti-inflammatory, antioxidant, and antibacterial functions. However, excessive tea saponin reacts with cholesterol components that make up animal red blood cell membranes, which can damage cell membranes, cause hemolysis, and lead to poor feed palatability, thus limiting the application of camellia seed meal in feed. Therefore, tea saponin in camellia seed meal needs to be degraded before it can be used as a feed ingredient.

[0004] Previously, there have been many studies on the degradation of tea saponins by microbial fermentation. Among the reported studies, there are few bacterial strains that can degrade tea saponins. The main degrading strains are molds, such as Aspergillus niger, Penicillium citrinum, and Citrobacter. Among them, there are many Aspergillus niger strains, but the degradation rate does not reach 100%, and they do not show the function of improving antibacterial properties and eliminating hemolytic properties.

[0005] Therefore, how to screen a multifunctional Aspergillus niger strain that can efficiently degrade tea saponins is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a tea saponin degrading strain and its application, so as to overcome the shortcomings of the prior art.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A tea saponin-degrading strain, namely Aspergillus niger AN-8, is deposited at the China General Microbiological Culture Collection Center on June 5, 2025, with accession number CGMCC NO.41992.

[0009] This invention also seeks protection for the use of the above-mentioned tea saponin-degrading strains in the degradation of tea saponins.

[0010] This invention also seeks protection for the use of the above-mentioned tea saponin-degrading strains in fermented camellia seed meal.

[0011] This invention also seeks protection for the use of the above-mentioned tea saponin-degrading strains in the preparation of broiler feed.

[0012] This invention provides a strain of Aspergillus niger that can simultaneously and efficiently degrade tea saponins, enhance their antibacterial activity, and improve hemolytic properties. This greatly improves the degradation rate of tea saponins, reaching 70% after 144 hours of fermentation and 100% after 216 hours. Furthermore, the antibacterial properties are significantly improved and the hemolytic properties are significantly reduced after fermentation, thus greatly enhancing the bioactivity and safety of tea saponins.

[0013] The isolation and purification steps of Aspergillus niger AN-8 in this invention are as follows: Take 100g each of tea seed meal, tea root soil, tea fruit, and leaves, add 300mL of sterile water, stir evenly, shake at room temperature for 30min, and let stand for 10min. Take 10mL of the supernatant and inoculate it into 100mL of inorganic salt liquid culture medium containing tea saponin. Incubate at 180r / min in a constant temperature incubator for 24h with shaking. Spread 100μL of the culture solution onto Bengal Red solid plates and incubate in a constant temperature incubator for 36h. Separately, take 5mL of tea saponin culture solution and incubate at 80℃ for 10min. After cooling to room temperature, spread 100μL of the culture solution onto LB solid plates and incubate in a constant temperature incubator for 36h. Select single colonies with different appearances and streak them twice in tea saponin inorganic salt solid culture medium for purification.

[0014] The screening steps for Aspergillus niger AN-8 of this invention are as follows: Single colonies were selected on a solid culture medium containing tea saponin inorganic salts and inoculated onto a liquid culture medium containing a high concentration of tea saponin inorganic salts. The cultures were then shaken at 180 r / min in a constant temperature incubator. Fermentation broth was collected at 48 h, 96 h, 144 h, 192 h, and 240 h, filtered through a 0.22 μm filter membrane, and the tea saponin content was determined using a Waters high-performance liquid chromatography (HPLC) instrument. Each strain was repeated three times, and the average value was taken. Using uninoculated tea saponin inorganic salt liquid culture medium as a control, the strain with the highest degradation rate was selected as the target strain, and Aspergillus niger AN-8 and Aspergillus niger AN-5 were obtained through screening.

[0015] The Aspergillus niger AN-8 of this invention was isolated and purified from tea seed meal. When cultured on PDA medium at 28°C and 88% humidity for 48 hours, it was mostly white, fluffy, round colonies, which later turned bright yellow and then black and fluffy, covering the entire plate.

[0016] The DNA gene sequence of Aspergillus niger AN-8 of this invention is shown in the sequence listing, as follows:

[0017] .

[0018] The sequencing results of the ITS of Aspergillus niger AN-8 in this invention were analyzed for homology in the NCBI database, yielding highly similar sequences. A phylogenetic tree was constructed using MEGA11 software, showing that the ITS sequence of AN-8 shared 100% homology with Aspergillus niger. Based on morphological observation and ITS sequencing results, this Aspergillus niger AN-8 strain can be identified as an Aspergillus niger strain, and is named Aspergillus niger AN-8.

[0019] One loopful of Aspergillus niger AN-8 mycelium was inoculated onto an inorganic salt liquid culture medium containing a high concentration of tea saponin, and cultured with shaking at 180 rpm in a constant temperature incubator. 200 μL of the Aspergillus niger AN-8 culture medium was then used for antibacterial and hemolytic activity tests.

[0020] The antibacterial performance testing steps for Aspergillus niger AN-8 of this invention were as follows: Escherichia coli K12 and Staphylococcus aureus were inoculated onto LB liquid medium and cultured at a constant temperature with shaking at 180 rpm for 12 h. 100 μL of the diluted pathogenic bacterial solution was then evenly spread onto an LB solid plate. Sterile Oxford cups were then placed upright on the plates, and 200 μL of each of the following fermentation broths (from the "screening" step): Aspergillus niger AN-8 fermentation broth, Aspergillus niger AN-5 fermentation broth, sterile tea saponin inorganic salt medium, and Aspergillus niger AN-8 PDA culture medium and Aspergillus niger AN-5 PDA culture medium were added, with three replicates for each sample. The results showed that Aspergillus niger strain AN-8 significantly enhanced the antibacterial performance against Escherichia coli K12 and Staphylococcus aureus.

[0021] The hemolytic performance testing procedure for Aspergillus niger AN-8 of this invention is as follows: The Aspergillus niger AN-8 and Aspergillus niger AN-5 fermentation broths from the "screening" step were inactivated by incubating in an 80℃ water bath for 15 minutes. Oxford cups were placed upright on fresh sheep blood agar plates, and 200 μL of inactivated Aspergillus niger AN-8 fermentation broth, Aspergillus niger AN-5 fermentation broth, and sterile tea saponin inorganic salt medium were added respectively. Each sample was tested in triplicate, and the plates were incubated at a constant temperature for 24 hours. The results showed that the hemolytic performance of the Aspergillus niger AN-8 fermentation broth disappeared, while AN-5 still exhibited a small amount of hemolytic zone.

[0022] Furthermore, the composition of the above-mentioned tea saponin inorganic salt liquid culture medium is as follows: 10g of crude tea saponin extract, 0.18g / L K2HPO4, 0.18g / L MgSO4·H2O, 0.35g / L KCl, 0.35g / L (NH4)2SO4, 0.015g / L CaCl2, 0.013g / L FeCl3·6H2O, 0.013g / L ZNSO4, 0.011g / L CuSO4, 1000mL of water, sterilized at 121℃ for 15min.

[0023] Furthermore, the concentration of the above-mentioned tea saponin inorganic salt culture medium is 5–12 mg / mL.

[0024] Furthermore, the temperature of the aforementioned constant temperature incubator is 25–38°C.

[0025] Furthermore, the dilution factor of the aforementioned pathogens was 10. -2 ~10 -4 times.

[0026] Furthermore, the fermentation broth was cultured for 48–240 hours.

[0027] As can be seen from the above technical solution, compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] 1. This invention uses Aspergillus niger AN-8 as a strain for degrading tea saponins. This strain has a strong ability to degrade tea saponins, providing a strain source for tea saponin degradation and showing good application prospects.

[0029] 2. The Aspergillus niger AN-8 of this invention can efficiently degrade tea saponins. The tea saponin degradation rate reaches 70% after 144 hours of fermentation and 100% after 216 hours of fermentation.

[0030] 3. The tea saponin fermented with Aspergillus niger AN-8 of this invention can enhance the antibacterial properties of tea saponin. The diameter of the inhibition zone of Escherichia coli K12 is 1.5 times that of unfermented tea saponin, and the inhibition diameter increases from 8mm to 12mm. However, the antibacterial properties of Aspergillus niger AN-8PDA culture medium against Escherichia coli are relatively low. The diameter of the inhibition zone of Staphylococcus aureus is twice that of unfermented tea saponin, and the inhibition diameter increases from 9mm to 18mm. At the same time, the antibacterial properties are higher than those of AN-8PDA culture medium.

[0031] 4. The tea saponin fermented by Aspergillus niger AN-8 of this invention can eliminate the hemolytic properties of tea saponin, and the diameter of the hemolytic zone is reduced from 12 mm before fermentation to 0 mm.

[0032] 5. The process conditions of Aspergillus niger AN-8 of the present invention are easy to control, reducing operating costs and significantly improving the bioactivity of tea saponins. Attached Figure Description

[0033] Figure 1 This is a colony morphology diagram of Aspergillus niger AN-8.

[0034] Figure 2 Phylogenetic tree diagram of Aspergillus niger AN-8;

[0035] Figure 3 Liquid phase spectrum of tea saponin fermentation medium by Aspergillus niger AN-8;

[0036] Figure 4 The antibacterial properties of Aspergillus niger AN-8 fermentation broth;

[0037] Figure 5 The hemolytic properties of Aspergillus niger AN-8 fermentation broth. Detailed Implementation

[0038] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] Example 1

[0040] The isolation, purification, and screening of Aspergillus niger strain AN-8 specifically includes the following steps:

[0041] (1) Take 100g each of tea seed meal, soil from the roots of tea trees in Ningxiang, Hunan, tea fruit, and leaves, add 300mL of sterile water, stir well, shake at room temperature for 30min, and let stand for 10min. Take 10mL of the upper layer liquid and inoculate it into 100mL of inorganic salt liquid culture medium containing tea saponin. Place it in a constant temperature incubator at 32℃ and shake at 180r / min for 24h. Take 100μL of the culture solution and spread it on Bengal Red solid plates. Place it in a constant temperature incubator at 32℃ and let it stand for 36h. Take another 5mL of tea saponin culture solution and incubate it in an 80℃ water bath for 10min. After it is placed at room temperature, take 100μL of the culture solution and spread it on LB solid plates. Place it in a constant temperature incubator at 32℃ and let it stand for 36h. Select single colonies with different appearances and streak them twice in tea saponin inorganic salt solid culture medium for purification.

[0042] (2) Single colonies were selected on the solid culture medium containing tea saponin inorganic salts and inoculated onto the liquid culture medium containing high concentration of tea saponin inorganic salts. The cultures were shaken at 180 r / min in a constant temperature incubator at 32℃. Fermentation broth was collected at 48 h, 96 h, 144 h, 192 h, and 240 h and filtered through a 0.22 μm filter membrane. The tea saponin content was determined using a Waters high performance liquid chromatograph. Each strain was repeated three times and the average value was taken. The strain with the highest degradation rate was selected as the target strain, and Aspergillus niger AN-8 was obtained.

[0043] Example 2

[0044] Identification of Aspergillus niger strain AN-8

[0045] 1. Morphological observation:

[0046] Aspergillus niger strain AN-8 was inoculated onto PDA plates and cultured at 28°C for 5 days. The growth of the strain on the plates was observed. Initially, the mycelium was white, then turned green, and finally matured into black spores that coalesced into sheets. Figure 1 The colony morphology after the formation of black spores.

[0047] 2. ITS sequence analysis:

[0048] DNA was extracted from the tested strains using a fungal DNA extraction kit (Tiangen Biotech Co., Ltd.).

[0049] ITS primers: ITS1 (5ˊ-TCCGTAGGTGAACCTGCGG) and ITS4 (5ˊ-TCCTCCGCTTATTGATATGC).

[0050] PCR reaction system (50μL): 2×PCR Mix 25μL, 2.5μL each of 20mol / L ITS1 / ITS4 primers, 2μL template DNA, 18μL ddH2O.

[0051] The AN-8 ITS sequence showed 100% homology with the Aspergillus niger sequence. Combined with morphological analysis, this strain was identified as Aspergillus niger and named Aspergillus niger AN-8. The phylogenetic tree is as follows: Figure 2 As shown.

[0052] Example 3

[0053] The application of Aspergillus niger AN-8 in the degradation of tea saponins includes the following steps:

[0054] Activated Aspergillus niger AN-8 mycelia were picked and inoculated onto inorganic salt liquid medium containing 10 mg / mL high concentration of tea saponin, and cultured by shaking at 180 r / min in a constant temperature incubator at 32℃.

[0055] The results are as follows Figure 3 As shown.

[0056] Depend on Figure 3 It is known that the Aspergillus niger AN-8 of the present invention can efficiently degrade tea saponins, with a tea saponin degradation rate of 70% after 144 hours of fermentation and 100% after 216 hours of fermentation.

[0057] Example 4

[0058] The antibacterial performance testing of Aspergillus niger AN-8 includes the following steps:

[0059] Inoculate *Escherichia coli* K12 and *Staphylococcus aureus* onto LB liquid medium and incubate with shaking at 180 rpm for 12 h at a constant temperature. Take a 10⁻⁶ dose of the diluted medium. -2 100 μL of the pathogenic bacteria culture was evenly spread on an LB solid plate. Then, a sterile Oxford cup was placed upright on the plate, and 200 μL of Aspergillus niger AN-8 fermentation high-concentration tea saponin culture medium, AN-8PDA fermentation broth, and sterile tea saponin inorganic salt culture medium were added. Each sample was repeated in triplicate.

[0060] The results are as follows Figure 4 As shown.

[0061] Depend on Figure 4It can be seen that the tea saponin culture medium of Aspergillus niger strain AN-8 significantly improved the antibacterial performance of Escherichia coli K12 and Staphylococcus aureus. The diameter of the inhibition zone of Escherichia coli K12 was 1.5 times that of the unfermented tea saponin, and the inhibition diameter increased from 8 mm to 12 mm. However, the antibacterial performance of Aspergillus niger AN-8PDA culture medium was relatively low against Escherichia coli. The diameter of the inhibition zone of Staphylococcus aureus was twice that of the unfermented tea saponin, and the inhibition diameter increased from 9 mm to 18 mm. At the same time, the antibacterial performance was higher than that of the AN-8PDA culture medium.

[0062] Example 5

[0063] The hemolytic performance test of Aspergillus niger AN-8 includes the following steps:

[0064] Place the Oxford cup upright on a fresh sheep blood plate. Inactivate the cultured Aspergillus niger strain AN-8 in a tea saponin inorganic salt medium at 80°C for 15 minutes. Use uninoculated tea saponin medium as a control. Take 200 μL of each sample and add it to the Oxford cup. Repeat the process three times for each sample. Let the samples stand at a constant temperature for 24 hours.

[0065] The PDA agar medium consisted of 200g peeled potatoes, 20g glucose, 18g agar, and 1000mL water, sterilized at 121℃ for 15min.

[0066] PTB liquid culture medium: 200g peeled potato, 20g glucose, 1000mL water, sterilized at 121℃ for 15min.

[0067] High-concentration tea saponin inorganic salt culture medium: 10g crude tea saponin extract, 0.18g / L K2HPO4, 0.18g / L MgSO4·H2O, 0.35g / L KCl, 0.35g / L (NH4)2SO4, 0.015g / L CaCl2, 0.013g / L FeCl3·6H2O, 0.013g / L ZNSO4, 0.011g / L CuSO4, 1000mL water, sterilized at 121℃ for 15min.

[0068] The results are as follows Figure 5 As shown.

[0069] Depend on Figure 5 It is known that the fermentation of tea saponins by Aspergillus niger AN-8 in this invention can eliminate the hemolytic properties of tea saponins, and the diameter of the hemolytic zone is reduced from 12 mm before fermentation to 0 mm.

[0070] Example 6

[0071] Solid-state fermentation of camellia seed meal was carried out using Aspergillus niger AN-8. Camellia seed meal fermented with different proportions of Aspergillus niger AN-8 was added to broiler feed, and its effect on broiler growth performance was observed.

[0072] The solid-state fermentation steps are as follows: weigh 100g of camellia seed meal into a fermentation bag, inoculate 10mL of Aspergillus niger AN-8 bacterial solution that has been cultured for 7 days into the culture medium, add 70mL of water, culture at 32℃, turn the material over once every 24 hours, ferment for 10 days, and dry the fermented material in a 65℃ constant temperature drying oven.

[0073] The feeding trial was designed as follows: 216 one-day-old male Ross 308 broiler chickens were randomly divided into 6 treatment groups, with 6 replicates per treatment group and 6 chickens per replicate. They were fed 0.5%, 1.5%, and 2.5% unfermented camellia seed meal, and fermented camellia seed meal, respectively. The experimental diet formulations and nutrient levels are detailed in Tables 1 and 2. The nutritional standards were based on the chicken feeding standard "GB / T 5916-2020 Compound Feed for Laying Hens and Broilers," divided into two stages: 1–21 days old and 21–42 days old. Broiler chickens were fed only through free access to feed and water. Feed was collected weekly, and the amount of feed given and any uneaten feed was recorded.

[0074] Table 1 Feed formulation for broiler chickens aged 1-21 days

[0075]

[0076] Table 2 Feed formulation for broiler chickens aged 22–42 days

[0077]

[0078]

[0079] The results are shown in Table 3.

[0080] Table 3 Effects of fermented camellia seed meal on broiler growth performance

[0081]

[0082] As shown in Table 3, the growth performance of broilers in the fermented camellia seed meal group was significantly higher than that in the unfermented camellia seed meal group. When the addition amount reached 2.5%, the mortality rate of broilers was significantly reduced.

[0083] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A tea saponin-degrading strain, characterized in that, This strain is Aspergillus niger ( Aspergillus niger AN-8 is deposited at the China General Microbiological Culture Collection Center on June 5, 2025, with accession number CGMCCNO.41992.

2. The application of the tea saponin-degrading strain as described in claim 1 in the degradation of tea saponin.

3. The application of the tea saponin-degrading strain as described in claim 1 in fermented camellia seed meal.

4. The application of the tea saponin-degrading strain as described in claim 1 in the preparation of broiler feed.

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