Aperbacillus pallidofulvus Z-12 strain and application thereof in preparation of hypoglycemic active matter by fermenting tea seed meal

Abstract

The invention discloses an Apertus pallidofulvus Z-12 strain which is preserved in the Guangdong Microbial Culture Collection Center (GDMCC) on July 2, 2025, and the preservation number of the Apertus pallidofulvus Z-12 strain is GDMCC No: 66621. The Apertus pallidofulvus Z-12 strain is named as Apertus pallidofulvus Z-12 strain. According to the invention, several strains are separated from naturally stored tea seed meal, fresh tea seed meal is inoculated for fermentation, and in combination with an alpha-glucosidase inhibition rate, a new strain Aperbacillus pallidofulvus Z-12 for high yield of hypoglycemic active matters from fermented tea seed meal is screened and identified. Meanwhile, the Z-12 strain and the protease are innovatively and synergistically fermented under the optimal process condition, and the quality of the blood sugar reducing active matter in the tea seed meal is remarkably improved. The technology not only fills the technical blank of efficient utilization of high-quality protein of the tea seed meal, but also provides a brand new thought for industrial utilization of the tea seed meal.

Description

Technical Field

[0001] This invention relates to the field of bacterial fermentation technology, specifically, to a... Aspergillus pallidofulvus Z-12 strain and its application in the preparation of hypoglycemic active ingredients from fermented tea seed meal. Background Technology

[0002] Diabetes has become the third leading cause of disease threatening human health. Type 2 diabetes (T2MD) is the most common type of diabetes, accounting for over 90% of cases. Dietary carbohydrates are primarily absorbed in the intestines in the form of monosaccharides. After ingested polysaccharides such as starch are broken down into oligosaccharides and disaccharides by α-amylase in saliva and pancreas, they still require enzymatic breakdown into glucose by α-glucosidase before absorption. Therefore, α-glucosidase is an effective target for inhibiting carbohydrate absorption. Currently, the main α-glucosidase inhibitors used clinically to lower blood sugar include acarbose, voglibose, and miglitol; however, almost every drug causes certain adverse reactions or side effects.

[0003] Bioactive peptides generally refer to a collective term for various peptides, ranging from simple dipeptides to complex linear and cyclic structures, that possess specific physiological functions. In vivo, bioactive peptides exert their physiological effects by acting directly as neurotransmitters or indirectly stimulating the secretion of receptor hormones or enzymes, exhibiting functions such as lowering blood pressure, blood sugar, and cholesterol, and regulating the body's immunity. Numerous studies have shown that plant-derived bioactive peptides are one of the many options for oral or intravenous administration of diabetes medications, offering advantages such as high tissue affinity and specificity, and low side effects.

[0004] Tea seed meal, a major byproduct of tea oil processing, is still primarily used as crude feed and fertilizer, resulting in low economic benefits. Tea seed meal protein accounts for approximately 20% of its total weight, with a balanced amino acid composition, making it a high-quality plant protein resource and a potential high-quality source for preparing bioactive peptides. From a circular economy perspective, fully utilizing the high-quality protein in tea seed meal is of great significance for the resource-added transformation and utilization of production waste and byproducts. The article "Preparation, Physicochemical Properties and Hypoglycemic Activity of Tea Seed Meal Peptides" discloses the enzymatic hydrolysis of tea seed meal (protein content 28.81%±1.14%) from Dabie Mountain No. 4 tea seeds using alkaline protease. It was found that after 5 hours of hydrolysis, the degree of protein hydrolysis reached 15.84%±0.52%, and the corresponding hydrolysate (2 mg / mL) inhibited α-glucosidase at a rate of 22.54%±1.13%. Aspergillus pallidofulvus Aspergillus ( ) Aspergillus A relatively new strain of this bacterium, reportedly, can promote the biosynthesis of gallic acid during the microbial fermentation of Pu-erh tea. However, no evidence has yet been found... Aspergillus pallidofulvusStrain fermentation of tea seed meal to obtain high-efficiency inhibition - Fermentation products of glucosidase. SUMMARY

[0005] The present application aims to overcome the above-mentioned defects and deficiencies in the prior art, and provides a strain Aspergillus pallidofulvus The Z-12 strain is deposited in the Guangdong Microbial Culture Collection Center (GDMCC) on July 2, 2025, and the deposit number is GDMCC No: 66621.

[0006] The second object of the present application is to provide a microbial agent containing the above-mentioned Aspergillus pallidofulvus Z-12 strain, and its culture solution, bacterial suspension and bacterial body.

[0007] The third object of the present application is to provide the above-mentioned Aspergillus pallidofulvus Z-12 strain or the above-mentioned microbial agent in the preparation of hypoglycemic active substances by fermenting tea seed meal.

[0008] The fourth object of the present application is to provide the above-mentioned Aspergillus pallidofulvus Z-12 strain or the above-mentioned microbial agent in combination with protease in the preparation of hypoglycemic active substances by fermenting tea seed meal.

[0009] The fifth object of the present application is to provide a method for preparing hypoglycemic active substances by fermenting tea seed meal.

[0010] The sixth object of the present application is to provide a hypoglycemic active substance prepared according to the above-mentioned method.

[0011] The above-mentioned objects of the present application are realized by the following technical solutions: The present application provides a strain Aspergillus pallidofulvus The Z-12 strain is deposited in the Guangdong Microbial Culture Collection Center (GDMCC) on July 2, 2025, and the deposit number is GDMCC No: 66621. The address is No. 59 Building, 5th Floor, Institute of Martyrs, Guangzhou, Guangdong Province, China, with a postcode of 510070.

[0012] The present application isolates 25 strains of fungi from naturally stored tea seed meal by dilution and coating plate method. All strains are fermented separately with fresh tea seed meal, and the inhibition activity of the fermentation product on alpha-glucosidase is determined. The specific activity is determined by the inhibition activity of alpha-glucosidase and the polypeptide content in the fermentation product. It is found that the fungus Z-12 shows higher inhibition activity on alpha-glucosidase, which is 65.355 (%·mL·mg -1), which is significantly higher than other strains. The Z-12 strain is smooth and flat on the surface, with a silky appearance, and the conidia are spherical and oval in shape, with a light yellow color. After staining, the strain mycelium is observed under a 100x microscope, and it is found that the strain mycelium is developed, with no foot cells, and long strip-shaped conidia. The 16S rDNA fragment of the strain is subjected to PCR, sequencing and construction of a phylogenetic tree, and it is found that the strain Z-12 is in the same branch as Aspergillus pallidofulvus , with a confidence of 72, and the result is reliable, and therefore the strain is named Aspergillus pallidofulvus Z-12.

[0013] The application also provides a microbial agent containing one or more of the Z-12 strain and culture solution, microbial suspension and microbial bodies thereof. Aspergillus pallidofulvus The preparation method of the culture solution, microbial suspension and microbial bodies is inoculating and culturing the Z-12 strain to obtain the culture solution, centrifuging the culture solution to obtain the microbial suspension, and centrifuging the culture solution to obtain the microbial bodies. Aspergillus pallidofulvus

[0014] The application also provides the application of the Z-12 strain or the microbial agent in the preparation of hypoglycemic active substances by fermenting tea seed meal. Aspergillus pallidofulvus Microbial fermentation for producing products with biological activity is a process of using various enzymes such as proteases produced in the microbial fermentation process to decompose and transform complex substrates such as proteins into small molecular peptides, free amino acids and other secondary metabolites with potential physiological activity. The enzymes produced in the fermentation process can not only degrade proteins, but also complex carbohydrates and lipids. The enzymatic hydrolysis method mainly uses proteases to hydrolyze protein macromolecules to generate components with smaller molecular weight such as polypeptides. Some components (such as small molecular peptides) generated by this method usually have the advantages of good solubility and high stability. Therefore, the application provides a microbial enzyme synergistic fermentation technology, which combines the advantages of microbial fermentation and enzymatic hydrolysis technology. Enzymatic hydrolysis can provide easily available nutrients such as nitrogen source and carbon source for the microbial fermentation process, and promote the growth and metabolism of microorganisms. At the same time, the microbial fermentation process can overcome the problems such as single active component and lack of functional elements caused by simple enzymatic hydrolysis, and generate more abundant active substances through complex biological transformation. The organic combination of the two can not only reduce the production cost of high-activity fermentation products, but also play a role in raw material detoxification and bitterness removal in production, and finally obtain fermentation products with high hypoglycemic activity.

[0015] Therefore, the application also provides the application of the combination of the Z-12 strain or the microbial agent and proteases in the preparation of hypoglycemic active substances by fermenting tea seed meal.

[0016] Therefore, the application also provides the application of the combination of the Z-12 strain or the microbial agent and proteases in the preparation of hypoglycemic active substances by fermenting tea seed meal. Aspergillus Therefore, the application also provides the application of the combination of the Z-12 strain or the microbial agent and proteases in the preparation of hypoglycemic active substances by fermenting tea seed meal.​

[0017] Further, the blood sugar reducing active substance has an inhibitory activity on α-glucosidase.

[0018] The present application also provides a method for preparing a blood sugar reducing active substance by fermenting tea seed meal, wherein the tea seed meal is fermented by inoculating the Z-12 strain or the bacterial agent as described above. pallidofulvus Aspergillus pallidofulvus The Z-12 strain or the bacterial agent as described above is inoculated into fresh defatted tea seed meal for fermentation.

[0019] Further, the method further comprises adding a protease for fermentation at the initial stage of the fermentation system.

[0020] According to the single factor experiment results, the ratio of material to liquid (tea seed meal content), fermentation temperature, and the amount of added compound enzyme are fixed, (1) the inoculation amount (A), enzyme addition time (B), and fermentation time (C) are selected as three factors to design a three-factor three-level experiment to study the effects of different conditions on the tea seed meal enzyme synergistic solid-state fermentation. With the inhibitory activity of α-glucosidase (Y1) as the index, each treatment has three replicates, and the optimal combination is screened out. Through the Design-Expert 13 software for data fitting analysis, it is found that the contour plot of enzyme addition time and fermentation time is a flat ellipse, and the interaction is strong, which has a greater impact on the α-glucosidase inhibition rate. The contour plot of enzyme addition amount and inoculation amount tends to be circular, indicating that the interaction of the two factors is small, and the impact on the α-glucosidase inhibition rate is small. Through the Design-Expert 13 for optimal solution of the regression equation, the optimal process for the α-glucosidase inhibition rate of the tea seed meal enzyme synergistic fermentation product is obtained as follows: enzyme addition after 8.91 h of fermentation, fermentation time of 74.47 h, and strain inoculation amount of 15.31%, and the α-glucosidase inhibition rate of the product is 69.722%. The actual verification value is close to the predicted value, indicating that the model prediction of the optimal fermentation conditions for the α-glucosidase inhibition rate of the tea seed meal enzyme synergistic fermentation product is reliable.

[0021] Further, the fermentation time is 48-96 h.

[0022] Further, the strain or bacterial agent inoculation amount is 10-20%.

[0023] Further, the fermentation temperature is 32-42℃.

[0024] Further, the initial stage of fermentation is 4-12 h of fermentation.

[0025] Specifically, the initial stage of fermentation is only inoculation Aspergillus The Z-12 strain or the bacterial agent is inoculated into fresh defatted tea seed meal for fermentation for 9 h.

[0026] Further, the enzyme addition amount is 3-5%.

[0027] As a preferred implementation manner, the present application also provides a method for producing a blood glucose-lowering active product by fermenting tea seed meal with the Z-12 strain and a protease. pallidofulvus Aspergillus pallidofulvus The method for producing a blood glucose-lowering active product by fermenting tea seed meal with the Z-12 strain and a protease comprises the following steps: inoculating 15% of the Z-12 bacterial solution into a tea seed meal solution with a solid-liquid ratio of 1:5, fermenting at 37°C for 9 h, adding 4% of the protease, and then fermenting again for 65 h to obtain the bacterial-enzyme synergistic fermentation active product. Aspergillus pallidofulvus The method for producing a blood glucose-lowering active product by fermenting tea seed meal with the Z-12 strain and a protease comprises the following steps: inoculating 15% of the Z-12 bacterial solution into a tea seed meal solution with a solid-liquid ratio of 1:5, fermenting at 37°C for 9 h, adding 4% of the protease, and then fermenting again for 65 h to obtain the bacterial-enzyme synergistic fermentation active product.

[0028] The present application also provides a blood glucose-lowering active product prepared by the above method.

[0029] Compared with the prior art, the present application has the following beneficial effects: The present application provides a method for producing a blood glucose-lowering active product by fermenting tea seed meal with the Z-12 strain and a protease. Aspergillus pallidofulvus The Z-12 strain is preserved in the Guangdong Microbial Culture Collection Center (GDMCC) on July 2, 2025, and the preservation number is GDMCC No: 66621. The present application isolates several strains from naturally stored tea seed meal, screens and identifies a new strain with high yield of blood glucose-lowering active product by fermenting fresh tea seed meal through reverse inoculation and combining with the α-glucosidase inhibition rate. Figure 1 Z-12. Meanwhile, the present application innovatively synergistically ferments Z-12 and a protease under optimal process conditions, significantly improves the quality of the active product in tea seed meal. The technology not only fills the technical gap of high-efficiency utilization of high-quality protein in tea seed meal, but also provides a new idea for the industrialized utilization of tea seed meal, and has important application value. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 2 Figure 1 is a dilution plate diagram of the fungal colony 10. -2 Figure 2 is a dilution plate diagram of the fungal colony 10.

[0031] Figure 3 Figure 3 is a comparison of the effects of fermentation products of different fungal strains and tea seed meal on the α-glucosidase activity inhibition rate.

[0032] Figure 4 Figure 4 is a comparison of the ratio of the α-glucosidase activity inhibition rate to the product polypeptide concentration of the fermentation products of three fungal strains and tea seed meal.

[0033] Figure 5 Figure 5 is a micrograph of the fungal Z-12 strain.

[0034] Figure 6 Figure 6 is a growth curve diagram of the fungal Z-12 strain.

[0035] Figure 7Phylogenetic tree of strain Z-12 based on 16S rDNA gene sequence. Note: The number of branch points indicates the reliability of the branch.

[0036] Figure 8 Figure 6 is a graph showing the effect of products obtained from tea seed meal by separate enzymatic hydrolysis, separate strain fermentation, and enzyme-strain synergistic fermentation on α-glucosidase.

[0037] Figure 9 Figure 7 is a graph showing the effect of different processing techniques on the α-glucosidase activity inhibitory activity of products obtained from tea seed meal by enzyme-strain synergistic fermentation.

[0038] Figure 1 Figure 8 is a response surface plot and contour plot showing the effect of the interaction between enzyme addition time, enzyme-strain synergistic fermentation time, and strain inoculation amount on the α-glucosidase inhibitory rate of products obtained from tea seed meal by enzyme-strain synergistic fermentation. DETAILED DESCRIPTION

[0039] The present application is further illustrated by the following description in conjunction with the accompanying drawings and specific examples, which do not limit the present application in any form. Unless otherwise specified, the reagents, methods, and equipment used in the present application are conventional reagents, methods, and equipment in the technical field.

[0040] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0041] Example 1 Preparation of fermentation products of tea seed meal 1. Isolation of strains Take 10 g of naturally stored dry tea seed meal and add to a conical flask containing 50 mL of physiological saline. Then, take 1 mL of mixed bacterial solution (tea seed meal mixture) from the conical flask and perform gradient dilution with physiological saline, and spread 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 μL of each gradient dilution onto PDA solid plates, and place in an incubator at about 28-30°C to obtain white point-like strains. Then, randomly pick different single colonies from the plates with an inoculation loop and perform streaking purification on slant solid medium. After culturing at about 30°C for 48-72 h to obtain yellow or black spores, add an equal volume of 50% glycerol to the spores, and store at about 4°C for later use.

[0042] 10 -2 The dilution plate diagram of fungal colonies at a dilution of 10 Figure 2 The colonies were enriched and stored, and a total of 25 fungal strains were isolated from naturally stored tea seed meal. These strains will be used for subsequent separate fermentation of fresh tea seed meal.

[0043] 2. Preparation of tea seed meal fermentation product The 25 strains of fungi obtained by the above separation were inoculated into PDA solid culture medium respectively, and incubated at 30°C. When the spores were grown, the spores were scraped and adjusted to 1 x 10 7 ~ 2 x 10 7 CFU / mL, and inoculated into 25 mL of tea seed meal liquid medium at a 10% (v / v) inoculation amount, and incubated at 28°C for 48 h to prepare a fermentation seed liquid. The fermentation seed liquid was centrifuged (4000 rpm, 15 min), and the precipitate was used as the bacterial cells and unhydrolyzed tea seed meal components. A microporous filter membrane was placed in a filter membrane of a suction filtration device, and the supernatant was filtered (0.22 μm). The filtrate was the tea seed meal fermentation product, which was freeze-dried for later use.

[0044] The tea seed meal liquid medium was prepared as follows: fresh tea seed meal was defatted, i.e., the tea seed meal was ground to 40-60 mesh (uniform particles, increasing the solvent contact area) using a grinder. 50-100 g of the sample was weighed, and the initial weight was recorded. Hexane or petroleum ether was selected and mixed with the tea seed meal at a ratio of 1:8 or 1:10 (magnetic stirring in a closed container for 2 h). Then, the mixture was allowed to stand for 30 min, and the upper layer containing the oil solvent was decanted into a recovery bottle. An equal amount of fresh solvent was added, and the stirring, standing, and decanting steps were repeated. The defatted tea seed meal was transferred to a centrifuge tube, and centrifuged at 4000 rpm for 15 min to completely remove the residual solvent. The defatted tea seed meal was placed in a fume hood at about 30°C for 24 h. 20 g of the defatted tea seed meal and 100 mL of tertiary water were added at a solid-liquid ratio of 1:5, and 0.3% of glucose powder was added. The mixture was stirred uniformly and sterilized at 75°C for 2 h.

[0045] Example 2: Screening of a strain with high yield of hypoglycemic active substances 1. Method for determining α-glucosidase activity (α-GIA) The α-glucosidase and p-nitrophenyl-β-D-glucopyranoside (PNPG) were respectively configured into 1 U / mL and 5 mg / mL solutions using a 0.2 M, pH 6.8 sodium phosphate buffer. 50 μL of 10 mg / mL fermentation product (tea seed meal fermentation product prepared in Example 1) and 100 μL of PNPG were mixed using a pipette, and incubated at 37°C for 10 min. Then, 100 μL of the α-glucosidase solution (1 U / mL) was added, and the mixture was incubated at 37°C for 30 min. The reaction was stopped by adding 1 mL of sodium carbonate (0.1 M). 200 μL of the mixture was taken and added to a 96-well plate, and the α-glucosidase activity was determined at 405 nm.

[0046] ​

[0047] OD A : Absorbance of the control group, and absorbance of the supernatant of the fermentation product with an equal amount of buffer solution (other components are the same as the sample group). OD B : Absorbance of the supernatant containing the fermentation product and α-glucosidase solution replaced by the same amount of buffer solution (other components are the same as the standard group). OD S The absorbance of the sample group reflects the absorbance of the system. OD Sb : Absorbance of the blank sample group, absorbance of the reaction system in which the α-glucosidase solution is replaced by the same amount of buffer solution (other components are the same as the sample group).

[0048] Depend on Figure 3 It can be seen that when the concentration of fermentation products is 10 mg / mL, the fungal strain and the fermentation products of tea seed meal have different effects on... The inhibition rate of β-glucosidase ranged from 22.43% to 71.61%, with fungi 10, 11, and 12 showing better inhibitory activity, significantly higher than the unfermented group. p <0.05), there was no significant difference between them ( p >0.05).

[0049] 2. To investigate how the products of fungal fermentation of tea seed meal affect... - Inhibition of glucosidase production was used to measure the polypeptide content in fermentation products. The BCA method was used for assay. The kit was purchased from Yitao Biotechnology Co., Ltd., catalog number EK-5001. The BCA working solution was prepared by mixing BCA reagent A (EK-5001A) and reagent B (EK-5001B) at a ratio of 50:1 and mixing thoroughly. The working solution is stable at room temperature for several days. For standard preparation: an appropriate amount of bovine serum albumin (BSA) standard was diluted with the same diluent as the test sample to prepare standard protein solutions of different concentrations. 10 μL of each standard and test sample were added to microplates, and 200 μL of BCA working solution was added to each well. The plates were thoroughly mixed by pipetting, sealed, and incubated at 37°C for 30 minutes. After cooling to room temperature, the absorbance was measured at 562 nm using a microplate reader. A standard curve was plotted with the standard concentration on the x-axis and absorbance on the y-axis. The corresponding concentration of the sample was determined from the standard curve based on its absorbance.

[0050] The peptide content in the fermentation products of selected fungi No. 10, 11, and 12 and tea seed meal was determined. The inhibition rate of each peptide on α-glucosidase was obtained by comparing the inhibition rate with the peptide content. The results are as follows: Figure 4As shown, three strains of fungi with high α-glucosidase inhibition rate were compared, and one strain with high specific activity of α-glucosidase inhibition was screened, named Z-12, with a specific activity of 65.355 (%·mL·mg -1 )。

[0051] Example 3 Identification of Fungus Z-12 1. Morphological observation of the strain A small amount of preserved first-generation strain Z-12 was inoculated on PDA solid medium, and after sealing, it was placed at 28°C for 72 h of static culture, and the morphology was observed. A small amount of bacterial solution was used to stain the strain with lactic acid phenol cotton blue staining solution and observe the morphology of the bacterial body.

[0052] 2. Determination of the growth curve of the strain The strain was spotted in the center of the PDA medium plate, and after a certain period of growth, the plate was placed on the microscope stage, and the ocular micrometer of the microscope used was calibrated, and the length of each grid was calculated. The tip of a single hypha at the edge of the colony was focused under low magnification. Then the ocular micrometer was parallel to the hypha, and the position where the hypha started to branch was coincided with a line on the ocular micrometer, which was the "reference point". The length of the hyphal growth was measured at certain time intervals, and after several observations, a growth curve was drawn according to the time and the length of the tip extension, and the growth rate (cm / day) was calculated.

[0053] The results of the plate streaking observation of the preserved Z-12 strain are shown in Figure 5 , and it was found that the three strains had smooth and flat surfaces, a velvety appearance, spherical and oval-shaped conidia, and a light yellow color; after staining, the strain hyphae were observed under a 100x microscope, and they were well developed, without foot cells, and had long, single or short chain-shaped conidia. After secondary activation, the growth rate of the strain Z-12 was 1.85 cm / day Figure 6 ).

[0054] 3. Identification results of the strain The purified strain was subjected to DNA extraction by SDS method, and the conserved sequence 16S rDNA fragment of the bacterial body was amplified using universal primers. The amplified product was sent to a sequencing company for sequencing, and the obtained sequence was spliced. The spliced sequence was compared in the NCBI database, and a phylogenetic tree was constructed.

[0055] Through Blast comparison in NCBI, the results are shown in Aspergillus , and it was found that the Z-12 strain had the highest homology with the strain pallidofulvus spergillus pallidofulvus , reaching 97%. The strain Z-12 belongs to A Aspergillus pallidofulvusBranch, confidence 72, named Figure 7 Z-12. The strain was deposited at the Guangdong Microbial Culture Collection Center (GDMCC) on July 2, 2025, with the accession number GDMCC No: 66621. The address is No. 59, Building 5, 100, Junyi Road, Guangzhou, Guangdong Province, China, with a postcode of 510070.

[0056] Example 4. Obtaining high hypoglycemic activity fermentation product by using synergistic solid-state fermentation of tea seed meal Microbial fermentation to produce bioactive products is a process that uses various enzymes such as proteases produced during microbial fermentation to decompose and transform complex substrate molecules such as proteins into small molecular peptides, free amino acids, and other secondary metabolites with potential physiological activity. Enzymes produced during fermentation can not only degrade proteins, but also complex carbohydrates and lipids. Enzymatic hydrolysis mainly uses proteases to hydrolyze protein macromolecules to generate smaller polypeptides and other components by breaking peptide bonds. Some components produced by this method (such as small molecular peptides) usually have good solubility and high stability. The present application provides a synergistic fermentation technology that combines the advantages of microbial fermentation and enzymatic hydrolysis. Enzymatic hydrolysis can provide easily available nutrients such as nitrogen and carbon sources for microbial fermentation, promoting microbial growth and metabolism; at the same time, the fermentation process of microorganisms can overcome the problems of single active component or lack of functional elements that may be caused by simple enzymatic hydrolysis, and produce more abundant active substances through complex biological transformation. The organic integration of the two not only reduces the production cost of high-activity fermentation products, but also plays a role in raw material detoxification and bitterness removal in production, ultimately obtaining fermentation products with high hypoglycemic activity.

[0057] I. Experimental methods 1. Separate enzymatic hydrolysis, separate strain fermentation, and synergistic fermentation The effects of products obtained by separate enzymatic hydrolysis, separate strain fermentation, and synergistic fermentation of tea seed meal on α-glucosidase were compared; the enzymatic hydrolysis was carried out by using complex protease (Cangzhou Xiasheng Industry Group Co., Ltd., product number SFG-2444) under certain fermentation conditions, and the types and activities of the proteases are shown in Table 1 (a small amount of sterile water was added to dissolve the protease, which was then placed in a 50°C water bath for 30 min to prepare an enzyme solution for use).

[0058] Table 1. Types and activities of proteases in complex protease

[0059] (1) The inhibitory effect of the hydrolysate obtained by hydrolyzing tea seed meal with complex protease on alpha-glucosidase was evaluated: 3% complex protease was added to fresh rapeseed meal liquid medium (as in Example 1), and enzymolysis was carried out at 50°C under natural pH, the enzymolysis time was 4 h, and after the enzymolysis was completed, enzyme inactivation was carried out in a 90°C water bath for 10 min, centrifugation was carried out at 4000 rpm for 15 min, 0.22 um water system membrane filtration was carried out, and after freeze-drying, the product was reserved.

[0060] (2) The inhibitory effect of the fermentation product obtained by fermenting tea seed meal with Z-12 strain alone on alpha-glucosidase was evaluated: 10% Z-12 bacterial liquid was added to fresh rapeseed meal liquid medium, the fermentation temperature was 28°C, the fermentation time was 48 h, centrifugation was carried out at 4000 rpm for 15 min, 0.22 um water system membrane filtration was carried out, and after freeze-drying, the product was reserved.

[0061] (3) The inhibitory effect of the fermentation product obtained by fermenting tea seed meal with bacteria and enzymes synergistically on alpha-glucosidase was evaluated: 10% Z-12 bacterial liquid was added to fresh rapeseed meal liquid medium, 3% complex protease prepared was added after 6 h of fermentation, and uniform stirring was carried out with a sterile glass rod, and then bacteria and enzymes were synergistically fermented after being sealed with a sealing film, the fermentation temperature was 32°C, the fermentation time was 42 h, and the fermentation was carried out under natural pH. After the fermentation was completed, enzyme inactivation was carried out in a 90°C water bath for 10 min, centrifugation was carried out at 4000 rpm for 15 min, 0.22 um water system membrane filtration was carried out, and after freeze-drying, the product was reserved.

[0062] 2. Single factor experiment (1) The ratio of material to liquid (the content of tea seed meal) is one of important factors affecting the production of active substances by synergistically fermenting tea seed meal with bacteria and enzymes. In the present application, complex protease is added, the addition time of the enzyme is only 6 h after the addition of bacterial liquid, the inoculation amount is 10%, the amount of added enzyme is 3%, the fermentation time is 48 h in total, and the fermentation temperature is 32°C. The effects of the ratio of material to liquid of 1:2, 1:3, 1:4, 1:5 and 1:6 on the inhibition of alpha-glucosidase activity and the production of active substances by synergistically fermenting tea seed meal were explored.

[0063] (2) The addition time of complex enzyme is one of important factors affecting the production of active substances by synergistically fermenting tea seed meal with bacteria and enzymes. Therefore, in the present application, the ratio of material to liquid is 1:5, the inoculation amount is 10%, the amount of added enzyme is 3%, the fermentation time is 48 h in total, and the fermentation temperature is 32°C. The effects of the addition time of complex enzyme, which is before fermentation (complex enzyme is added in the first 12 h for enzymolysis under the same conditions, and no bacterial liquid is added at this time), at the beginning of fermentation (only bacterial liquid is added for fermentation for 6 h), at the middle of fermentation (only bacterial liquid is added for fermentation for 24 h), at the end of fermentation (only bacterial liquid is added for fermentation for 42 h), and after the end of fermentation (only bacterial liquid is added for fermentation for 48 h, and enzyme inactivation is carried out for 12 h after the fermentation is stopped), on the inhibition of alpha-glucosidase activity and the production of active substances by synergistically fermenting tea seed meal were explored.

[0064] (3) The inoculation amount is one of the important factors affecting the active substances obtained by the synergistic fermentation of tea seed meal by bacteria and enzymes. Therefore, the time for adding the complex enzyme is fixed at 6 h after adding the bacterial liquid, the solid-liquid ratio is 1:5, the enzyme addition amount is 3%, the fermentation time is 48 h, the fermentation temperature is 32℃, and the effects of inoculation amounts of 5%, 10%, 15%, 20% and 25% on the inhibition of α-glucosidase activity and the production of active substances by the synergistic fermentation of tea seed meal by bacteria and enzymes are explored.

[0065] (4) The enzyme addition amount is one of the important factors affecting the active substances obtained by the synergistic fermentation of tea seed meal by bacteria and enzymes. Therefore, the time for adding the complex enzyme is fixed at 6 h after adding the bacterial liquid, the solid-liquid ratio is 1:5, the inoculation amount is 10%, the fermentation time is 48 h, the fermentation temperature is 32℃, and the effects of enzyme addition amounts of 2%, 3%, 4%, 5% and 6% on the inhibition of α-glucosidase activity and the production of active substances by the synergistic fermentation of tea seed meal by bacteria and enzymes are explored.

[0066] (5) The fermentation time is one of the important factors affecting the active substances obtained by the synergistic fermentation of tea seed meal by bacteria and enzymes. Therefore, the time for adding the complex enzyme is fixed at 6 h after adding the bacterial liquid, the solid-liquid ratio is 1:5, the enzyme addition amount is 3%, the inoculation amount is 10%, the fermentation temperature is 32℃, and the effects of fermentation times of 12 h, 24 h, 48 h, 72 h and 96 h on the inhibition of α-glucosidase activity and the production of active substances by the synergistic fermentation of tea seed meal by bacteria and enzymes are explored.

[0067] (6) The fermentation temperature is one of the important factors affecting the active substances obtained by the synergistic fermentation of tea seed meal by bacteria and enzymes. Therefore, the time for adding the complex enzyme is fixed at 6 h after adding the bacterial liquid, the solid-liquid ratio is 1:5, the enzyme addition amount is 3%, the inoculation amount is 10%, the fermentation time is 48 h, and the effects of fermentation temperatures of 27℃, 32℃, 37℃, 42℃ and 47℃ on the inhibition of α-glucosidase activity and the production of active substances by the synergistic fermentation of tea seed meal by bacteria and enzymes are explored.

[0068] 3. Response surface optimization of the synergistic fermentation process According to the Box-Behnken design principle, combined with the single factor test results, the solid-liquid ratio (tea seed meal content), fermentation temperature and the amount of added complex enzyme are fixed, the inoculation amount (A), enzyme addition time (B) and fermentation time (C) are selected as three factors to design a three-factor and three-level test to study the effects of different conditions on the synergistic solid-state fermentation of tea seed meal by bacteria and enzymes. The inhibition of α-glucosidase activity (Y) is taken as the index, and each treatment has 3 replicates to screen the better combination.

[0069] Table 2 Response surface design table for the synergistic fermentation of tea seed meal by bacteria and enzymes to obtain blood glucose-lowering active substances

[0070] II. Experimental results 1. By Figure 8 It can be seen that there was no significant difference in the inhibition rate of α-glucosidase between the fermentation group of strain Z-12 and the product obtained by the complex enzyme digestion. p >0.05), the fermentation group and enzymatic hydrolysis group obtained hypoglycemic active ingredients superior to the original meal during the fermentation process. The fermentation group using strain Z-12 and the compound enzyme showed the best inhibitory activity, with an inhibition rate significantly higher than other groups. p <0.05), possibly because the microbial-enzyme co-fermentation technology combines the advantages of both microbial fermentation and enzymatic hydrolysis. Enzymatic hydrolysis can provide basic nutrients such as nitrogen and carbon sources for the fermentation process of microorganisms. At the same time, fermentation technology can overcome the problems of functional element loss caused by simple enzymatic hydrolysis, which greatly improves production efficiency.

[0071] 2. By Figure 8 As shown in A, with the increase of inoculum amount, the inhibition rate of α-glucosidase by the substrate of bacterial-enzyme co-fermentation gradually increases. The highest α-glucosidase inhibition rate is found when the inoculum amount reaches 15%. This may be because the appropriate amount of bacterial strain can co-ferment with the complex enzyme to produce active polysaccharides with α-glucosidase inhibitory activity and substances with α-glucosidase inhibitory activity. Among them, the optimal inhibitory activity is found when the bacterial strain inoculum amount reaches 15%.

[0072] Depend on Figure 8 As shown in section B, the highest α-glucosidase inhibition rate was observed at a substrate-to-liquid ratio of 1:5. There was no significant difference in substrate-to-liquid ratios between 1:4 and 1:6. This is likely because a substrate-to-liquid ratio of 1:5 allows for sufficient contact between the substrate, bacteria, and enzyme, which is beneficial for the formation of active metabolites. Therefore, a substrate-to-liquid ratio of 1:5 was chosen as the optimal ratio.

[0073] Depend on Figure 8 C indicates that the highest α-glucosidase inhibition rate was observed in the early stage of fermentation (enzyme was added after only 6 hours of fermentation with bacterial solution), and there was no significant difference in α-glucosidase inhibition activity between adding enzyme to hydrolyze tea seed meal first and then adding the bacterial strain for fermentation. p <0.05), but as fermentation progressed (24-60 h), the inhibition rate of the bacterial enzyme fermentation product against α-glucosidase decreased significantly with the addition of compound enzymes. This may be because the initial addition of enzymes can enzymatically hydrolyze the substrate and create a favorable environment for microbial fermentation. It can also prevent excessive microbial growth, which would lead to insufficient nutrients and the initiation of fermentation and decomposition of exogenous enzymes. Adding enzymes in the middle, late and post-fermentation stages did not achieve the effect of co-fermentation, and the post-fermentation environment may not provide a suitable enzymatic hydrolysis environment for exogenous enzymes.

[0074] Depend on Figure 8DIt was found that the α-glucosidase inhibitory activity of the product increased with the increase of enzyme dosage, and then decreased. The highest α-glucosidase inhibitory activity was obtained when the enzyme dosage was 4%. The enzyme dosage of 3-5% had no significant effect on the α-glucosidase inhibitory activity of the product (p<0.05). The moderate enzyme dosage (≤4%) could fully hydrolyze the tea seed meal substrate, release more active substances, and enhance the inhibitory effect of the product. However, the excessive enzyme dosage (>4%) could degrade the active ingredients, inhibit the growth of the bacteria, or produce competitive substrates, resulting in the decrease of the activity.

[0075] By Figure 8 EIt was found that the α-glucosidase inhibitory activity of the product was the highest when the fermentation temperature was 37°C, but there was no significant difference between the 37°C group and the 32°C group (p>0.05). The growth of microorganisms was inhibited at high temperatures, and the α-glucosidase inhibitory activity of the product was significantly reduced. Therefore, 37°C was selected as the optimal fermentation temperature. p >0.05), temperature over high, microbial growth was inhibited, the α-glucosidase inhibitory activity of the product was significantly reduced, therefore, 37°C was selected as the optimal fermentation temperature.

[0076] By Figure 9 FIt was found that the α-glucosidase inhibitory activity increased with the increase of fermentation time when the fermentation time was less than 72 h, and reached the peak at 72 h. However, the inhibitory activity began to decrease with the prolongation of fermentation time. This may be due to the decrease of substrate nutrients, the decomposition of small molecular active substances by microorganisms, and the gradual decrease of α-glucosidase inhibitory activity.

[0077] 3、On the basis of single factor experiment, enzyme addition time (A), fermentation time (B) and inoculum size (C) were selected as independent variables, and the α-glucosidase inhibitory activity of tea seed meal enzyme fermentation product was selected as response value (Y). The response surface experiment of 3 factors and 3 levels was designed to optimize the enzyme hydrolysis process. The experimental design and results are shown in Table 3 and Table 4.

[0078] Table 3 Box-Behnken experimental design and results of tea seed meal enzyme fermentation

[0079] Table 4 Analysis of variance of regression model

[0080] Note: P <0.05, significant difference, represented by *; P <0.01, extremely significant difference, represented by **; P <0.0001, extremely significant difference, represented by ***; P >0.05, not significant difference.

[0081] The regression model and variance analysis were used to analyze the data in Table 3 by Design-Expert 13 software for multiple quadratic regression fitting analysis, and the quadratic regression equation of enzyme adding time (A), fermentation time (B), inoculum size (C) and the alpha-glucosidase inhibitory rate (Y) of the tea seed meal enzyme fermentation product was obtained as follows: Y = 69.64 + 0.3425A + 0.8987B + 0.3162C + 0.2425AB + 0.2625AC - 0.795BC - 1.19A2 - 4.32B2 - 2.24C2.

[0082] As shown in Table 4, the model F value was 30.13, P <0.0001, indicating that the design model had extremely significant difference, and the test method had high reliability; the lack of fit was 0.4696, which was greater than 0.05, and was not significant, indicating that the test value of the model was linearly related to the predicted value, and had good fitting degree. The correlation coefficient R 2 = 0.9748 > 0.9, indicating that the model had good correlation. The correction coefficient R 2 adj = 0.9425, indicating that the model had high reliability, and indicating that the model could predict the alpha-glucosidase inhibitory activity of the tea seed meal enzyme fermentation product. According to the F value, the order of the influence of each factor on the alpha-glucosidase inhibitory activity was fermentation time > enzyme adding time > inoculum size. According to the P value, the primary term fermentation time, the quadratic terms A2, B2 and C2 had extremely significant influence on the alpha-glucosidase inhibitory rate (P P <0.01), and the other influences were not significant (P P > 0.05).

[0083] The response surface graph and contour graph of the interaction between enzyme adding time, enzyme fermentation time and strain inoculum size on the alpha-glucosidase inhibitory rate of the tea seed meal enzyme fermentation product are shown in Figs. 1-3. ​As shown in the results, the interaction of the three factors of enzyme addition time, fermentation time and inoculum size showed a trend of first increasing and then decreasing, indicating that there was a maximum value within the test range. The greater the curvature of the response surface of the interaction of the three factors, the greater the influence of the interaction of the two factors on the α-glucosidase inhibitory rate of the tea seed meal enzyme fermentation product. The results showed that the contour plot of enzyme addition time and fermentation time was flat and oval, indicating a strong interaction and a greater influence on the α-glucosidase inhibitory rate. In the response surface plot, the curvature was greater with the change of fermentation time, indicating that the fermentation time had a more significant influence on the α-glucosidase inhibitory rate. The contour plot of enzyme addition amount and inoculum size tended to be circular, indicating a smaller interaction of the two factors and a smaller influence on the α-glucosidase inhibitory rate, which was consistent with the results of the variance analysis in Table 3.

[0084] The optimal solution of the regression equation was obtained by Design-Expert 13, and the optimal process for the α-glucosidase inhibitory rate of the tea seed meal enzyme fermentation product was obtained as follows: enzyme addition after 8.91 h of fermentation, fermentation time of 74.47 h, and inoculum size of 15.31%, and the α-glucosidase inhibitory rate of the product was 69.722%. In order to facilitate practical operation, the optimal fermentation conditions were revised as enzyme addition after 9 h of fermentation, fermentation time of 74 h, and inoculum size of 15%, and under these conditions, 3 sets of parallel experiments were repeated to verify the α-glucosidase inhibitory rate, which was 67.872%, close to the predicted value, indicating that the model predicted the optimal fermentation conditions for the α-glucosidase inhibitory rate of the tea seed meal enzyme fermentation product were reliable.

Claims

1. A plant Apergillus pallidofulvus Z-12 strain, characterized in that, The strain was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on July 2, 2025, with accession number GDMCC No: 66621.

2. A microbial agent, characterized in that, The microbial agent contains the active ingredient as described in claim 1. Apergillus pallidofulvus Z-12 strain and one or more of its culture medium, bacterial suspension, and bacterial cells; the preparation method of the culture medium, bacterial suspension, and bacterial cells is as follows: Apergillus pallidofulvus The Z-12 strain was inoculated and cultured to obtain a culture medium; the culture medium was centrifuged and resuspended to obtain a bacterial suspension; the culture medium was centrifuged to obtain bacterial cells.

3. The claim 1 Apergillus pallidofulvus Application of strain Z-12 or the microbial agent according to claim 2 in the preparation of hypoglycemic active ingredients from fermented tea seed meal.

4. The claim 1 Apergillus pallidofulvus Application of strain Z-12 or the combination of the bacterial agent and protease described in claim 2 in the preparation of hypoglycemic active ingredients from fermented tea seed meal.

5. The application according to claim 3 or 4, characterized in that, The hypoglycemic active ingredient has the activity of inhibiting α-glucosidase.

6. A method for preparing hypoglycemic active ingredients from fermented tea seed meal, characterized in that, The claim 1 Apergillus pallidofulvus The Z-12 strain or the inoculum agent described in claim 2 is inoculated into fresh defatted tea seed meal for fermentation to obtain the product.

7. The method according to claim 6, characterized in that, The method also includes adding protease during the initial fermentation stage.

8. The method according to claim 7, characterized in that, The fermentation time is 48–96 h.

9. The method according to claim 7, characterized in that, The claim 1 Apergillus pallidofulvus The inoculation amount of strain Z-12 or the inoculum agent according to claim 2 is 10-20%.

10. A hypoglycemic active ingredient prepared by the method according to any one of claims 6 to 9.

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