Multifunctional lactiplantybacillus plantarum and its applications

Abstract

This invention discloses the multifunctional Lactiplantybacillus plantarum and its applications. The Lactiplantybacillus plantarum strain LY4, which was the subject of research in this invention, was deposited with the Guangdong Provincial Center for Microbial Species Preservation on April 19, 2023. Its accession number is GDMCC NO:63373, and its whole genome sequence registration number is SRR24579381. As demonstrated in the research of this invention, the LY4 strain has high inhibitory activity against α-glucosidase and α-amylase, the main enzymes that cause elevated blood glucose levels, with inhibition rates reaching up to 100%, achieving more than 96% of the efficacy of the diabetes drug acarbose. It can also break down lipopolysaccharides. At the same time, the LY4 strain exhibits excellent acid and bile salt resistance, as well as superior safety and heat stability, meeting the survival requirements of probiotics in the human gastrointestinal tract. The LY4 strain is a highly effective strain for blood glucose control, and by alleviating diabetes while breaking down lipopolysaccharides, it provides a new option for blood glucose control in diabetic patients.

Description

Technical Field

[0001] The present invention belongs to the technical field of microorganisms. More specifically, it relates to multifunctional Lactiplantibacillus plantarum and its applications.

Background Art

[0002] Diabetes is a general term for metabolic disorders caused by insulin deficiency or insulin resistance due to various etiologies and pathogenesis mechanisms, and is mainly characterized by abnormal glucose metabolism. Diabetes is accompanied by many complications, bringing great inconvenience to the lives of patients and imposing a huge economic burden on families and society. It is the third chronic non-infectious disease threatening human health and life after malignant tumors and cardiovascular diseases. Therefore, effective prevention and treatment of diabetes have become important issues. α-Glucosidase is a hydrolase present in the brush border of the small intestinal mucosa, which has a catalytic effect of hydrolyzing carbohydrates (such as oligosaccharides and disaccharides) into monosaccharides and releasing glucose into the bloodstream, thereby causing an increase in blood glucose levels. According to existing research, by inhibiting the activity of α-glucosidase, the conversion of carbohydrates into monosaccharides can be delayed, the absorption rate of glucose can be reduced, the postprandial blood glucose level of diabetic patients can be maintained low, the blood glucose level can be controlled, or by inhibiting α-amylase to block the conversion of starch into sugar, thereby reducing the glucose in the plasma and controlling the blood glucose level.

[0003] Lipopolysaccharides (LPS) are cell wall components secreted after rupture by Gram-negative bacteria and are a major inducing factor of inflammation. While healthy individuals have an abundance of Gram-positive bacteria, the intestines of patients with type 2 diabetes (T2DM) are rich in Gram-negative bacteria. These Gram-negative bacteria release large amounts of LPS when they die, and some of it is absorbed into the bloodstream, resulting in significantly elevated blood LPS concentrations in T2DM patients. LPS binds to receptor proteins on the small intestinal epithelial cell membrane, inhibiting intestinal permeability and activating transcription factors such as NF-κB to induce an immune response and trigger inflammatory reactions. LPS also binds to CD14 / Toll-like receptor 4 (TLR4), inhibiting pancreatic β-cell expression via the NF-κB pathway. Simultaneously, LPS activates the MAPK pathway, affecting the expression of insulin receptor substrates and inducing insulin resistance. Disruption of the gut microbiota leads to competition with Gram-negative bacteria, resulting in elevated LPS concentrations. High concentrations of LPS activate TLR4, releasing inflammatory factors such as interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), triggering a chronic inflammatory response in the body, ultimately leading to insulin resistance.

[0004] In recent years, with the advancement of biotechnology, research on probiotics has attracted increasing attention and is becoming more important. Probiotics are live microorganisms that are beneficial to the host's health when added to food in certain amounts. Probiotics have biological functions such as antibacterial, antiviral, cholesterol-lowering, antioxidant, and immune-enhancing effects. As many studies have shown, probiotics, prebiotics, or synbiotics can clearly alleviate the clinical symptoms of diabetic patients. According to a literature report, LI et al. found that the cell-free supernatants of Lactobacillus plantarum (formerly known as Lactobacillus plantarum) X1, CCFM12, CCFM30, CCFEM236, CCFM311, and RS4 all had an inhibitory effect on α-glucosidase, with inhibition rates of 41.81%, 27.14%, 24.96%, 31.18%, 27.83%, and 35.68%, respectively. Zhang Cho et al. screened for Lactobacillus plantarum NX-1 strain from naturally fermented kimchi liquid, which exhibits superior inhibition of α-glucosidase activity. The α-glucosidase inhibition rate with its uninactivated fermented supernatant was 33.13%. Compared to chemical drugs, probiotics are safer and have fewer toxic side effects, making biological therapies more promising and attractive. However, currently disclosed probiotics have relatively low inhibitory effects on α-glucosidase and α-amylase, and further improvements are needed.

[0005] Currently, there is a shortage of drugs that can simultaneously inhibit α-glucosidase / α-amylase in terms of both blood glucose lowering and lipopolysaccharide degradation. Since lipopolysaccharides can promote the release of major inducers of insulin resistance, there is an urgent need to discover bacterial strains or drugs that can simultaneously inhibit α-glucosidase / α-amylase and degrade lipopolysaccharides. [Overview of the project] [Problems that the invention aims to solve]

[0006] The technical problem that this invention aims to solve is to overcome the drawbacks and deficiencies of conventional probiotics, which inhibit α-glucosidase and α-amylase, and to provide Lactiplantibacillus plantarum and its applications that can inhibit the activity of α-glucosidase and α-amylase and decompose lipopolysaccharides.

[0007] The first object of the present invention is to provide a multifunctional Lactiplantibacillus plantarum LY4 strain.

[0008] A second object of the present invention is to provide applications for the Lactiplantibacillus plantarum LY4 strain.

[0009] A third object of the present invention is to provide an inhibitor of α-glucosidase and / or α-amylase.

[0010] A fourth object of the present invention is to provide a product for the alleviation or treatment of diabetes or for the control of blood glucose levels.

[0011] A fifth object of the present invention is to provide a method for degrading lipopolysaccharides by inhibiting α-glucosidase and / or α-amylase.

[0012] The above objectives of the present invention are achieved by the following technical solutions.

[0013] This invention provides a multifunctional Lactiplantibacillus plantarum LY4 strain that inhibits the activity of α-glucosidase and α-amylase and further degrades lipopolysaccharides. This strain was deposited with the Guangdong Provincial Center for Species Preservation on April 19, 2023, under accession number GDMCC NO:63373. The whole genome sequence of the LY4 strain has been uploaded to NCBI, with registration number SRR24579381.

[0014] The LY4 strain provided in this invention was isolated from rice wine fermentation mash. The LY4 strain has a high inhibitory effect on α-glucosidase, an important enzyme that causes elevated blood glucose levels. The α-glucosidase inhibition rate of the fermentation supernatant of the LY4 strain can reach more than 96% of that of the diabetes drug acarbose. The LY4 strain also has excellent inhibitory activity against α-amylase, with the α-amylase inhibition rate of the fermentation supernatant reaching 100%. Its effect is not affected by high temperatures and it has good thermal stability. Its inhibitory effect is very close to that of acarbose, but it is safer and has no toxic side effects. On the other hand, the LY4 strain can decompose lipopolysaccharide, achieving a removal rate of 29.91% of 10¹ EU / mL of lipopolysaccharide within 4 hours. In other words, the LY4 strain can efficiently inhibit both α-glucosidase and α-amylase. By inhibiting the activity of α-glucosidase and α-amylase and breaking down lipopolysaccharides, this strain slows down sugar conversion and absorption, lowering postprandial blood glucose levels, suppressing postprandial hyperglycemia, and alleviating diabetes. Furthermore, this strain exhibits excellent resistance to acids and bile salts, meeting the survival requirements for probiotics in the human gastrointestinal tract.

[0015] Therefore, the present invention provides the following applications of the multifunctional Lactiplantibacillus plantarum LY4 strain and its bacterial solution.

[0016] Applications in the inhibition of α-glucosidase and / or α-amylase for the purpose of non-disease diagnosis and treatment.

[0017] Applications in the degradation of lipopolysaccharides for the purpose of non-disease diagnosis and treatment.

[0018] Applications in the manufacture of products that inhibit α-glucosidase and / or α-amylase.

[0019] Applications in the manufacture of products that break down lipopolysaccharides.

[0020] Use in the manufacture of a product for the alleviation or treatment of diabetes or for the control of blood glucose levels.

[0021] <{ Preferably, the product is a food, a dietary supplement, or a pharmaceutical.

[0022] The present invention provides an inhibitor of α-glucosidase and / or α-amylase, comprising Lactiplantibacillus plantarum LY4 strain and / or its bacterial solution.

[0023] Preferably, the bacterial solution is a bacterial fermentation broth obtained by inoculating the LY4 strain into a medium at an inoculation rate of 1-5% and culturing statically at 35-40 °C for 24-72 hours.

[0024] More preferably, the fermentation broth is a fermentation supernatant or a bacterial suspension.

[0025] The present invention provides a product for the alleviation, treatment of diabetes or control of blood glucose levels, comprising Lactiplantibacillus plantarum LY4 strain and / or its bacterial solution.

[0026] [[ID=*24]]Preferably, the concentration of the LY4 strain is 2×10 , , ,

[0030] cfu / mL or more.

[0027] Preferably, the bacterial solution is a bacterial fermentation broth obtained by inoculating the LY4 strain into a medium at an inoculation rate of 1-5% and culturing statically at 35-40 °C for 24-72 hours.

[0028] <{ More preferably, the LY4 strain is inoculated into the medium at an inoculation rate of 1%, and the fermentation broth is obtained by culturing statically at 37 °C for 24 hours.

[0029] The present invention provides a method for inhibiting α-glucosidase and / or α-amylase and degrading lipopolysaccharide, in which a sample is treated with Lactiplantibacillus plantarum LY4 strain and / or its bacterial solution.

[0030] The present invention has the following beneficial effects. This invention provides the multifunctional Lactiplantibacillus plantarum LY4 strain. The LY4 strain exhibits a high inhibitory effect on α-glucosidase, an important enzyme that causes elevated blood glucose levels. The α-glucosidase inhibition rate of the fermented supernatant of the LY4 strain can reach more than 96% of that of the diabetes drug acarbose. The LY4 strain also exhibits excellent inhibitory activity against α-amylase, and the α-amylase inhibition rate of its fermented supernatant can reach 100%. This inhibitory effect is unaffected by high temperatures or proteases, and its efficacy is comparable to that of acarbose, but it is safer and has no toxic side effects. Furthermore, the LY4 strain can degrade lipopolysaccharides, achieving a 29.91% removal rate of 10¹ EU / mL of lipopolysaccharides in 4 hours. This means that the LY4 strain can degrade lipopolysaccharides, further slowing down sugar conversion and absorption, thereby lowering postprandial blood glucose levels and suppressing postprandial hyperglycemia. In addition, the LY4 strain provided by this invention possesses excellent acid and bile salt resistance, meeting the survival requirements for probiotics in the human gastrointestinal tract.

[0031] The LY4 strain provided by this invention inhibits the activity of α-glucosidase and α-amylase, and by breaking down lipopolysaccharides, it slows down the conversion and absorption of sugars, thereby lowering postprandial blood glucose levels, suppressing postprandial hyperglycemia, alleviating diabetes, and playing an active role in the prevention and treatment of diabetes. Furthermore, Lactipruntilabacillus plantarum is included in the national list of edible probiotics. This strain is highly safe, has no toxic side effects, and is highly resistant to stress. The Lactipruntilabacillus plantarum LY4 of this invention can be applied to fermented foods, nutritional supplements, and pharmaceuticals, providing a new option for the daily blood glucose control of diabetic patients. [Brief explanation of the drawing]

[0032] [Figure 1] This is a diagram illustrating the cellular morphology of *Lactiplantybacillus plantarum* LY4. [Figure 2]This is a phylogenetic tree of *Lactiplantybacillus plantarum* LY4 constructed using the neighbor-joining method. [Figure 3] This is a circular genome map of *Lactiplantybacillus plantarum* LY4. [Figure 4] This figure shows the results of an acid resistance experiment on Lactiplantybacillus plantarum LY4. [Figure 5] This figure shows the results of a bile salt resistance experiment with Lactiplantibacillus plantarum LY4. [Figure 6] This figure shows the inhibition rate of α-glucosidase activity by Lactiplantibacillus plantarum LY4. [Figure 7] This figure shows the results of an experiment to inhibit α-glucosidase activity using a time gradient of the fermentation supernatant of Lactiplantybacillus plantarum LY4. [Figure 8] This figure shows the results of an experiment to inhibit α-glucosidase activity using a temperature gradient in the fermentation supernatant of Lactiplantybacillus plantarum LY4. [Figure 9] This figure shows the results of an experiment to inhibit α-glucosidase activity by protease treatment of the fermentation supernatant of Lactiplantybacillus plantarum LY4. [Figure 10] This figure shows the results of an experiment inhibiting α-amylase activity using Lactiplantybacillus plantarum LY4. [Figure 11] This figure shows the results of a lipopolysaccharide decomposition experiment using the fermentation supernatant of Lactiplantybacillus plantarum LY4. [Modes for carrying out the invention]

[0033] The present invention will be further described below in conjunction with the accompanying drawings and specific examples, but the examples will not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and apparatus used in the present invention are conventional reagents, methods, and apparatus in the art.

[0034] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0035] Example 1: Screening and identification of bacterial strains 1. Isolation and purification of bacterial strains: The sample was taken from rice brewing mash. An appropriate amount of rice brewing mash was drawn up and added to 99 mL of Grade III sterile water, to which an appropriate amount of sterile glass beads was added, and the mixture was shaken at room temperature in a shaker at 200 rpm / min for 6 hours to completely separate the microorganisms from the mash and completely disperse them, and the concentration of the bacterial suspension in this Erlenmeyer flask was 10 times that of the original sample. -2 The concentration doubled. The bacterial suspension was appropriately serially diluted using the serial dilution method, spread onto MRS medium, inverted the plate, and incubated in a 37°C incubator for 48 hours. Based on the colony morphology, the strain was purified by streaking multiple times on MRS medium to obtain a purified strain, which was named strain LY4. 2. Morphological observation of the bacterial cells: The LY4 strain purified as described above was inoculated onto an MRS agar plate and cultured inverted at 37°C for 48 hours, and the colony morphology was observed. The morphology of the strain is shown in Figure 1; the colonies were milky white and opaque, with a smooth and moist surface. This strain was Gram-positive. 3. Molecular biological identification of bacteria: The purified bacterial DNA was extracted using the SDS method, and the conserved bacterial sequence was amplified using universal primers 27f (5'-CCTGGCTCAGAGAGTTTGAT-3') and 1492r (5'-GGTTACCTTGTTACGACTT-3'). The amplified primers were then sent to a sequencing service provider for sequencing, and the resulting sequences were concatenated and compared using BLAST in the NCBI database. Figure 2 shows the results of constructing phylogenetic tree components using the neighbor-joining method of Mega software. According to the comparison results, strain LY4 is Lactiplantibacillus plantarum CGMCC10331 TIt showed 99% homology with strain (KR106205.1). Based on these identification results, the final taxonomic position of strain LY4 was determined to be Lactiplantibacillus plantarum, and it was named Lactiplantibacillus plantarum strain LY4. This strain was deposited with the Guangdong Provincial Center for Microbial Species Preservation on April 19, 2023. Its accession number is GDMCC NO:63373, and its address is 5th Floor, Building 59, Dayuan, 100 Xianlie Middle Road, Guangzhou. 4. Whole-genome sequencing of bacteria: The experimental process was carried out according to the standard protocol provided by PacBio. The experimental process included sample quality testing, library construction, library quality testing, and sequencing, and the genome assembly software used was hifiasm v0.12. Genome assembly results showed that the total length of the genome was 3,270,221 bp, it was a circular sequence, and the GC content was 44.44%. Non-coding RNA analysis revealed that the Lactiplantybacillus plantarum LY4 genome contains 68 tRNAs and 16 rRNAs (of which 6 are 5S rRNAs, 5 are 16S rRNAs, and 5 are 23S rRNAs). As shown in Figure 3, a circular genome map was plotted using Circos v0.66 software, utilizing genomic information obtained from assembly and prediction, such as tRNA, rRNA, repetitive sequences, GC content, and gene function information. The whole genome sequence of Lactiplantibacillus plantarum strain LY4 has been uploaded to NCBI, with registration number SRR24579381.

[0036] Example 2: Measurement of bile salts and pH tolerance of Lactiplantibacillus plantarum LY4 1. Measurement of pH tolerance: The activated LY4 bacterial suspension was centrifuged at 4°C and 10,000 r / min for 8 minutes. The supernatant was discarded, and the samples were inoculated into MRS liquid media at pH 2.0, pH 3.0, and pH 4.0, respectively, and incubated at 37°C. Standard MRS medium was used as a control. At 0 and 4 hours after inoculation, the samples were diluted with sterile physiological saline using a 10-fold serial dilution method to create an appropriate gradient. 10 μL of the diluted liquid was spotted onto an MRS plate and incubated at 37°C. The number of viable cells was measured, and the acid-tolerant survival rate was calculated by substituting the number of viable cells into the following formula. Survival rate (%)=(log N1 / log N0 ) × 100% Here, N1 represents the number of viable cells in the experimental group, and N0 represents the number of viable cells at 0 hours in standard MRS medium. 2. Measurement of bile salt resistance: The activated LY4 bacterial suspension was centrifuged at 4°C and 10,000 r / min for 8 minutes. The supernatant was discarded, and the samples were inoculated into 0.1% (w / v), 0.3% (w / v), and 0.5% (w / v) bile salt MRS liquid medium, respectively. These were incubated statically at 37°C. Standard MRS medium was used as a control. At 0 and 4 hours after inoculation, the samples were diluted with sterile physiological saline using a 10-fold serial dilution method to create an appropriate gradient. 10 μL of the diluted liquid was spotted onto an MRS plate and incubated in a 37°C incubator. The number of viable cells was measured, and the bile salt resistance survival rate was calculated by substituting the viable cell count into the survival rate calculation formula. 3. Experimental results: Figure 4 shows the pH tolerance of Lactiplantybacillus plantarum LY4. In pH tolerance tests, the survival rate of Lactiplantybacillus plantarum LY4 was 53.3% at pH=2, reached 90.4% at pH=3, and exceeded 100%, reaching 105% at pH=4. Figure 5 shows the bile salt resistance of Lactipruntivebacillus plantarum LY4. In the bile salt resistance test, the survival rate of Lactipruntivebacillus plantarum LY4 was 97.9% when 0.1% bile salt was added, reached 96.2% when 0.3% bile salt was added, and maintained an 84% survival rate even when the amount of added bile salt was increased to 0.5%. Therefore, the present invention demonstrates good resistance to acids and bile salts in Lactiplantibacillus plantarum LY4.

[0037] Example 3: Inhibition of α-glucosidase activity by Lactiplantibacillus plantarum LY4 1. Preparation of different Lactiplantibacillus plantarum samples: Strain activation: The LY4 strain was removed from a -20°C refrigerator, inoculated into an MRS test tube at a 1% inoculation rate, activated, and used after two generations of activation. Preparation of fermentation liquid: The fermentation liquid of the activated Lactiplantybacillus plantarum LY4 strain described above was inoculated into 100 mL of liquid culture medium at a 1% inoculation rate, and the medium was incubated at 37°C for 24 hours to obtain the fermentation liquid. Preparation of fermentation supernatant / inactivated fermentation supernatant: The fermentation liquid prepared above was centrifuged at 4°C and 10,000 r / min for 10 minutes, the supernatant was collected, and filtered through a 0.22 μm filter membrane to obtain a fermentation supernatant sample. The fermentation supernatant was boiled at 100°C for 20 minutes to obtain an inactivated fermentation supernatant sample. It was stored refrigerated at 4°C. Preparation of live bacterial suspension / inactivated bacterial suspension: The fermentation liquid prepared above was centrifuged at 4°C and 10,000 r / min for 10 minutes to collect the bacterial cells, washed with 0.1 mol / L PBS (pH=6.8), and then the bacterial cells were resuspended in PBS and OD 600 The bacterial suspension concentration was adjusted to 1.0 to obtain a live bacterial suspension. The live bacterial suspension was boiled at 100°C for 20 minutes to obtain an inactivated bacterial suspension. Preparation of extracellular supernatant (CFS): The fermentation broth prepared above was centrifuged at 4°C and 10,000 r / min for 10 minutes to collect the bacterial cells, washed with PBS, and then the bacterial cells were resuspended in PBS. 600 The bacterial suspension was adjusted to a concentration of 1.0, then cultured with shaking at 37°C and 180 r / min for 24 hours. The supernatant was collected by centrifugation at 4°C and 10000 r / min for 10 minutes, filtered through a 0.22 μm filter membrane to obtain the extracellular supernatant, and stored refrigerated at 4°C. Preparation of intracellular extract (CFE): The fermentation broth prepared above was centrifuged at 4°C and 10,000 r / min for 10 minutes to collect the bacterial cells, washed with PBS, and then the bacterial cells were resuspended in PBS and OD (Oxygen-Draining). 600 The bacterial suspension was adjusted to a concentration of 1.0, lysozyme was added, and the mixture was reacted at 37°C for 3 hours. After sonication under ice bath conditions, the liquid was centrifuged at 4°C and 10,000 r / min for 10 minutes, the supernatant was collected, filtered through a 0.22 μm filter membrane to obtain a cell-free extract, which was stored refrigerated at 4°C. 3. Experimental method: The fermentation supernatant, inactivated fermentation supernatant, live bacterial suspension, inactivated bacterial suspension, extracellular supernatant, and intracellular extract from each group prepared above were aspirated in 25 μL portions and added to a 96-well plate with 25 μL of 20 mmol / L PNPG. After incubation at 37°C for 10 minutes, the samples were removed, 50 μL of α-glucosidase solution (0.2 U / mL) was added, and the mixture was reacted at 37°C for 20 minutes. The samples were then removed, 100 μL of 0.1 mol / L Na2CO3 was added to stop the reaction, and the absorbance was measured at a wavelength of 405 nm. Acarbose was used as a positive control. The formula for calculating the α-glucosidase inhibition rate is as follows:

number

[0038] Example 4: Inhibition of α-amylase activity by Lactiplantibacillus plantarum LY4 1. Sample preparation: Experimental Samples: The preparation methods for the LY4 strain fermentation supernatant, inactivated fermentation supernatant, live bacterial suspension, inactivated bacterial suspension, extracellular supernatant, intracellular extract, acarbose group, PBS group, and MRS group were the same as in Example 3. 2. Experimental method: A small amount of starch medium was poured into a sterile plate. Once solidified, an Oxford cup was placed on the surface of the medium using tweezers. The medium was then poured back into the plate, and once solidified, the Oxford cup was removed. Three Oxford cups were placed on each plate. 50 μL of the above sample and 50 μL of 1 mg / L α-amylase solution were taken, mixed, and added to the wells. As a blank control, 50 μL of PBS and 50 μL of α-amylase solution were mixed. As a negative control, 50 μL of MRS and 50 μL of α-amylase solution were mixed. As a positive control, 50 μL of acarbose and 50 μL of α-amylase solution were mixed. The samples were incubated in a 37°C incubator for 24 hours. The samples were removed, stained with diluted iodine solution, observed, and the size of the clear zone was measured using calipers. 3. Experimental results: Figure 10 shows the inhibitory effects of different sample groups on α-amylase. The acarbose-positive group showed 100% inhibition of α-amylase, and no clear zone was formed. No clear zone was formed in the fermentation supernatant group or the inactive fermentation supernatant group either. The diameter of the clear zone in the live bacterial suspension group was 22 mm, in the inactivated bacterial suspension group was 23 mm, in the extracellular supernatant group was 23 mm, in the intracellular extract group was 23 mm, in the MRS-negative control group was 23 mm, and in the blank PBS control group was 23 mm. This indicates that the MRS component in the fermentation broth does not have an inhibitory effect on α-amylase and does not affect the experimental results. The fermentation supernatants and inactivated fermentation supernatants of Lactiplantybacillus plantarum LY4 showed α-amylase inhibitory effects equivalent to acarbose, inhibiting α-amylase activity by 100%. However, live bacterial suspensions, inactivated bacterial suspensions, intracellular metabolites, and intracellular extracts did not show significant inhibitory effects on α-amylase.

[0039] Example 5: Degradation of lipopolysaccharides by Lactiplantibacillus plantarum LY4 1. Sample preparation: After activating Lactiplantybacillus plantarum LY4 strain, 5 mL of the cultured bacterial suspension was aspirated and transferred to a pyrogen-free centrifuge tube. After centrifugation at 4000 r / min for 10 minutes, the suspension was resuspended three times with LPS-free sterile water to remove residual medium, and the volume was increased to 5 mL with lipopolysaccharide-free sterile water to prepare the bacterial suspension. The supernatant was then aspirated separately and transferred to pyrogen-free test tubes to prepare the supernatant. 2. Experimental method: The bacterial suspension supernatant and bacterial suspension precipitate were collected separately and placed in TSB medium to prepare TSB blank medium. 1 mL of 90 EU lipopolysaccharide was added to each, and the mixtures were reacted at 37°C for 0 hours and 4 hours, respectively. The solutions after 0 hours and 4 hours were diluted 400-fold, 800-fold, and 1600-fold, respectively. Next, 0.1 mL of each dilution was added to a pyrogen-free microplate, with 3 wells for each concentration. Then, 0.1 mL of Limulus amoeba cytolysate reagent was added to each well, and the mixture was mixed by shaking at medium speed for 10 seconds. The microplates were placed in a preheated ELx808 endotoxin analyzer for detection. Simultaneously, standard curves were created using standard endotoxins at concentrations of 0.005 EU / mL, 0.05 EU / mL, 0.5 EU / mL, and 5 EU / mL. The endotoxin solution was placed in at least 3 parallel wells for each concentration, and in 2 parallel wells for the negative control group. The detection results were the average of the 3 parallel wells. 3. Experimental results: Figure 11 shows the effect of the fermented supernatant of Lactiplantybacillus plantarum LY4 on the degradation of lipopolysaccharides. After 4 hours, the removal rate of 101 EU / mL of lipopolysaccharide reached 29.91%, which was a statistically significant value (p ≤ 0.01). This indicates that the fermented supernatant of Lactiplantybacillus plantarum LY4 degrades lipopolysaccharides, suppresses inflammatory responses, and lowers blood glucose levels.

[0040] As can be seen from the above, Lactipruntilabacillus plantarum LY4 provided by the present invention has excellent inhibitory activity against α-glucosidase and α-amylase. By inhibiting the activity of α-glucosidase and α-amylase, it slows down the conversion and absorption of sugars, while simultaneously breaking down lipopolysaccharides and lowering postprandial blood glucose levels. This suppresses postprandial hyperglycemia, alleviates diabetes, and has a positive effect on the prevention and treatment of diabetes, making it applicable to products that lower blood glucose levels. Currently, Lactipruntilabacillus plantarum is registered as an edible bacterium in many countries and regions, and is highly safe. Providing Lactipruntilabacillus plantarum in various product forms to diabetic patients offers more treatment options and has a positive impact on blood glucose control and maintaining a healthy and good mental state in diabetic patients. Furthermore, the fermentation conditions for Lactipruntilabacillus plantarum are simple, making industrialization easy and low-cost, and it is a probiotic strain with great market potential.

[0041] While the above embodiments represent preferred embodiments of the present invention, the embodiments of the present invention are not limited to these embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principles of the present invention shall be deemed equivalent substitutions and shall be within the scope of protection of the present invention.

Claims

1. A multifunctional Lactiplantibacillus plantarum LY4 strain, characterized by being deposited with the Guangdong Provincial Microbial Species Preservation Center on April 19, 2023, under accession number GDMCC NO: 63373.

2. Application of the Lactiplantybacillus plantarum LY4 strain and / or its bacterial suspension as described in claim 1, in the inhibition of α-glucosidase and / or α-amylase for the purpose of non-disease diagnosis and treatment.

3. Application of the Lactiplantybacillus plantarum LY4 strain and / or its bacterial suspension as described in claim 1, in the degradation of lipopolysaccharides for the purpose of non-disease diagnosis and treatment.

4. Application of the Lactiplantybacillus plantarum LY4 strain and / or its bacterial suspension described in claim 1 to the production of a product that inhibits α-glucosidase and / or α-amylase.

5. Application of the Lactiplantybacillus plantarum LY4 strain and / or its bacterial suspension described in claim 1 to the production of a lipopolysaccharide-degrading product.

6. Application of the lactiplantybacillus plantarum LY4 strain and / or its bacterial suspension as described in claim 1, in the manufacture of a product for the alleviation or treatment of diabetes, or a product for the control of blood glucose levels.

7. An inhibitor of α-glucosidase and / or α-amylase, characterized by comprising the Lactiplantybacillus plantarum LY4 strain and / or its bacterial suspension as described in claim 1.

8. A product for the alleviation or treatment of diabetes or for the control of blood glucose levels, characterized by comprising the lactiplantybacillus plantarum LY4 strain and / or its bacterial solution as described in claim 1.

9. The concentration of the aforementioned LY4 strain is 2 × 10 ９ The inhibitor according to claim 7 or the product according to claim 8, characterized in that it is cfu / mL or higher.

10. A method for inhibiting α-glucosidase and / or α-amylase and decomposing lipopolysaccharide, characterized in that the sample is treated with the Lactiplantibacillus plantarum LY4 strain and / or its bacterial suspension as described in claim 1.

Images