Multifunctional plant lactobacillus plantarum and use thereof

Abstract

A multifunctional plant lactobacillus plantarum and a use thereof. The plant lactobacillus plantarum LY4 strain was stored in the Guangdong Microbial Culture Collection Center on 19 April, 2023, with the strain deposit number of: GDMCC N0: 63373. The LY4 strain has high inhibition capabilities with regard to key enzymes α-glucosidase and α-amylase which cause blood sugar to rise, with an inhibition rate reaching as high as 100%, an effect reaching 96% or more of the diabetes drug Acarbose, and the ability to degrade lipopolysaccharides; moreover, the LY4 strain has good resistance to acid and bile salt, improved safety and thermal stability, can be used as an efficient strain for controlling blood sugar, can degrade lipopolysaccharides while relieving diabetes, and provides a new choice for controlling blood sugar in diabetes patients.

Description

A multifunctional Lactobacillus plantarum strain and its application Technical Field

[0001] The present invention belongs to the field of microbial technology and more specifically relates to a multifunctional Lactobacillus plantarum strain and its application. Background Art

[0002] Diabetes is a general term for metabolic disorders caused by insulin deficiency or insulin resistance, with varying etiologies and pathogenesis, primarily characterized by abnormal glucose metabolism. Diabetes presents numerous complications, causing significant inconvenience to patients and placing a significant economic burden on families and society. It is the third most common chronic, non-communicable disease threatening human health and well-being, after malignant tumors and cardiovascular disease. Therefore, effective prevention and treatment of diabetes is a critical task. α-glucosidase is a hydrolase found in the brush border of the small intestinal mucosa that catalyzes the hydrolysis of carbohydrates (such as oligosaccharides and disaccharides) into monosaccharides, releasing glucose into the bloodstream and leading to elevated blood sugar levels. Existing research suggests that inhibiting α-glucosidase activity can slow the conversion of carbohydrates into monosaccharides, reducing the rate of glucose absorption and maintaining low plasma glucose levels in diabetic patients after meals, thus regulating blood sugar. Alternatively, inhibiting α-amylase can block the conversion of starch into sugars, thereby lowering plasma glucose levels and achieving blood sugar control.

[0003] Lipopolysaccharides (LPS) are cell wall components secreted by Gram-negative bacteria after their rupture and are a major inducing factor in the initiation of inflammation. While Gram-positive bacteria are abundant in healthy individuals, Gram-negative bacteria are abundant in the intestines of patients with type 2 diabetes mellitus (T2DM). Upon death, these bacteria release large amounts of LPS, some of which is absorbed into the bloodstream. Consequently, LPS concentrations in the blood of patients with T2DM are significantly elevated. LPS can bind to receptor proteins on the membranes of small intestinal epithelial cells, disrupting intestinal permeability and activating transcription factors such as NF-κB to generate an immune response and induce inflammation. LPS can also bind to the CD14 / Toll-like receptor 4 (TLR4) receptor, inhibiting pancreatic β-cell expression through the NF-κB pathway. LPS can also activate the MAPKs pathway, affecting the expression of insulin receptor substrates and leading to insulin resistance. Intestinal flora imbalance can lead to a competition for the number of Gram-negative bacteria, thereby increasing the concentration of LPS. High concentrations of LPS activate TLR4, releasing inflammatory factors such as interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), etc., thereby triggering a chronic inflammatory response in the body and ultimately leading to insulin resistance.

[0004] In recent years, with the development of biotechnology, research on probiotics has attracted increasing attention and recognition. Probiotics are live microorganisms that, when added to food at a certain dose, are beneficial to the host's health. Probiotics possess biological functions such as antibacterial, antiviral, cholesterol-lowering, antioxidant, and immune-enhancing properties. Existing studies have shown that probiotics, prebiotics, or synbiotics can significantly alleviate the clinical symptoms of diabetic patients. According to literature reports, 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 inhibited α-glucosidase, with inhibition rates of 41.81%, 27.14%, 24.96%, 31.18%, 27.83%, and 35.68%, respectively. Zhang Zhao et al. isolated a strain of Lactobacillus plantarum NX-1 from naturally fermented kimchi liquid, which exhibited excellent α-glucosidase inhibition. The uninactivated fermentation supernatant exhibited an α-glucosidase inhibition rate of 33.13%. Compared to chemical drugs, probiotics offer greater safety and minimal toxicity, making them a promising and attractive biotherapy option. However, the inhibitory effects of currently available probiotics on both α-glucosidase and α-amylase are relatively low, and this remains to be seen.

[0005] Currently, there is a lack of drugs that can simultaneously inhibit the blood sugar lowering effects of α-glucosidase / α-amylase and the degradation of lipopolysaccharide. Lipopolysaccharide can stimulate the release of key inducing factors of insulin resistance. Therefore, it is urgent to find strains or drugs that can simultaneously inhibit α-glucosidase / α-amylase and degrade lipopolysaccharide content.

[0006] Summary of the Invention

[0007] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the existing probiotics in inhibiting α-glucosidase and α-amylase, and to provide a plant lactobacillus that has the ability to inhibit the activity of α-glucosidase and α-amylase and degrade lipopolysaccharide and its application.

[0008] The first object of the present invention is to provide a multifunctional Lactobacillus plantarum LY4 strain.

[0009] The second object of the present invention is to provide the application of Lactobacillus plantarum LY4 strain.

[0010] The third object of the present invention is to provide an α-glucosidase and / or α-amylase inhibitor.

[0011] The fourth object of the present invention is to provide a product for alleviating / treating diabetes or controlling blood sugar.

[0012] The fifth object of the present invention is to provide a method for inhibiting α-glucosidase and / or α-amylase and degrading lipopolysaccharide.

[0013] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0014] The present invention provides a multifunctional Lactobacillus plantarum LY4 strain, which can inhibit the activity of α-glucosidase and α-amylase and degrade lipopolysaccharide. The strain was deposited with the Guangdong Provincial Culture Collection Center on April 19, 2023, with the culture collection number: GDMCC NO: 63373. The full gene sequence of the LY4 strain has been uploaded to NCBI with the accession number SRR24579381.

[0015] The LY4 strain provided by the present invention is isolated from rice wine fermentation mash. The LY4 strain has a high inhibitory effect on α-glucosidase, a key enzyme that causes elevated blood sugar. The inhibition rate of the fermentation supernatant of the LY4 strain on α-glucosidase can reach more than 96% of the efficiency of the diabetes drug acarbose. The LY4 strain also has excellent inhibitory ability against α-amylase. The inhibition rate of its fermentation supernatant on α-amylase can reach 100%, and the effect is not affected by high temperature and has good thermal stability. The inhibitory effect is very close to that of acarbose, but it has better safety than acarbose and no toxic side effects. At the same time, the LY4 strain can degrade lipopolysaccharide, with a lipopolysaccharide removal rate of 29.91% for 101EU / mL in 4 hours. That is, the LY4 strain can efficiently inhibit α-glucosidase and α-amylase, and can degrade lipopolysaccharide by inhibiting the activity of α-glucosidase and α-amylase, slowing the conversion and absorption of carbohydrates, lowering postprandial blood sugar levels, thereby suppressing postprandial hyperglycemia and alleviating diabetes. At the same time, the strain has good acid and bile salt resistance, which meets the survival requirements of probiotics in the human gastrointestinal tract.

[0016] Therefore, the present invention provides the following applications of the multifunctional Lactobacillus plantarum LY4 strain and / or its bacterial solution:

[0017] Application in the inhibition of α-glucosidase and / or α-amylase for the purpose of non-disease diagnosis and treatment.

[0018] Application in the degradation of lipopolysaccharide for the purpose of non-disease diagnosis and treatment.

[0019] Application of the invention in the preparation of products for inhibiting α-glucosidase and / or α-amylase.

[0020] Application in the preparation of products for degrading lipopolysaccharides.

[0021] Application in the preparation of products for alleviating or treating diabetes or controlling blood sugar.

[0022] Preferably, the product is a food, a health product or a medicine.

[0023] The invention provides an alpha-glucosidase and / or alpha-amylase inhibitor containing Lactobacillus plantarum LY4 strain and / or its bacterial liquid.

[0024] Preferably, the bacterial liquid is a fermentation liquid of the strain, which is obtained by inoculating 1 to 5% of the LY4 strain into a culture medium and culturing it at 35 to 40° C. for 24 to 72 hours.

[0025] More preferably, the fermentation broth is a fermentation supernatant or a bacterial suspension.

[0026] The present invention provides a product for alleviating / treating diabetes or controlling blood sugar, comprising Lactobacillus plantarum LY4 strain and / or its bacterial liquid.

[0027] Preferably, the concentration of the LY4 strain is not less than 2×10 9 cfu / mL.

[0028] Preferably, the bacterial liquid is a fermentation liquid of the strain, which is obtained by inoculating 1 to 5% of the LY4 strain into a culture medium and culturing it at 35 to 40° C. for 24 to 72 hours.

[0029] More preferably, the LY4 strain is inoculated into the culture medium at a 1% inoculum rate, and the culture is statically cultured at 37° C. for 24 hours to obtain the fermentation broth.

[0030] The invention provides a method for inhibiting alpha-glucosidase and / or alpha-amylase and degrading lipopolysaccharide. The method comprises adopting Lactobacillus plantarum LY4 strain and / or bacterial liquid thereof to process a sample.

[0031] The present invention has the following beneficial effects:

[0032] The present invention provides a multifunctional Lactobacillus plantarum LY4 strain, which has a high inhibitory effect on α-glucosidase, a key enzyme that causes elevated blood sugar. The inhibition rate of the fermentation supernatant of the LY4 strain on α-glucosidase can reach more than 96% of the efficiency of the diabetes drug acarbose. The LY4 strain also has excellent inhibitory ability on α-amylase, and its fermentation supernatant can inhibit α-amylase by 100%. It is also unaffected by high temperature and protease, and its effect is comparable to that of acarbose, but it has better safety than acarbose and no toxic side effects. At the same time, the LY4 strain can degrade lipopolysaccharide, with a lipopolysaccharide removal rate of 29.91% for 101EU / mL in 4 hours. That is, the LY4 strain can degrade lipopolysaccharide, better slowing down the conversion and absorption of sugars to reduce postprandial blood sugar levels, thereby suppressing postprandial hyperglycemia. The LY4 strain provided by the present invention has good acid and bile salt resistance, meeting the survival requirements of probiotics in the human gastrointestinal tract.

[0033] The LY4 strain provided by the present invention can slow down the conversion and absorption of carbohydrates by inhibiting the activity of α-glucosidase and α-amylase and degrading lipopolysaccharides, thereby reducing postprandial blood sugar levels, thereby suppressing postprandial hyperglycemia, alleviating diabetes, and having a positive effect on the prevention and treatment of diabetes. In addition, plant lactobacillus has been included in the national edible probiotic list, and the strain is highly safe, has no toxic side effects, and is highly resistant to stress. Plant lactobacillus LY4 of the present invention can be applied to fermented foods, health products, and medicines, providing new options for daily blood sugar control in diabetic patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 is a morphological diagram of Lactobacillus plantarum LY4 bacteria;

[0035] FIG2 is a phylogenetic tree of Lactobacillus plantarum LY4 constructed using the neighbor-joining method;

[0036] Figure 3 is a circle map of the Lactobacillus plantarum LY4 genome;

[0037] FIG4 is a graph showing the results of an acid tolerance experiment of Lactobacillus plantarum LY4;

[0038] FIG5 is a graph showing the results of a bile salt tolerance experiment with Lactobacillus plantarum LY4;

[0039] FIG6 is a graph showing the inhibition rate of α-glucosidase activity by Lactobacillus plantarum LY4;

[0040] FIG7 is a graph showing the results of an experimental inhibition experiment on α-glucosidase activity by a time gradient of the fermentation supernatant of Lactobacillus plantarum LY4;

[0041] FIG8 is a graph showing the results of an experimental study on the inhibition of α-glucosidase activity by a temperature gradient in the fermentation supernatant of Lactobacillus plantarum LY4;

[0042] FIG9 is a graph showing the results of an experimental inhibition experiment on α-glucosidase activity by protease treatment of Lactobacillus plantarum LY4 fermentation supernatant;

[0043] FIG10 is a graph showing the results of an experiment on the inhibition of α-amylase activity by Lactobacillus plantarum LY4;

[0044] FIG11 is a graph showing the experimental results of degradation of lipopolysaccharide by the fermentation supernatant of Lactobacillus plantarum LY4. DETAILED DESCRIPTION

[0045] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0046] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0047] Example 1 Screening and identification of strains

[0048] 1. Isolation and purification of strains:

[0049] The sample comes from rice wine fermentation mash. Take an appropriate amount of rice wine fermentation mash and place it in 99mL of sterile tertiary water. Add an appropriate amount of sterile glass beads and place it in a shaker at room temperature and 200rpm / min for 6 hours to fully separate the microorganisms from the lees and fully disperse the microorganisms. The bacterial concentration of the bacterial suspension in this triangular flask is 10% of the original sample. -2 The gradient dilution method was used to spread the appropriate gradient dilution bacterial suspension on MRS medium. The plate was inverted and placed in a 37°C incubator for 48 hours. Based on the colony morphology, the strain was streaked multiple times on MRS medium to obtain a purified strain, which was named LY4 strain.

[0050] 2. Observation of bacterial morphology:

[0051] The purified LY4 strain was inoculated on an MRS medium plate and inverted for 48 h at 37° C. to observe its colony morphology. The strain morphology is shown in FIG1 . The colony was opaque milky white with a smooth and moist surface. The strain was Gram-positive.

[0052] 3. Molecular biological identification of bacteria:

[0053] DNA was extracted from the purified bacteria using the SDS method. Universal primers 27f (5'-CCTGGCTCAGAGAGTTTGAT-3') and 1492r (5'-GGTTACCTTGTTACGACTT-3') were used to amplify the conserved bacterial sequence. The amplified primers were then sent to a sequencing company for sequencing. The resulting sequences were then spliced ​​and compared against the NCBI database using BLAST.

[0054] At the same time, the results of the phylogenetic tree construction using the neighbor-joining method of Mega software are shown in Figure 2. The comparison results show that the LY4 strain and Lactiplantibacillus plantarum CGMCC10331 T (KR106205.1) strain similarity reached 99%. Based on the above identification results, the final classification status of the LY4 strain was determined to be Lactiplantibacillus plantarum, and was named Lactiplantibacillus plantarum LY4 strain. It was deposited in the Guangdong Provincial Microbial Culture Collection on April 19, 2023, with the culture collection number: GDMCC N0: 63373, and the storage address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0055] 4. Whole-genome sequencing of bacteria:

[0056] The experimental process was carried out according to the standard protocol provided by PacBio, including sample quality testing, library construction, library quality testing and sequencing. The genome assembly software was HIF-IASM v0.12.

[0057] The genome assembly results showed that the genome was 3270221 bp long, circular, and had a GC content of 44.44%.

[0058] The results of non-coding RNA analysis showed that there were 68 tRNAs and 16 rRNAs on the genome of B. plantarum LY4, including 6 5S rRNAs, 5 16S rRNAs, and 5 23S rRNAs.

[0059] Using the assembled and predicted genomic information, such as tRNA, rRNA, repeat sequences, GC content, and gene function, the Circos v0.66 software was used to create a genome circle map, as shown in Figure 3. The complete genome sequence of Lactiplantibacillus plantarum strain LY4 has been uploaded to NCBI with accession number SRR24579381.

[0060] Example 2 Bile Salt and pH Tolerance Determination of Lactobacillus plantarum LY4

[0061] 1. pH tolerance determination:

[0062] The activated LY4 bacterial solution was centrifuged at 4°C, 10000r / min for 8min, and the supernatant was discarded. The solution was inoculated into MRS liquid culture medium with pH 2.0, pH 3.0, and pH 4.0, respectively, and cultured at 37°C. Ordinary MRS culture medium was used as a control. At 0h and 4h after inoculation, 10 μL of the appropriate gradient was diluted with sterile physiological saline using a ten-fold gradient dilution method. The viable bacteria were counted after culture in a 37°C incubator. The number of viable bacteria was substituted into the following formula to calculate the acid-resistant survival rate. Survival rate (%) = (log N1 / log N0 )×100%

[0063] Where: N1: the number of viable bacteria in the experimental group culture, N0: the number of viable bacteria at 0h after culture in ordinary MRS medium.

[0064] 2. Bile salt tolerance test:

[0065] The activated LY4 bacterial liquid was centrifuged at 4°C and 10,000 r / min for 8 minutes, and the supernatant was discarded. The culture medium was inoculated into 0.1% (w / v), 0.3% (w / v), and 0.5% (w / v) bile salt MRS liquid culture medium, respectively, and cultured at 37°C. Ordinary MRS culture medium was used as a control. At 0h and 4h after inoculation, 10 μL of the appropriate gradient was diluted with sterile physiological saline using a ten-fold gradient dilution method and spotted on an MRS plate. After culture in a 37°C incubator, the viable bacteria were counted, and the number of viable bacteria was substituted into the survival rate formula to calculate the bile salt-resistant survival rate.

[0066] 3. Experimental results:

[0067] The pH tolerance of Lactobacillus plantarum LY4 is shown in FIG4 . In the pH tolerance test, when pH = 2, the survival rate of Lactobacillus plantarum LY4 is 53.3%; when pH = 3, the survival rate of Lactobacillus plantarum LY4 can reach 90.4%; when pH = 4, the survival rate of Lactobacillus plantarum LY4 exceeds 100%, reaching 105%.

[0068] The bile salt tolerance of Lactobacillus plantarum LY4 is shown in FIG5 . In the bile salt tolerance assay, when 0.1% bile salt was added, the survival rate of Lactobacillus plantarum LY4 reached 97.9%, when 0.3% bile salt was added, the survival rate of Lactobacillus plantarum LY4 reached 96.2%, and when the amount of bile salt added was increased to 0.5%, Lactobacillus plantarum LY4 still maintained a survival rate of 84%.

[0069] Therefore, it is shown that the Lactobacillus plantarum LY4 of the present invention has good acid resistance and bile salt resistance.

[0070] Example 3 Inhibition of α-glucosidase activity by Lactobacillus plantarum LY4

[0071] 1. Preparation of different Lactobacillus plantarum samples:

[0072] Strain activation: Take out the LY4 strain from the -20°C refrigerator and inoculate it into an MRS test tube at a 1% inoculum for activation. Use it after two generations of activation.

[0073] Preparation of fermentation broth: The activated Lactobacillus plantarum LY4 strain fermentation broth was inoculated into 100 mL of liquid culture medium at a rate of 1%, and cultured at 37° C. for 24 h to obtain fermentation broth.

[0074] Preparation of fermentation supernatant / inactivated fermentation supernatant: Centrifuge the fermentation broth prepared above at 4°C, 10,000 rpm for 10 minutes. Collect the supernatant and filter through a 0.22 μm filter to obtain the fermentation supernatant sample. Boil the fermentation supernatant at 100°C for 20 minutes to obtain the inactivated fermentation supernatant sample. Refrigerate at 4°C until needed.

[0075] Preparation of live bacterial suspension / inactivated bacterial suspension: The fermentation broth prepared above was centrifuged at 4°C and 10,000 rpm for 10 min to collect the bacteria. After washing with 0.1 mol / L PBS (pH = 6.8), the bacteria were resuspended in PBS and the concentration of the bacterial suspension was adjusted to OD 600 The viable bacterial suspension was boiled at 100°C for 20 min to obtain an inactivated bacterial suspension.

[0076] Preparation of extracellular supernatant (CFS): The fermentation broth prepared above was centrifuged at 4°C and 10,000 rpm for 10 min, the cells were collected, washed with PBS, and resuspended in PBS. The concentration of the bacterial solution was adjusted to OD 600 The supernatant was collected by centrifugation at 4°C and 10,000 r / min for 10 minutes, and the extracellular supernatant was obtained by filtration using a 0.22 μm filter membrane and stored at 4°C for later use.

[0077] Preparation of intracellular extract (CFE): The fermentation broth prepared above was centrifuged at 4°C and 10,000 rpm for 10 min to collect the cells. After washing with PBS, the cells were resuspended in PBS and the concentration of the cell solution was adjusted to OD 600The concentration of the supernatant was 1.0, lysozyme was added, the mixture was reacted at 37°C for 3 h, and then ultrasonically disrupted in an ice bath. The ultrasonically disrupted liquid was centrifuged at 4°C and 10,000 r / min for 10 min, the supernatant was collected, and the cell-free extract was obtained by filtering with a 0.22 μm filter membrane, which was refrigerated at 4°C for later use.

[0078] 3. Experimental methods:

[0079] 25 μL of each of the fermentation supernatant, inactivated fermentation supernatant, live bacterial suspension, inactivated bacterial suspension, extracellular supernatant, and intracellular extract prepared above was added to a 96-well plate along with 25 μL of 20 mmol / L PPG. The plates were incubated at 37°C for 10 min, removed from the plate, and then 50 μL of α-glucosidase solution (0.2 U / mL) was added. The plates were incubated at 37°C for 20 min, removed from the plate, and terminated with 100 μL of 0.1 mol / L Na₂CO₃. The absorbance was measured at 405 nm. Acarbose was used as a positive control.

[0080] The calculation formula for the inhibition rate of α-glucosidase is as follows:

[0081] Wherein: A: absorbance with α-glucosidase but without sample; B: absorbance with sample but without α-glucosidase; C: absorbance with α-glucosidase and sample; D: absorbance with sample but without α-glucosidase.

[0082] 4. Experimental results:

[0083] The results are shown in Figure 6. The inhibition rate of α-glucosidase in the acarbose positive control group was 100%, while the inhibition rate of the Lactobacillus plantarum LY4 fermentation supernatant was 96%, the inhibition rate of the inactivated fermentation supernatant was 90%, the inhibition rate of the live bacterial suspension was 14%, the inhibition rate of the inactivated bacterial suspension was 7%, the inhibition rate of the extracellular supernatant was 15%, and the inhibition rate of the intracellular extract was 6%. This shows that different components of Lactobacillus plantarum LY4 all have an inhibitory effect on α-glucosidase.

[0084] In order to further illustrate the inhibitory effect of Lactobacillus plantarum LY4 fermentation supernatant on α-glucosidase, a time gradient experiment was performed on the Lactobacillus plantarum LY4 fermentation supernatant, and the supernatant was cultured for 8 h, 24 h, 48 h, and 72 h, respectively. The results are shown in Figure 7. When fermented for 8 h, the inhibition rate of Lactobacillus plantarum LY4 fermentation supernatant on α-glucosidase was 4.65%, the inhibition rate reached 91.48% when fermented for 24 h, the inhibition rate was 92.17% when fermented for 48 h, and the inhibition rate was 93.61% when fermented for 72 h, indicating that Lactobacillus plantarum LY4 can achieve a higher inhibition rate of α-glucosidase when cultured for 24 h, and as the culture time increases, the inhibition rate is higher.

[0085] A temperature gradient experiment was further performed on the fermentation supernatant of Lactobacillus plantarum LY4. The fermentation broth after 24 hours of culture was centrifuged at 4°C and 10,000 r / min for 10 minutes, the supernatant was collected, filtered using a 0.22 μm filter membrane, and then bathed in water baths at 20°C, 40°C, 60°C, 80°C, and 100°C for 30 minutes. After the water bath, it was cooled to room temperature and its inhibitory effect on α-glucosidase was determined using the same method as above.

[0086] The results are shown in FIG8 . The inhibition rate of the fermentation supernatant on α-glucosidase did not change significantly at different temperatures of 20-100° C., and the inhibition rate was around 95%, indicating that the fermentation supernatant had good thermal stability and could withstand high temperatures.

[0087] The supernatant of Lactobacillus plantarum LY4 fermentation was further treated with protease. The fermentation broth after culturing for 24 hours was centrifuged at 4°C and 10,000 r / min for 10 minutes, and the supernatant was collected. After filtering with a 0.22 μm filter membrane, pepsin and trypsin (both at a final concentration of 1 mg / mL) were used in a water bath at 37°C for 3 hours. After the water bath, the supernatant was cooled to room temperature and its inhibitory effect on α-glucosidase was determined using the same method as above.

[0088] The results are shown in Figure 9. After treatment with pepsin and trypsin, the inhibition rate of the fermentation supernatant on α-glucosidase did not change significantly. The inhibition rate of the untreated fermentation supernatant was 95.79%, the inhibition rate of the fermentation supernatant treated with pepsin was 96.54%, and the inhibition rate of the fermentation supernatant treated with trypsin was 93.72%, indicating that the active ingredient in the fermentation broth that inhibits α-glucosidase can be ruled out as a protein substance.

[0089] Example 4 Inhibition of α-amylase activity by Lactobacillus plantarum LY4

[0090] 1. Sample preparation:

[0091] Experimental samples: fermentation supernatant of LY4 strain, inactivated fermentation supernatant, live bacterial suspension, inactivated bacterial suspension, extracellular supernatant, intracellular extract, acarbose group, PBS group, and MRS group were prepared in the same manner as in Example 3.

[0092] 2. Experimental methods:

[0093] Pour a small amount of starch culture medium onto a sterile plate. Once solidified, use tweezers to place an Oxford cup on the surface of the culture medium. Pour the culture medium onto the plate again. Once solidified, remove the Oxford cup. Place three Oxford cups on each plate. Mix 50 μL of each sample with 50 μL of 1 mg / L α-amylase solution and add to each well. A blank control group consists of 50 μL of PBS and 50 μL of α-amylase solution; a negative control group consists of 50 μL of MRS and 50 μL of α-amylase solution; and a positive control group consists of 50 μL of acarbose and 50 μL of α-amylase solution. Incubate the plate in a 37°C incubator for 24 hours. Remove the plate, stain with dilute iodine solution, and measure the size of the clearing zone using a vernier caliper.

[0094] 3. Experimental results:

[0095] Figure 10 shows the inhibitory effects of different sample groups on α-amylase. The acarbose-positive group exhibited 100% inhibition of α-amylase, with no clearing zones produced. The fermentation supernatant and inactivated fermentation supernatant groups also exhibited no clearing zones. The clearing zone diameters for the live bacterial suspension group, the inactivated bacterial suspension group, the extracellular supernatant group, and the intracellular extract group were 23 mm. The clearing zone diameters for the MRS-negative control group and the blank PBS control group were 23 mm, indicating that the MRS component in the fermentation broth had no inhibitory effect on α-amylase and did not interfere with the experimental results. The fermentation supernatant and inactivated fermentation supernatant groups of Lactobacillus plantarum LY4 exhibited the same inhibitory effect on α-amylase as acarbose, achieving 100% inhibition of α-amylase activity. However, the live bacterial suspension, inactivated bacterial suspension, intracellular metabolites, and intracellular extracts showed no significant inhibitory effect on α-amylase.

[0096] Example 5 Degradation of lipopolysaccharide by Lactobacillus plantarum LY4

[0097] 1. Sample preparation:

[0098] After activating the Lactobacillus plantarum LY4 strain, 5 mL of the cultured bacterial solution was pipetted into a pyrogen-free centrifuge tube and centrifuged at 4000 r / min for 10 min. After centrifugation, the culture medium was resuspended three times with LPS-free sterile water to wash away the residual culture medium, and the volume was made up to 5 mL with lipopolysaccharide-free sterile water to prepare a bacterial suspension. In addition, the supernatant was pipetted into a pyrogen-free test tube to prepare a supernatant.

[0099] 2. Experimental methods:

[0100] The supernatant of the bacterial solution was taken separately, and the bacterial solution sediment was placed in TSB medium to prepare TSB blank medium. 1 mL of 90 EU lipopolysaccharide was added to each well and reacted at 37°C for 0 h and 4 h respectively. The 0 h reaction solution and the 4 h reaction solution were diluted 400 times, 800 times and 1600 times respectively. Then 0.1 mL of the diluted solution was taken and added to the pyrogen removal microplate, adding 3 wells for each concentration. Then 0.1 mL of Limulus amebocyte lysate was added to each well. After mixing by medium speed shaking for 10 s, the microplate was placed in a preheated endotoxin automatic analyzer ELx808 for detection. At the same time, a standard curve was established using standard endotoxins at concentrations of 0.005 EU / mL, 0.05 EU / mL, 0.5 EU / mL and 5 EU / mL. At least 3 parallel wells were used for each concentration of endotoxin solution, and 2 parallel wells were used for the negative control. The test results were the average of the results of the three parallel treatments.

[0101] 3. Experimental results:

[0102] The degradation effect of Lactobacillus plantarum LY4 fermentation supernatant on lipopolysaccharide is shown in Figure 11. The removal rate of 101EU / mL lipopolysaccharide reached 29.91% in 4 hours, and the effect was significant (p≤0.01), indicating that Lactobacillus plantarum LY4 fermentation supernatant can degrade lipopolysaccharide, reduce inflammatory response, and assist in lowering blood sugar.

[0103] In summary, plant lactobacillus LY4 provided by the invention all has good inhibitory effect to alpha-glucosidase, alpha-amylase, can slow down the conversion and absorption of carbohydrate by the activity that suppresses alpha-glucosidase and alpha-amylase, can degrade lipopolysaccharide simultaneously, reduce postprandial blood sugar level, thus suppress postprandial hyperglycemia, alleviate diabetes, the prevention and treatment of diabetes are had a positive effect, can be applied to reduce the product of blood sugar level.At present, plant lactobacillus has been listed in the edible fungi list by a plurality of countries or regions, safety is high, plant lactobacillus can be impelled to be presented in front of diabetic patients with various product forms, give them more treatment selection, no matter this has positive significance in terms of diabetic patients' blood sugar control or its keeping healthy and good psychological state, in addition, plant lactobacillus fermentation condition is simple, be easy to industrialization, cost is low, will be a strain of probiotic strains with huge market potential.

[0104] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A multifunctional Lactobacillus plantarum LY4 strain, characterized in that: The strain was deposited in Guangdong Provincial Microbiological Culture Collection Center on April 19, 2023, with the culture collection number: GDMCC NO:63373.

2. Use of the Lactobacillus plantarum LY4 strain and / or its bacterial solution according to claim 1 in inhibiting α-glucosidase and / or α-amylase for the purpose of non-disease diagnosis and treatment.

3. Use of the Lactobacillus plantarum LY4 strain and / or its bacterial solution as claimed in claim 1 in degrading lipopolysaccharide for the purpose of non-disease diagnosis and treatment.

4. Use of the Lactobacillus plantarum LY4 strain and / or its bacterial solution according to claim 1 in the preparation of products that inhibit α-glucosidase and / or α-amylase.

5. Use of the Lactobacillus plantarum LY4 strain and / or its bacterial solution according to claim 1 in preparing products that degrade lipopolysaccharides.

6. Use of the Lactobacillus plantarum LY4 strain and / or its bacterial solution according to claim 1 in preparing products for alleviating or treating diabetes or controlling blood sugar.

7. An α-glucosidase and / or α-amylase inhibitor, characterized in that: Containing the Lactobacillus plantarum LY4 strain and / or its bacterial liquid as described in claim 1.

8. A product for alleviating / treating diabetes or controlling blood sugar, characterized in that: Containing the Lactobacillus plantarum LY4 strain and / or its bacterial liquid as described in claim 1.

9. The inhibitor according to claim 7 or the product according to claim 8, characterized in that The concentration of LY4 strain is not less than 2×10 9 cfu / mL.

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

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