Pediococcus acidilactici H-3 for producing tannase and application thereof

Abstract

The invention belongs to the technical field of biology, and provides pediococcus acidilactici H-3 for producing tannase and application of the pediococcus acidilactici H-3. The invention discloses pediococcus acidilactici H-3, the Latin of the pediococcus acidilactici H-3 is pediococcus acidilactici, the preservation name of the pediococcus acidilactici H-3 is pediococcus acidilactici, the preservation place is China General Microbiological Culture Collection Center, the preservation date is January 13, 2026, and the preservation number is CGMCC No.37372. The invention further discloses a preparation method of the pediococcus acidilactici H-3. According to the invention, an excellent strain resource is provided for improving the flavor of fruits with high tannin content and developing the functional characteristics of the fruits, and a new direction is provided for the application of the strains in plant matrix fermentation.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to a tannin-producing strain of Pediococcus lactis H-3 and its applications. Background Technology

[0002] Black-fruited glandular-ribbed chokeberry ( Aronia melanocarpa Also known as the ageless berry, it belongs to the Rosaceae family and is a deciduous shrub. It is particularly valuable due to its unique polyphenolic composition, which includes various active ingredients such as proanthocyanidins, anthocyanins, flavonoids, and phenolic acids. Studies have shown that black chokeberry has antioxidant, anticancer, anti-inflammatory, and hypoglycemic effects. In recent years, research on black chokeberry has shown great potential in various fields. However, due to the high tannin content in its fruit, the fresh fruit has a strong astringent taste; therefore, de-astringency treatment is necessary to improve its flavor.

[0003] Tannins are polyphenolic compounds, classified into hydrolyzable tannins (HT) and condensed tannins (CT). Condensed tannins consist of polymers of 2 to 50 (or more) flavonoid units linked by carbon-carbon bonds. Condensed tannins are the main component of higher plants such as black chokeberry. High tannin content not only affects the quality of food but also produces an unpleasant taste. Methods for removing tannins mainly include chemical, physical, and biological methods. Chemical methods primarily involve deastringents binding with tannins through specific chemical bonds to form insoluble complexes, thus achieving efficient tannin removal. Common examples include bovine serum albumin, polyvinylpyrrolidone (PVP), and gelatin. Physical methods often utilize physical adsorption deastringents. Studies have shown that using activated carbon combined with ultrasound to treat chokeberry juice significantly improves the deastringency, but this compromises juice stability and leads to nutrient loss. Biological methods mainly employ enzyme preparations and microorganisms for deastringency. Tannin enzymes, characterized by high efficiency, specificity, and no side effects, can be used to treat the astringency caused by tannins in natural foods. Tannin enzymes (tannin ester acyl hydrolases) are crucial for improving the quality of black chokeberry. As an inducible enzyme, tannin enzymes specifically hydrolyze the ester and phenolic acid bonds of polyphenols, effectively reducing the content of ester-type catechins and thus alleviating the astringency in black chokeberry. However, there are currently no reports on the application of tannin-producing lactic acid bacteria in the astringency removal of black chokeberry. Therefore, there is an urgent need in this field for a lactic acid bacteria strain that can efficiently produce tannins. Summary of the Invention

[0004] The purpose of this invention is to provide a tannin-producing strain of *Pediococcus lactis* H-3 and its application. This tannin-producing lactic acid bacteria is used to ferment and remove astringency from *Sorbus nigra*, improving its astringent taste. Based on single-factor experiments, response surface methodology was used to optimize the astringency removal conditions of *Sorbus nigra*. The main components, such as total polyphenols and total flavonoids, as well as DPPH, ABTS, and hydroxyl radical scavenging rates, were analyzed to explore the changes in these main components during fermentation and their correlation with antioxidant activity. This aims to provide a theoretical basis for the development of *Sorbus nigra*.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a tannin-producing Pediococcus acidilactic strain H-3, whose Latin name is Pediococcus acidilactic, its preservation name is Pediococcus acidilactic, its deposit location is the China General Microbiological Culture Collection Center, its deposit date is January 13, 2026, and its accession number is CGMCC No. 37372.

[0006] The present invention also provides the application of the aforementioned *Pediococcus lactis* H-3 in the deastringency removal of black chokeberry fruit.

[0007] The present invention also provides a bacterial solution containing the aforementioned *Pediococcus lactis* H-3.

[0008] The present invention also provides a method for preparing the bacterial culture, comprising the following steps: inoculating the *Pediococcus lactis* H-3 into tannin selection medium and culturing it at 35-39°C for 12-16 hours.

[0009] Preferably, the pH of the tannin screening medium is 3.5-4.5, and the tannin screening medium is prepared by adding 0.002-0.006 g of bromophenol blue and 0.8-1.2 g of tannic acid to 90-110 mL of MRS agar medium and sterilizing at 115-125 °C for 15-25 min.

[0010] The present invention also provides the application of the bacterial solution in the deastringency removal of black chokeberry fruit.

[0011] The present invention also provides a method for removing astringency from black chokeberry fruit using the bacterial solution, comprising the following steps: Mix the bacterial culture with the pulp of black chokeberry fruit and ferment at 31-43℃ for 1-5 days.

[0012] Preferably, the viable count of *Pediococcus lactis* H-3 in the bacterial solution is 1 × 10⁻⁶. 7 ~1×10 9CFU / mL, the inoculation amount of the bacterial solution is 2-3 vt of black chokeberry pulp.

[0013] The beneficial effects of this invention are as follows: This invention isolates a tannin-producing lactic acid bacterium, *Pediococcus lactis* H-3, from naturally fermented *Sorbus nigra* pulp. Its extracellular enzyme activity is 3.41 U / mL, with an optimal pH of 4.0 and an optimal temperature of 37 °C. Cultivation at 37 °C for 14 h resulted in the transition from the logarithmic growth phase to the stationary phase. Response surface methodology was used to optimize the fermentation process for deastringency removal from *Sorbus nigra*. The optimal fermentation conditions were: inoculum size 2.5 vt%, fermentation time 4 days, and fermentation temperature 37 °C. Under these optimized conditions, the condensed tannin content in the fermentation broth decreased to 2.98 g / L, a significant reduction compared to pre-fermentation levels. P <0.05) decreased by 52.54%. During the fermentation of black chokeberry pulp by strain H-3, pH, reducing sugar, TSS, and anthocyanin content continuously decreased; total polyphenols, total acid content, and SOD enzyme activity generally increased; total flavonoid content showed an initial increase followed by a decrease, with a maximum value of 4.23 mg / mL; and the maximum values ​​of DPPH, ABTS, and hydroxyl radical scavenging rates of the fermented pulp reached 61.34%, 80.12%, and 80.23%, respectively, indicating significantly enhanced antioxidant activity. P <0.05. Total polyphenols, SOD enzyme, and antioxidant activity showed a highly significant positive correlation ( P <0.01). It is evident that the results of this invention provide excellent microbial resources for improving the flavor of fruits with high tannin content and developing their functional properties, and offer a new direction for the application of this type of strain in plant substrate fermentation.

[0014] Preservation Instructions

[0015] Pediococcus acidilactic, whose Latin name is Pediococcus acidilactic, is deposited at the China General Microbiological Culture Collection Center (CGMCC) on January 13, 2026. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The deposit date is CGMCC No. 37372. Attached Figure Description

[0016] Figure 1 This is a growth curve of strain H-3; Figure 2 To locate the tanninase produced by strain H-3, different lowercase letters represent significant differences between groups. P <0.05); Figure 3 The optimal temperature and pH for strain H-3 are shown in the figure. a - optimal temperature; b - optimal pH. Different lowercase letters on the same curve in the figure indicate significant differences. P <0.05); Figure 4 The plate morphology and Gram staining results for strain H-3 are shown in Figure a. Plate morphology; Figure b. Gram staining (100x magnification). Figure 5 Phylogenetic tree of strain H-3; Figure 6 The effects of inoculum size, fermentation time, and fermentation temperature on the content of condensed tannins are shown in Figure 1. a - inoculum size; b - fermentation time; c - fermentation temperature. Figure 7 This is a response surface plot of the effects of various factors on the content of condensed tannins. Different lowercase letters on the same curve in the figure indicate significant differences. P <0.05); Figure 8 The graph shows the changes in TSS and reducing sugar content during the fermentation of black chokeberry. Different lowercase letters on the same curve indicate significant differences. P <0.05); Figure 9 The graph shows the changes in total acid content and pH during the fermentation of black chokeberry. Different lowercase letters on the same curve indicate significant differences. P <0.05); Figure 10 The graph shows the changes in total polyphenol and total flavonoid content during the fermentation of black chokeberry. Different lowercase letters on the same curve indicate significant differences. P <0.05); Figure 11 The graph shows the changes in condensed tannin content during the fermentation of black chokeberry. Different lowercase letters on the curves indicate significant differences. P <0.05); Figure 12 The graph shows the changes in anthocyanin and SOD enzyme content during the fermentation of black chokeberry. Different lowercase letters on the same curve indicate significant differences. P <0.05); Figure 13 The changes in antioxidant capacity during the fermentation of black chokeberry were investigated, including α-DPPH free radical scavenging rate, β-ABTS free radical scavenging rate, and β-hydroxyl free radical scavenging rate. Figure 14 Correlation analysis of the main components and antioxidant activities during the fermentation of black chokeberry. Detailed Implementation

[0017] This invention provides a tannin-producing Pediococcus acidilactic strain H-3, whose Latin name is Pediococcus acidilactic, its preservation name is Pediococcus acidilactic, its deposit location is the China General Microbiological Culture Collection Center, its deposit date is January 13, 2026, and its accession number is CGMCC No. 37372.

[0018] The present invention also provides the application of the aforementioned *Pediococcus lactis* H-3 in the deastringency removal of black chokeberry fruit.

[0019] The present invention also provides a bacterial solution containing the aforementioned *Pediococcus lactis* H-3.

[0020] The present invention also provides a method for preparing the bacterial culture, comprising the following steps: inoculating the *Pediococcus lactis* H-3 into tannin selection medium and culturing it at 35-39°C for 12-16 hours.

[0021] In this invention, the pH of the tannin screening medium is preferably 3.5-4.5, more preferably 4.0. The preferred method for preparing the tannin screening medium is as follows: 0.002-0.006 g of bromophenol blue and 0.8-1.2 g of tannic acid are added to 90-110 mL of MRS agar medium, and sterilized at 115-125 °C for 15-25 min. More preferably, 0.004 g of bromophenol blue and 1 g of tannic acid are added to 100 mL of MRS agar medium, and sterilized at 121 °C for 20 min. The culture temperature is further preferably 37 °C, and the culture time is further preferably 14 h.

[0022] The present invention also provides the application of the bacterial solution in the deastringency removal of black chokeberry fruit.

[0023] The present invention also provides a method for removing astringency from black chokeberry fruit using the bacterial solution, comprising the following steps: Mix the bacterial culture with the pulp of black chokeberry fruit and ferment at 31-43℃ for 1-5 days.

[0024] In this invention, the viable count of *Pediococcus lactis* H-3 in the bacterial solution is preferably 1 × 10⁻⁶. 7 ~1×10 9 CFU / mL, further preferably 1×10⁻⁶ 8 The inoculum concentration is CFU / mL, the preferred inoculum amount is 2-3 vt of black chokeberry pulp, more preferably 2.5 vt of black chokeberry pulp, the preferred fermentation temperature is 37°C, and the preferred fermentation time is 4 days.

[0025] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0026] Example 1: Materials and Methods

[0027] 1. Materials and Instruments

[0028] Black chokeberry fruit, Yanbian Black Fruit Technology Development Co., Ltd.; Celluclast 1.5L cellulase (enzyme activity 10000 U / mL), Pectinex XXL pectinase (enzyme activity 10000 U / mL) U / mL), Novozymes (China) Biotechnology Co., Ltd.; MRS broth, Beijing Aoboxing Biotechnology Co., Ltd.; MRS agar, Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd.; Folin-Ciocalteu, DPPH, ABTS, vanillin, tannic acid, Shanghai Maclean Biochemical Technology Co., Ltd.; SOD kit, Nanjing Jiancheng Bioengineering Institute; Rutin, gallic acid, glucose, catechin standards, Shanghai Chunyou Biotechnology Co., Ltd.; Rhodanine, propyl gallate, Shanghai Aladdin Biochemical Technology Co., Ltd.; Magnesium sulfate, potassium chloride, potassium hydroxide, sodium nitrite, disodium hydrogen phosphate, sodium bicarbonate, aluminum nitrate, hydrogen peroxide, ferrous sulfate, Liaoning Quanrui Reagent Co., Ltd.; Sulfuric acid, Tianjin Kemeio Chemical Reagent Co., Ltd.; Potassium persulfate, sodium hydroxide, anhydrous sodium carbonate, methanol, sodium nitrate, Tianjin Damao Chemical Reagent Factory; Calcium carbonate, Tianjin Yongda Chemical Reagent Co., Ltd.

[0029] Tannin selection medium: Add 0.004 g of bromophenol blue and 1 g of tannic acid to 100 mL of MRS agar medium, and sterilize at 121℃ for 20 min.

[0030] Enzyme-producing culture medium: Add 2% tannic acid, 1% sucrose, 0.3% sodium nitrate, 0.1% dipotassium hydrogen phosphate, 0.05% potassium chloride, and 0.05% MgSO4·7H2O to 100 mL of distilled water, and sterilize at 121 °C for 20 min.

[0031] SQ810C Vertical Pressure Steam Sterilizer, Zhiwei (Xiamen) Instrument Co., Ltd.; CTH1850R Centrifuge, Hunan Xiangli Science Co., Ltd.; M-2800 UV Spectrophotometer, Shanghai Meixi Instrument Co., Ltd.; ZQWY-200V Shaking Incubator, Shanghai Zhichu Instrument Co., Ltd.; FS-450N Ultrasonic Processor, Shanghai Shengxi Ultrasonic Instrument Co., Ltd.; DK-600B Electric Thermostatic Water Bath, Shanghai Senxin Experimental Instrument Co., Ltd.; LB90A Handheld Saccharimeter, Guangzhou Mingrui Electronic Technology Co., Ltd.; pH Meter, Shanghai Instrument & Electronics Scientific Instrument Co., Ltd.

[0032] 2. Experimental Methods

[0033] 2.1 Isolation and purification of lactic acid bacteria

[0034] After washing, the fruits of *Sorbus nigra* were pulped with distilled water at a 1:1 ratio. Cellulase (3.2 mL / kg) and pectinase (6.4 mL / kg, enzyme ratio 1:2) were added based on fruit weight, and the mixture was reacted in a 45 ℃ water bath for 3.5 h. The enzymatically hydrolyzed pulp was then dispensed and naturally fermented at 25 ℃, 31 ℃, and 37 ℃ for 45 days. Samples were taken every 3 days for 15 consecutive days, serially diluted, and plated onto MRS agar containing 0.2 wt% calcium carbonate, and incubated at 37 ℃ for 48 h. Colonies with calcium dissolution zones were selected and purified by streak plating, then stored at 4 ℃ for later use.

[0035] 2.2 Screening of tannin-producing lactic acid bacteria

[0036] 2.2.1 Screening of tannin-tolerant strains

[0037] The pure bacterial culture was spot-inoculated onto tannin selection medium and incubated at 37 °C for 4 days. If a discolored zone appeared around the colony, it had the ability to degrade tannins and was named crude enzyme solution 1. Tannin enzyme activity was subsequently measured.

[0038] 2.2.2 Tanninase Activity Assay

[0039] Gallic acid standard solutions with concentrations of 60, 80, 100, 120, and 140 μmol / L were prepared using a citric acid buffer solution at pH 5.0. 0.5 mL of each gallic acid standard solution was taken and 0.3 mL of a 0.05 mol / L methanol-tannin solution was added, and the mixture was reacted at 30 °C for 5 min. Then, 0.2 mL of a 0.5 mol / L KOH aqueous solution was added, and the mixture was kept at 30 °C for 5 min. Finally, 4 mL of distilled water was added to dilute the solution, using distilled water as a blank. The absorbance of the gallic acid standard solutions was measured at a wavelength of 520 nm, yielding the standard curve equation: y = 0.0014x - 0.0172. R 2 =0.9908.

[0040] Three clean test tubes were labeled as blank, test, and control. 0.25 mL (2.35 mmol / L) of propyl gallate solution was added to each tube. 0.25 mL (pH 5.0) of citrate buffer was added to the blank tube, and 0.25 mL of crude enzyme solution 1 was added to the test tube. The tubes were incubated at 30 °C for 5 min. Then, 0.3 mL (0.05 mol / L) of methanol-to-tannin solution was added to each tube, and the reaction was incubated at 30 °C for 5 min. Next, 0.2 mL (0.5 mol / L) of KOH aqueous solution was added, and the reaction was incubated at 30 °C for 5 min. Afterward, only the control tube was incubated with 0.25 mL of crude enzyme solution 1. Finally, each tube was diluted with 4 mL of distilled water, incubated at 30 °C for 5–10 min, and the absorbance of the reaction mixture was measured at 520 nm using distilled water as a blank. The change in absorbance at 520 nm (ΔA) was recorded. 520 Tanninase activity was calculated using a standard curve.

[0041] 2.3 Growth curve of tannin-producing lactic acid bacteria

[0042] The target strain was inoculated into MRS liquid medium at a ratio of 2 vt% and cultured at 37 °C for 24 h. Samples were taken every 2 h, and the OD was measured. 600 The growth curve of the target strain was measured.

[0043] 2.4 Enzyme-producing characteristics of tannin-producing lactic acid bacteria

[0044] 2.4.1 Enzyme localization of the target strain

[0045] Take 30 mL of the stable bacterial culture (14 h after inoculation into MRS liquid medium), centrifuge (8000 r / min, 15 min), and collect 2 mL of the supernatant for later use. Wash the resulting bacterial sludge twice with sterile phosphate buffer (pH 7.2), then resuspend it in 10 mL of PBS solution, and collect 2 mL of the suspension for later use. Disrupt the remaining mixture using an ultrasonic cell disruptor to obtain the lysate, and collect 2 mL of the lysate for later use. Measure the tanninase activity of the target bacterial culture supernatant, suspension, and lysate, respectively.

[0046] 2.4.2 Optimal temperature and optimal pH of the target strain

[0047] The activated target bacteria (live count of 1×10⁻⁶) 8CFU / mL was inoculated into the enzyme-producing medium at a ratio of 2 vt% and cultured at 37 °C for 48 h (100 r / min). After centrifugation, the supernatant was collected to obtain the crude enzyme solution. 1 mL of the crude enzyme solution was mixed with 2 mL of PBS buffer at pH 5.0 and incubated for 10 min at various temperature gradients (28 °C, 31 °C, 34 °C, 37 °C, 40 °C, 43 °C). 1 mL of the crude enzyme solution was then mixed with 2 mL of PBS buffer at different pH values ​​(3.0, 3.5, 4.0, 4.5, 5.0, 5.5). All mixtures were shaken to mix thoroughly and allowed to stand at room temperature for 10 min before tanninase activity was measured.

[0048] 2.5 Identification of tannin-producing lactic acid bacteria

[0049] 2.5.1 Morphological observation

[0050] Lactic acid bacteria were serially diluted and evenly spread on MRS agar plates, incubated at 37 °C for 24 h, and then Gram stained. The morphology and structure of the strains were observed using an optical microscope.

[0051] 2.5.2 Molecular biological identification

[0052] The bacterial culture was streaked and incubated at 37 °C for 24 h, then sequenced by Shanghai Sangon Biotech Co., Ltd. The sequencing results were compared with data in the NCBI database using the NCBI Blast program to identify the species of the strain. The corresponding sequence of the type strain with high homology to the 16S rDNA sequence of the target strain was selected, and a phylogenetic tree was constructed using MEGA 11 software.

[0053] 2.6 Optimization of the deastringency removal process of tannin-producing lactic acid bacteria in black chokeberry pulp

[0054] 2.6.1 Single-factor experiment

[0055] To investigate the effects of inoculum size, fermentation time, and fermentation temperature on the content of condensed tannins, single-factor experiments were conducted to optimize the deastringency process. With a fermentation time of 3 days and a fermentation temperature of 37 ℃, the content of condensed tannins in the fermentation broth was measured at inoculum sizes of 1 vt%, 1.5 vt%, 2 vt%, 2.5 vt%, 3 vt%, and 3.5 vt%. With an inoculum size of 2.5 vt% and a fermentation temperature of 36 ℃, the content of condensed tannins in the fermentation broth was measured at fermentation times ranging from 1 to 6 days. With an inoculum size of 2.5 vt% and a fermentation time of 4 days, the content of condensed tannins in the fermentation broth was measured at fermentation temperatures of 31 ℃, 34 ℃, 37 ℃, 40 ℃, and 43 ℃.

[0056] 2.6.2 Optimization of Astringency Removal Process Conditions for Black-fruited Sorrel Fruit Based on Response Surface Experimentation

[0057] Based on the single-factor model, three influencing factors were selected: A. inoculum size, B. fermentation time, and C. fermentation temperature. The condensed tannin content was used as the response value to conduct a response surface optimization experiment. The levels of each factor in the response surface experiment are shown in Table 1.

[0058] Table 1. Coding table of factors and levels in response surface methodology.

[0059] 2.7 Determination of major components during the fermentation of black chokeberry

[0060] 2.7.1 Determination of Soluble Solids (TSS) Content

[0061] The soluble solids content was determined using a handheld saccharimeter at 25 °C.

[0062] 2.7.2 Determination of reducing sugar content

[0063] Add 0, 0.10, 0.20, 0.30, 0.40, and 0.50 mL of 1 mg / mL glucose standard solution to six test tubes, respectively. Add deionized water to each tube to a final volume of 0.5 mL. Then add 1.5 mL of 3,5-dinitrosalicylic acid reagent and mix thoroughly. Boil in a water bath for 5 min, then rapidly cool under running water. Add 4 mL of deionized water to each test tube and mix well. Measure the OD value at 540 nm. The glucose standard curve equation is obtained as y = 2.5929x - 0.0714. R 2 =0.9962.

[0064] Take 0.5 mL of the supernatant from the pulp of *Sorbus nigra* (centrifuge at 4000 r / min for 15 min), dilute it 10-fold, and then add 1.5 mL of 3,5-dinitrosalicylic acid reagent, mixing thoroughly. Boil in a water bath for 5 min, then rapidly cool under running water, adding 4 mL of deionized water to each test tube and mixing well. Measure the OD value at 540 nm and calculate the reducing sugar content.

[0065] 2.7.3 pH Measurement

[0066] The pH was measured using a pH meter.

[0067] 2.7.4 Determination of total acid content

[0068] The total acid was determined according to the method in GB 12456-2021, "National Food Safety Standard - Determination of Total Acid in Food".

[0069] 2.7.5 Determination of total polyphenol content

[0070] Accurately measure 0.2, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, and 6.0 mL of 0.05 mg / mL gallic acid standard solution into 25 mL volumetric flasks. Add 1.0 mL of Folin-Ciocalteu reagent and 13 mL of 10 wt% Na₂CO₃ solution to each flask. Dilute to volume with distilled water, mix well, and let stand at room temperature for 60 min. Measure the absorbance at 760 nm. Plot a standard curve with gallic acid concentration (x) on the x-axis and absorbance value (y) on the y-axis. The equation for the gallic acid standard curve is y = 0.0719x - 0.0004. R 2 =0.9978.

[0071] Take 1 mL of supernatant from the pulp of *Sorbus nigra* (centrifuge at 4000 r / min for 15 min), dilute it 100 times, add 1.0 mL of Folin-Ciocalteu reagent and 13 mL of 10 wt% Na2CO3 solution, make up to volume with distilled water, mix well, let stand at room temperature for 60 min, measure the absorbance at 760 nm and calculate the total polyphenol content.

[0072] 2.7.6 Determination of total flavonoid content

[0073] Accurately weigh 5.1 mg of rutin standard and dilute to 25 mL with 50% ethanol to prepare a rutin standard solution with a concentration of 0.204 mg / mL. Accurately transfer 0, 1.0, 2.0, 3.0, 4.0, and 5.0 mL of the rutin standard solution to each volume, add 1 mL of 5wt% sodium nitrite solution, shake well, and let stand for 5 min. Then add 1 mL of 10wt% aluminum nitrate solution, shake well, and let stand for 5 min. Finally, add 10 mL of 10wt% sodium hydroxide solution, dilute to 25 mL with water, shake well, and let stand for 20 min. Measure the absorbance at a wavelength of 510 nm. Plot the rutin mass concentration (x) on the x-axis and the absorbance value (y) on the y-axis to obtain the rutin standard curve equation: y = 13.999x - 0.0019. R 2 =0.9997.

[0074] Take 1 mL of the supernatant of black chokeberry pulp (centrifuge at 4000 r / min for 15 min), dilute it 10 times, add the reagents sequentially according to the above method, measure the absorbance at 510 nm, and calculate the total flavonoid content.

[0075] 2.7.7 Determination of Condensed Tannin Content

[0076] Catechin standard solutions with concentrations of 0.01, 0.02, 0.04, 0.08, 0.10, 0.20, and 0.40 g / L were prepared. 2.5 mL of 3% vanillin-methanol solution was added, and after mixing, 2.5 mL of 30% sulfuric acid-methanol solution was added. The mixture was reacted at 30 °C in the dark for 20 min, and the absorbance was measured at 500 nm. The standard curve equation for catechin was obtained by plotting catechin mass concentration (x) on the x-axis and absorbance (y) on the y-axis: y = 0.004x + 0.0226. R 2 =0.9984.

[0077] Transfer 1 mL of the supernatant of black chokeberry pulp (centrifuge at 4000 r / min for 15 min), dilute it 10 times, take 1 mL of the supernatant, and determine and calculate the condensed tannin content according to the experimental method of the above-mentioned catechin standard curve.

[0078] 2.7.8 Anthocyanin content determination

[0079] Anthocyanin content was determined using a pH differential method. 1.00 mL of fermented *Sorbus nigra* pulp was diluted 30-fold with buffer solutions of pH 1.0 and pH 4.5, respectively, and mixed thoroughly. The absorbance values ​​were measured at 510 nm and 710 nm using a UV-Vis spectrophotometer. The anthocyanin content in the fermentation broth was calculated according to Equations 1 and 2.

[0080] Formula 1; Formula 2; In the formula: ΔA: absorbance; Mt: relative molecular mass of cyanidin-3-glucoside: 449.2 g / mol; DF: dilution factor; V: total volume of fermentation broth (mL); e: extinction coefficient of cyanidin-3-glucoside: 26900 L / mol·cm; b: optical path length of cuvette (cm): 1 cm.

[0081] 2.7.9 SOD Enzyme Activity Assay

[0082] SOD enzyme activity was determined using a reagent kit method developed by Nanjing Jiancheng Biotechnology Research Institute.

[0083] 2.8 Determination of antioxidant capacity during fermentation of black chokeberry

[0084] 2.8.1 DPPH free radical scavenging rate

[0085] Mix 0.5 mL of 0.5 mmol / mL DPPH solution with 2 mL of fermentation broth thoroughly, incubate at 37 °C in the dark for 20 min, and measure the absorbance A1 at 517 nm using anhydrous methanol as a blank. Mix 2 mL of anhydrous methanol with 0.5 mL of DPPH solution thoroughly and measure its absorbance A0. Mix 0.5 mL of anhydrous methanol with 2 mL of diluted fermentation broth thoroughly and measure its absorbance A2, then calculate according to Equation 3.

[0086] Formula 3; In the formula: A1, absorbance of the DPPH solution after the reaction with fermentation broth; A0, absorbance of DPPH after the addition of anhydrous methanol; A2, absorbance of the solution with or without fermentation broth.

[0087] 2.8.2 ABTS free radical scavenging rate

[0088] Take 10 mL each of 7 mmol / L ABTS stock solution and 2.45 mmol / L K2S2O8 stock solution, mix them, and incubate overnight at room temperature in the dark. Before the assay, dilute the ABTS free radical stock solution with anhydrous ethanol to an OD734 nm value of 0.700±0.002 to prepare ABTS working solution. Take 20 μL of sample (inoculum 2.5 vt%, fermentation temperature 37℃, fermentation time 0-5 days) into a test tube, add 2 mL of ABTS working solution, mix well, and incubate at 35℃ in the dark for 30 min. Use distilled water as a control and measure the absorbance at 734 nm. The ABTS free radical scavenging rate is calculated according to Equation 4.

[0089] Equation 4; In the formula: A0, absorbance of blank control solution; A1, absorbance of fermentation broth tube.

[0090] 2.8.3 Hydroxyl radical scavenging rate

[0091] Take 2 mL each of ferrous sulfate solution (6 mmol / L), salicylic acid solution (6 mmol / L), and H2O2 solution (6 mmol / L), and add 2 mL of sample solution (inoculum amount 2.5 vt%, fermentation temperature 37℃, fermentation time 0~5 d). Mix well and incubate in a 37℃ water bath for 30 min. Measure the absorbance at 510 nm as A1. Use distilled water instead of H2O2 as a blank group to measure A0. The formula for calculating the hydroxyl radical scavenging rate is shown in Equation 5.

[0092] Formula 5; In the formula: A1 is the absorbance of the sample solution; A0 is the absorbance of the blank solution with distilled water.

[0093] Example 2 Results and Analysis

[0094] One-way ANOVA and Pearson correlation analysis were performed using SPSS Statistics 27.0. Experimental data were analyzed and plotted using Design-Expert 12.0 and Origin 2025b. Developmental trees were plotted using MEGA 11. The significance level was 0.05. Each experiment was repeated three times. All results are expressed as mean ± standard deviation.

[0095] 1. Isolation and screening of tannin-producing lactic acid bacteria

[0096] Twenty-one lactic acid bacteria strains exhibiting calcium-dissolving zones were isolated from black chokeberry pulp fermented naturally for 45 days. They were numbered H-1 to H-21. The results showed that only one strain, H-3, grew on tannin-selective medium and exhibited a color change around its colonies.

[0097] 2. Growth curve of strain H-3

[0098] Figure 1 The growth curve of strain H-3 after incubation at 37 ℃ for 24 h is shown. Figure 1 As can be seen, strain H-3 was in the lag phase for the first 4 hours, then rapidly entered the logarithmic growth phase and multiplied rapidly. When the culture time reached 14 hours, the growth state of the strain transitioned from the logarithmic growth phase to the stationary growth phase, and tended to stabilize after 14 hours. Therefore, 14 hours was selected as the time for later activation of strain H-3.

[0099] 3. Enzyme production characteristics of strain H-3

[0100] 3.1 Enzyme localization of strain H-3

[0101] Depend on Figure 2 Enzyme activity was detected in multiple sites of strain H-3, with the highest activity (3.41 U / mL) in the supernatant of the bacterial culture. A certain level of enzyme activity was also detected in the bacterial sludge suspension, presumably because some tannins can be adsorbed onto the cell wall surface. After ultrasonic disruption, intracellular enzymes, cell membrane-bound enzymes, and cell wall-bound enzymes were fully released, and certain enzyme activity was also detected in the disruption solution. This result proves that a certain amount of tannins exists within the cells of this strain. The results indicate that the tannins produced by strain H-3 are mainly extracellular enzymes, suggesting that extracellular enzymes have greater advantages in food fermentation systems.

[0102] 3.2 Optimal temperature and optimal pH for strain H-3

[0103] Depend on Figure 3It can be seen that the enzyme activity of strain H-3 first increases and then decreases with increasing temperature and pH. The optimal temperature is 37 ℃, at which point the enzyme activity is 4.57 U / mL, and the optimal pH is 4.0, at which point the enzyme activity is 4.53 U / mL. The pH of *Sorbus nigra* is between 3.7 and 3.9, which is suitable for the action of tanninases; therefore, pH adjustment is not necessary during use.

[0104] Identification of strain H-3

[0105] 4.1 Morphological observation

[0106] Strain H-3 appears on MRS agar medium as a milky white, round, small, and raised pattern, such as... Figure 4 -a. Gram-positive, such as Figure 4 -b, which matches the appearance characteristics of lactic acid bacteria.

[0107] 4.2 Molecular biological identification

[0108] PCR amplification and sequencing of the 16S rDNA gene of strain H-3 yielded a 1513 bp 16S rDNA gene sequence. Alignment analysis of the 16S rDNA gene sequence of strain H-3 showed that it was similar to that of *Pediococcus lactis* (…). Pediococcus acidilactici The 16S rDNA gene sequence similarity reached over 99%. A phylogenetic tree of strain H-3 was constructed using the neighbor-joining method in MEGA 11 software (see below). Figure 5 Strain H-3 and Pediococcus lactis ( Pediococcus acidilactici The JCM8797 and LC097074.1 strains are on the same branch and have high homology.

[0109] Optimization of the deastringency removal process of strain H-3 in black chokeberry pulp

[0110] 2.5.1 Results of Single-Factor Experiment

[0111] like Figure 6 As shown in Figure a, when the inoculum amount is less than 2.5 vt%, the content of condensed tannins decreases, reaching a minimum of 3.35 g / L at 2.5 vt%. When the inoculum amount is greater than 2.5 vt%, the content increases. This is because the substrate is saturated with tanninase, or the tanninase decomposition products repolymerize into condensed tannins. Therefore, the deastringency effect is best when the inoculum amount is 2.5 vt%.

[0112] like Figure 6b shows that the condensed tannin content decreased from day 1 to day 4, reaching its lowest level of 3.04 g / L on day 4, before continuing to rise. This is because the activity of tanninase decreased and the decomposition products were resynthesized into condensed tannins. Throughout the entire process, the condensed tannin content was lowest on day 4 of fermentation.

[0113] from Figure 6 The curves showing the change in condensed tannin content versus temperature indicate that, before 37 ℃, the condensed tannin content gradually decreases with increasing temperature. The decomposition effect on condensed tannins is optimal at 37 ℃, with the lowest condensed tannin content at 2.98 g / L. Above 37 ℃, the condensed tannin content gradually increases with temperature. This is because higher temperatures inhibit the activity of tanninases, thus reducing their ability to decompose condensed tannins. This is consistent with the optimal temperature and enzyme activity results for strain H-3 mentioned above.

[0114] 5.2 Response Surface Optimization of De-stressing Process Conditions

[0115] 5.2.1 Results and Analysis of Response Surface Experiments

[0116] Table 2 Response Surface Design and Results

[0117] Table 3. Analysis of variance data for the regression equation

[0118] Note: P <0.01 (highly significant) ); P <0.05 (significant) ); P >0.05 (not significant).

[0119] Using Design-Expert 12.0 data analysis software, a multiple regression was performed on the index components (condensed tannin content) in Table 2, and the quadratic regression equation is as follows: Y=2.94-0.0175A+0.5138B-0.0763C+0.1025AB-0.0925AC-0.0800BC+0.7040A 2 +0.6715B 2 +0.2465C 2 .

[0120] As shown in Table 3, the variance of the regression model is... P <0.0001, Missing term P=0.0630>0.05, indicating that the regression model is highly significant and has no significant lack of fit, showing good goodness of fit and statistical significance. The coefficient of determination of the regression model... R 2 =0.9952, corrected correlation coefficient R 2 adj =0.9891, coefficient of variation (CV) = 1.86%. These parameters confirm a high degree of agreement between the actual and model-predicted values ​​of condensed tannin content, indicating that the regression model can be used for further analysis. F The order of influence of each factor on the condensed tannin content, determined by the magnitude of the values, is: fermentation time (B) > fermentation temperature (C) > inoculum size (A). In the model, the squared term A has a highly significant effect on the condensed tannin content after fermentation. 2 B 2 C 2 The interaction terms AB and AC, and the linear terms B and C have a significant impact. P <0.05, the combined effect of other factors on the content of condensed tannins in the system did not reach a significant level ( P >0.05).

[0121] 5.2.2 Analysis of the interaction of various factors on the response surface and study of optimal conditions

[0122] like Figure 7 The response surface methodology reveals that a stronger interaction between factors results in a more pronounced contour slope, indicating a more significant impact of that factor on the condensed tannin content. After optimization using the Box-Behnken response surface methodology, the optimal theoretical deastringency conditions for black chokeberry pulp were determined to be: inoculum size 2.398 vt%, fermentation time 3.810 d, and fermentation temperature 36.099℃, predicting a condensed tannin content of 2.940 g / L. To facilitate practical operation, three parallel experiments were conducted with an inoculum size of 2.5 vt%, a fermentation time of 4 d, and a fermentation temperature of 37℃. The average condensed tannin content was 2.98 g / L, less than 5% different from the model prediction, indicating the model's effectiveness.

[0123] 6. Changes in main components during the fermentation of black chokeberry

[0124] 6.1 Changes in TSS and reducing sugar content during the fermentation of black chokeberry

[0125] Figure 8This study investigated the changes in TSS and reducing sugar content during the fermentation of *Sorbus nigra*. After 5 days of fermentation, TSS and reducing sugar content decreased from the initial 13.1 wt% and 23.81 g / L to 11.0% and 21.28 g / L, respectively. This is because the TSS in the fermentation broth is primarily composed of sugars, which are the core substrates for microbial metabolism. During the fermentation process, strain H-3 relies on a carbon source for its growth, reproduction, and acid production metabolism, leading to a decrease in TSS and reducing sugar content.

[0126] 6.2 Changes in pH and total acid content during the fermentation of black chokeberry

[0127] Changes in pH and total acid content during the fermentation of black chokeberry are as follows: Figure 9 As shown, the pH gradually decreased during fermentation, from 3.84 to 3.44. This is because strain H-3 utilizes carbohydrates to produce lactic acid and other acids, leading to a decrease in pH. The total acidity gradually increased, rising from an initial acidity of 9.14 g / L to 11.53 g / L on day 5. This is because strain H-3 is highly acid-resistant and avoids being destroyed by its own produced acids as the pH of the fermentation broth gradually decreases, resulting in a continuous increase in the total acid content.

[0128] 6.3 Changes in total polyphenol and total flavonoid content during the fermentation of black chokeberry

[0129] Figure 10 The study investigated the changes in total polyphenol and total flavonoid content during the fermentation of black chokeberry. The total polyphenol content in the sample before fermentation was 4.4 mg / mL, and on day 5 of fermentation it was 5.14 mg / mL, representing a significant increase of 14.4% compared to before fermentation. P <0.05). This is related to the acidic environment of the fermentation system. Acidic conditions can promote the transformation of polyphenols from a bound state to a free state. At the same time, tannins, as an important component of phenols, can generate small molecule phenolic derivatives with low polymerization degree after being degraded by tanninase, thus realizing the transformation from a polymerized state to a free state. During the fermentation process, the total flavonoid content showed a trend of first increasing and then decreasing. On the second day of fermentation, the total flavonoid content reached the highest value (4.23 mg / mL). In the early stage of fermentation, the high osmotic pressure environment caused flavonoids to be gradually dissolved and released into the fermentation broth. After that, it began to decrease because the acid produced by lactic acid bacteria led to a decrease in the pH of the fermentation broth. This acidic environment affected the water solubility of flavonoids, thus reducing their content.

[0130] 6.4 Changes in the content of condensed tannins, anthocyanins, and SOD enzymes during the fermentation of black chokeberry.

[0131] Figure 11The changes in condensed tannin content during the fermentation of black chokeberry were observed. The condensed tannin content in the fermentation broth initially decreased and then increased. The lowest condensed tannin content was observed on day 4, at 2.98 g / L, a decrease of 52.54% compared to before fermentation. P <0.05). This is related to the fact that the tannic acid hydrolase produced by strain H-3 can degrade tannins into small molecule phenolic acids and polyols. The concentration subsequently increased to 3.23 g / L because the tannic acid enzyme activity decreased and the decomposition products were resynthesized into tannins.

[0132] Figure 12 This study describes the changes in anthocyanin and SOD enzyme content during the fermentation of black chokeberry. The anthocyanin content in the fermentation broth decreased from 706.56 mg / L to 372.58 mg / L during days 0-5. This is related to the instability of anthocyanins themselves and the adsorption by microorganisms. Furthermore, the growth and metabolism of strain H-3 consumed the sugars in the original substrate; as the sugar content decreased, the anthocyanin structure became unstable, leading to a decrease in its content. SOD enzyme activity showed an increasing trend during days 0-5, rising from 102.55 U / mL to 118.12 U / mL, an increase of 13.18%. P <0.05). This is related to the fact that microbial metabolism produces SOD enzymes or that the acidic environment created by microbial fermentation is conducive to maintaining SOD enzyme activity, indicating that *Pediococcus lactis* H-3 has a good ability to produce SOD enzymes.

[0133] 7. Determination of antioxidant capacity during fermentation of black chokeberry

[0134] Changes in antioxidant capacity during the fermentation of black chokeberry, such as Figure 13 As shown, during the 0-5 day fermentation process of black chokeberry, the DPPH free radical scavenging rate reached its maximum of 61.34%, the ABTS free radical scavenging rate increased from 60.72% to 80.12%, and the hydroxyl free radical scavenging rate increased from the initial 38.38% to 80.23%. This is because the action of lactic acid bacteria during fermentation increased the total polyphenols, SOD enzymes, and other antioxidant active substances in the pulp. In conclusion, *Pediococcus lactis* H-3 can enhance the antioxidant capacity of black chokeberry pulp during fermentation.

[0135] 8. Correlation analysis

[0136] Correlation analysis was performed on 11 indicators, including total polyphenols, SOD enzyme, DPPH, and ABTS free radical scavenging rate, during the fermentation of black chokeberry by *Pediococcus lactis* H-3. The results are as follows: Figure 14 As shown, the total acid, total polyphenols, SOD enzyme, DPPH, ABTS, and hydroxyl radical scavenging rates of the main components are positively correlated. P<0.05. pH, TSS, reducing sugar, condensed tannins, anthocyanins, and DPPH, ABTS, and hydroxyl radical scavenging rate were negatively correlated. P <0.05). The correlation between total flavonoids and antioxidant activity was weak. SOD enzyme showed a highly significant positive correlation with DPPH, ABTS, and hydroxyl radical scavenging rates (r=0.92, 0.965, 0.975, respectively). P <0.01), indicating that SOD has a significant impact on the antioxidant activity of black chokeberry pulp. The total polyphenols and DPPH free radical scavenging rate (r=0.929, P <0.01), ABTS free radical scavenging rate (r=0.95, P <0.01), hydroxyl radical scavenging rate (r=0.929, P The values ​​<0.01 showed a highly significant positive correlation, indicating that phenolic substances are also important substances affecting the antioxidant activity of black chokeberry pulp during fermentation. It was found that the higher the total phenol content, the stronger its ability to scavenge free radicals.

[0137] 9. Conclusion

[0138] This study isolated a tannin-producing lactic acid bacterium, *Pediococcus lactis* H-3, from naturally fermented *Sorbus nigra* pulp. Its extracellular enzyme activity was 3.41 U / mL, with an optimal pH of 4.0 and an optimal temperature of 37 ℃. Cultivation at 37 ℃ for 14 h resulted in the transition from the logarithmic growth phase to the stationary phase. Response surface methodology was used to optimize the fermentation process for deastringency removal from *Sorbus nigra*. The optimal fermentation conditions were: inoculum size 2.5 vt%, fermentation time 4 days, and fermentation temperature 37 ℃. Under these optimized conditions, the condensed tannin content in the fermentation broth decreased to 2.98 g / L, a significant reduction compared to pre-fermentation levels. P <0.05) decreased by 52.54%. During the fermentation of black chokeberry pulp by strain H-3, pH, reducing sugar, TSS, and anthocyanin content continuously decreased; total polyphenols, total acid content, and SOD enzyme activity generally increased; total flavonoid content showed an initial increase followed by a decrease, with a maximum value of 4.23 mg / mL; and the maximum values ​​of DPPH, ABTS, and hydroxyl radical scavenging rates of the fermented pulp reached 61.34%, 80.12%, and 80.23%, respectively, indicating significantly enhanced antioxidant activity. P <0.05. Correlation analysis showed that total polyphenols, SOD enzyme, and antioxidant activity were significantly positively correlated ( P <0.01). The findings of this study provide excellent microbial resources for improving the flavor of fruits with high tannin content and developing their functional properties, and offer a new direction for the application of this type of strain in plant substrate fermentation.

[0139] As shown in the above embodiments, this invention provides a tannin-producing *Pediococcus acidilactic* strain H-3 and its applications. The Latin name of the tannin-producing lactic acid bacteria H-3 is *Pediococcus acidilactic*, its preservation name is *Pediococcus acidilactic*, its deposit location is the China General Microbiological Culture Collection Center (CGMCC), its deposit date is January 13, 2026, and its accession number is CGMCC No. 37372. This invention provides an excellent strain resource for improving the flavor of fruits with high tannin content and developing their functional properties, and provides a new direction for the application of this type of strain in plant substrate fermentation.

[0140] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A strain of *Pediococcus lactis* H-3 that produces tanninase, characterized in that, The Latin name of the lactic acid cocci H-3 is Pediococcus acidilactic, the preservation name is Pediococcus acidilactic, the deposit location is China General Microbiological Culture Collection Center, the deposit date is January 13, 2026, and the deposit number is CGMCC No. 37372.

2. The application of the *Pediococcus lactis* H-3 as described in claim 1 in the deastringency removal of *Sorbus nigra* fruit.

3. A bacterial suspension containing the *Pediococcus lactis* H-3 as described in claim 1.

4. The method for preparing the bacterial solution according to claim 3, characterized in that, The procedure includes the following steps: inoculating the *Pediococcus lactis* H-3 of claim 1 into tannin selection medium and culturing it at 35-39°C for 12-16 hours.

5. The preparation method according to claim 4, characterized in that, The pH of the tannin screening medium is 3.5-4.

5. The preparation method of the tannin screening medium is as follows: add 0.002-0.006 g of bromophenol blue and 0.8-1.2 g of tannic acid to 90-110 mL of MRS agar medium, and sterilize at 115-125 °C for 15-25 min.

6. The application of the bacterial solution according to claim 3 in the deastringency removal of black chokeberry fruit.

7. A method for removing astringency from black chokeberry fruit using the bacterial solution described in claim 3, characterized in that, Includes the following steps: Mix the bacterial culture with the pulp of black chokeberry fruit and ferment at 31-43℃ for 1-5 days.

8. The method according to claim 7, characterized in that, The viable count of *Pediococcus lactis* H-3 in the bacterial solution was 1 × 10⁻⁶. 7 ~1×10 9 CFU / mL, the inoculation amount of the bacterial solution is 2-3 vt of black chokeberry pulp.

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