Lactobacillus crispatus SX-E770 and application of lactobacillus crispatus SX-E770 in preparation of hypoglycemic and / or antioxidant products

Abstract

The invention discloses a lactobacillus crispatus SX-E770 and application of the lactobacillus crispatus SX-E770 in preparation of products for reducing blood glucose and / or resisting oxidation, the preservation number of the lactobacillus crispatus SX-E770 is CCTCC NO: M 2025033, and the lactobacillus crispatus SX-E770 is preserved in China Center for Type Culture Collection on January 6, 2025. The novel lactobacillus crispatus strain SX-E770 provided by the invention simultaneously has excellent blood glucose reducing and oxidation resisting capabilities, can effectively reduce the content of glucose in a body, is good in safety, and has wide application prospects in the aspect of developing products such as probiotic preparations and medicines for regulating the blood glucose level, relieving oxidative damage and preventing and treating diabetes mellitus.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a strain of Lactobacillus crispatus SX-E770 and application thereof in preparing a hypoglycemic and / or anti-oxidative product. Background Art

[0002] Probiotics are a type of active microorganisms that are beneficial to the host. They mainly live in the host's intestines and reproductive tract. There are many types, including lactobacillus, bifidobacteria, yeast, Bacillus subtilis, etc. Probiotics play an important role in improving host immunity, maintaining intestinal microecological balance, lowering cholesterol, relieving allergies, losing weight, and lowering blood sugar. They have been widely used in the development of functional foods or dietary supplements. Lactobacillus crispatus ), as a member of the probiotic family, has attracted much attention due to its strong adhesion ability and good tolerance to acid and bile salts. It can grow slowly in an acidic environment of pH 3.5 and has the ability to degrade cholesterol. However, existing research has mostly focused on the single function of Lactobacillus crispatus (such as anti-infection or digestive regulation), and there is still a gap in the development of Lactobacillus crispatus strains that have both hypoglycemic and antioxidant effects.

[0003] Diabetes mellitus (DM) is a chronic metabolic disease characterized by hyperglycemia, caused by insufficient insulin secretion and utilization disorders. Its main manifestations are "three mores and one less", namely, polydipsia, polyuria, polyphagia and weight loss. Long-term diabetes may damage the heart, blood vessels, eyes, kidneys and nerves, and may even cause acute and severe metabolic disorders. Currently, diabetes is treated with oral hypoglycemic drugs or insulin injections. However, it is difficult to achieve high-quality blood sugar standards, and some clinical drugs such as insulin have limitations such as low injection acceptance and no protective effect on the heart and kidneys. At the same time, the comprehensive management of diabetes also faces many challenges.

[0004] Oxidative stress is an important cause of metabolic diseases such as diabetes. When the body is affected by factors such as environmental pollution, bad eating habits (high-sugar and high-fat diet), and long-term mental stress, it will produce excessive free radicals, destroying the ability of pancreatic beta cells to secrete insulin, causing insulin resistance, leading to blood sugar metabolism disorders, and ultimately causing diabetes. Once diabetes occurs, it will in turn aggravate oxidative stress. Antioxidants or free radical scavengers have a protective effect against oxidative damage, but most chemically synthesized or plant-extracted antioxidant drugs are not recommended for long-term use due to potential adverse reactions.

[0005] Although some Lactobacillus crispatus has been developed for hypoglycemic or digestive regulation, its application is mostly limited to a single function, and its potential in regulating blood sugar and anti-oxidation has not been fully explored. Therefore, those skilled in the art are in urgent need of developing Lactobacillus crispatus with both hypoglycemic and anti-oxidation functions to fill the technical gap in the current probiotic application field. Summary of the invention

[0006] In order to solve the problems existing in the prior art, the present invention provides a strain of Lactobacillus crispatus SX-E770 and application thereof in preparing a hypoglycemic and / or anti-oxidative product.

[0007] The first object of the present invention is to provide a strain of Lactobacillus crispatus ( Lactobacillus crispatus )SX-E770.

[0008] A second object of the present invention is to provide the Lactobacillus crispatus ( Lactobacillus crispatus )Application of SX-E770 in the preparation of hypoglycemic products.

[0009] A third object of the present invention is to provide the Lactobacillus crispatus ( Lactobacillus crispatus )Application of SX-E770 in the preparation of antioxidant products.

[0010] The fourth object of the present invention is to provide the Lactobacillus crispatus ( Lactobacillus crispatus )Application of SX-E770 in the preparation of hypoglycemic and antioxidant products.

[0011] A fifth object of the present invention is to provide the Lactobacillus crispatus ( Lactobacillus crispatus )Application of SX-E770 in the preparation of products for inhibiting amylase activity.

[0012] The sixth object of the present invention is to provide the Lactobacillus crispatus ( Lactobacillus crispatus )Application of SX-E770 in the preparation of free radical scavenging products.

[0013] The seventh object of the present invention is to provide the Lactobacillus crispatus ( Lactobacillus crispatus ) Use of SX-E770 in the preparation of products for preventing and / or treating diabetes.

[0014] The eighth object of the present invention is to provide a microbial agent.

[0015] In order to achieve the above object, the present invention is implemented by the following scheme: The present invention isolates a strain of Lactobacillus crispatus from fresh feces of centenarians in Suixi County, Zhanjiang City, Guangdong Province. The strain has excellent blood sugar lowering, antioxidant and adhesion abilities, and can significantly reduce the glucose content in the body of a zebrafish hyperglycemia model.

[0016] Therefore, the present invention requests protection for the following contents: A strain of Lactobacillus crispatus ( Lactobacillus crispatus ) SX-E770 The preservation number of the Lactobacillus crispatus SX-E770 is CCTCC NO: M 2025033, which was deposited in the China Center for Type Culture Collection on January 6, 2025. The preservation address is: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University, opposite the First Affiliated Primary School, China Center for Type Culture Collection, Postal Code: 430072, the preservation date is January 6, 2025, and the classification name is Lactobacillus crispatus ( Lactobacillus crispatus ), deposited under the name Lactobacillus crispatus SX-E770.

[0017] The Lactobacillus crispatus ( Lactobacillus crispatus ) Application of SX-E770 in the preparation of a hypoglycemic product. The hypoglycemic agent involved in the present invention refers to lowering blood sugar levels. The Lactobacillus crispatus ( Lactobacillus crispatus )SX-E770 is used for, including but not limited to, lowering glucose levels in the body or blood and inhibiting amylase activity.

[0018] The Lactobacillus crispatus ( Lactobacillus crispatus )Application of SX-E770 in the preparation of antioxidant products.

[0019] The Lactobacillus crispatus ( Lactobacillus crispatus )Application of SX-E770 in the preparation of products for inhibiting amylase activity.

[0020] The Lactobacillus crispatus ( Lactobacillus crispatus ) Application of SX-E770 in the preparation of a product for removing free radicals. The Lactobacillus crispatus described in the application ( Lactobacillus crispatus ) The free radicals that SX-E770 can scavenge include but are not limited to DPPH and hydroxyl radicals.

[0021] The Lactobacillus crispatus ( Lactobacillus crispatus )Application of SX-E770 in the preparation of products for scavenging DPPH and / or hydroxyl free radicals.

[0022] The Lactobacillus crispatus ( Lactobacillus crispatus ) Use of SX-E770 in the preparation of products for preventing and / or treating diabetes.

[0023] Preferably, the Lactobacillus crispatus ( Lactobacillus crispatus ) The 16S rDNA sequence of SX-E770 is shown in SEQ ID NO:3.

[0024] Preferably, the Lactobacillus crispatus ( Lactobacillus crispatus )SX-E770 includes the Lactobacillus crispatus ( Lactobacillus crispatus) at least one of live bacteria, culture solution or culture supernatant of SX-E770.

[0025] More preferably, it also includes the Lactobacillus crispatus ( Lactobacillus crispatus ) Lyophilized powder of SX-E770 bacteria.

[0026] More preferably, it also includes the Lactobacillus crispatus ( Lactobacillus crispatus ) Lyophilized powder of culture supernatant of SX-E770.

[0027] More preferably, it also includes the Lactobacillus crispatus ( Lactobacillus crispatus ) Bacterial suspension of SX-E770.

[0028] A microbial agent comprising the Lactobacillus crispatus ( Lactobacillus crispatus )SX-E770.

[0029] Preferably, the microbial agent includes Lactobacillus crispatus ( Lactobacillus crispatus ) at least one of live bacteria, culture solution or culture supernatant of SX-E770.

[0030] More preferably, the microbial agent further comprises the Lactobacillus crispatus ( Lactobacillus crispatus ) Lyophilized powder of SX-E770 bacteria.

[0031] More preferably, the microbial agent further comprises the Lactobacillus crispatus ( Lactobacillus crispatus ) Lyophilized powder of culture supernatant of SX-E770.

[0032] More preferably, the microbial agent further comprises the Lactobacillus crispatus ( Lactobacillus crispatus ) Bacterial suspension of SX-E770.

[0033] Preferably, the Lactobacillus crispatus ( Lactobacillus crispatus ) The content of SX-E770 is 1×10 6 CFU / mL~1×10 8 CFU / mL.

[0034] More preferably, the Lactobacillus crispatus ( Lactobacillus crispatus ) The content of SX-E770 is 1×10 6 CFU / mL.

[0035] The freeze-dried powder and bacterial suspension of the novel Lactobacillus crispatus SX-E770 of the present invention can significantly reduce the glucose content in zebrafish in vivo, and has a potential blood sugar-lowering effect.

[0036] Compared with the prior art, the present invention has the following beneficial effects: The new strain of Lactobacillus crispatus SX-E770 provided by the present invention has excellent blood sugar lowering and antioxidant abilities, can effectively reduce the glucose content in the body, has good safety, and has broad application prospects in the development of probiotic preparations, medicines and other products for regulating blood sugar levels, alleviating oxidative damage, and preventing and treating diabetes. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the colony morphology of Lactobacillus crispatus SX-E770.

[0038] Figure 2 This is a microscopic morphological observation of Lactobacillus crispatus SX-E770 after Gram staining, with a magnification of 10×100.

[0039] Figure 3 This is the comparison and identification result of the 16S rDNA sequence of Lactobacillus crispatus SX-E770 at NCBI.

[0040] Figure 4 This is a graph showing the results of an in vitro α-amylase activity inhibition experiment of Lactobacillus crispatus SX-E770. The dotted line in the graph indicates that the α-amylase inhibition rate is 50%.

[0041] Figure 5 The total antioxidant capacity of Lactobacillus crispatus SX-E770 is measured. The dotted line in the figure indicates Fe 2+ The concentration is greater than 1 nM.

[0042] Figure 6 This is a statistical result graph of the DPPH radical scavenging rate of Lactobacillus crispatus SX-E770. The dotted line in the figure indicates that the DPPH radical scavenging rate is 50%.

[0043] Figure 7 This is a statistical graph of the hydroxyl radical scavenging rate of Lactobacillus crispatus SX-E770. The dotted line in the figure indicates that the hydroxyl radical scavenging rate is 50%.

[0044] Figure 8 This is the Gram staining image of Lactobacillus crispatus SX-E770 after adhering to Caco2 cells, with a magnification of 10×40.

[0045] Figure 9 This is the statistical result of blood glucose concentration in zebrafish hypoglycemic experiment using Lactobacillus crispatus SX-E770. DETAILED DESCRIPTION

[0046] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0047] Example 1 Isolation, identification and preservation of Lactobacillus crispatus SX-E770 1. Isolation of strains Fresh stool samples from long-lived elderly people in Suixi County, Zhanjiang City, Guangdong Province were diluted in gradients and inoculated into MRS solid medium, cultured anaerobically at 37°C for 48 hours, and single colonies on the plate were picked for streaking. The streaking was performed three times to obtain the purified monoclonal strain. The monoclonal strain obtained by three generations of streaking was inoculated into MRS liquid medium, cultured anaerobically at 37°C for 48 hours, and 30v / v% glycerol (containing 0.5w / v% cysteine) was added. The bacterial liquid and 30v / v% glycerol were mixed in equal volumes and stored in a -80°C refrigerator.

[0048] The method for streaking the strain plate is as follows: in the anaerobic workstation, after mixing the bacterial solution, immerse the disposable inoculation loop into the bottom of the bacterial solution and scrape it repeatedly three times to ensure that the bacterial solution is smeared on the inoculation loop, and use the inoculation loop to streak three areas on the MRS solid culture medium plate and record the label.

[0049] 2. Morphological identification of strains (1) Colony morphology observation The isolated monoclonal strain was streaked on a plate and its colony morphology was as follows: Figure 1 As shown, the colonies are round, milky white, opaque, with a smooth and shiny surface and a moist texture.

[0050] (2) Gram staining and microscopic morphological observation After the isolated monoclonal strains were subjected to Gram staining and examined under an oil immersion microscope, Gram-positive strains appeared purple and Gram-negative strains appeared red. Figure 2 As shown, the strain is purple and is a Gram-positive bacillus.

[0051] 3. Molecular biological identification of strains The isolated monoclonal strain was subjected to full-length 16S rDNA amplification by PCR, and the amplification primers used were 16S universal primers: 1492R (5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO: 1)) and 27F (5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO: 2)), and then the amplified product was subjected to gel electrophoresis, and the amplified product was collected for sequencing. The 16S rDNA sequence of the monoclonal strain was obtained as follows:

[0052] The 16S rDNA sequence of the monoclonal strain (SEQ ID NO: 3) was compared with the standard bacterial species sequence at NCBI for identification. Figure 3 As shown, the comparison results showed that the monoclonal strain was Lactobacillus crispatus, named Lactobacillus crispatus SX-E770.

[0053] 4. Strain preservation The Lactobacillus crispatus SX-E770 obtained above has been deposited in the China Center for Type Culture Collection (CCTCC, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University, opposite the First Affiliated Primary School, zip code: 430072), the deposit date is January 6, 2025, and the classification name is Lactobacillus crispatus ( Lactobacillus crispatus ), the deposit number is CCTCC NO: M 2025033, the deposit name is Lactobacillus crispatus SX-E770.

[0054] Example 2 In vitro functional assay of Lactobacillus crispatus SX-E770 1. Preparation of bacterial suspension, culture supernatant and lyophilized powder of Lactobacillus crispatus SX-E770 The Lactobacillus crispatus SX-E770 obtained in Example 1 was resuspended in MRS medium and anaerobically cultured at 37°C until the OD600 value of the bacterial solution was 1.6 as measured by an ultraviolet spectrophotometer, and then the bacterial solution was inoculated into the high-pressure sterilized MRS medium at a ratio of 2v / v%, and anaerobically cultured at 37°C for 24 h; the cultured bacterial solution was collected and centrifuged at 8000 r / min for 8 min, the supernatant after centrifugation was collected, and filtered through a 0.22 µm microporous filter membrane to obtain the culture supernatant of Lactobacillus crispatus SX-E770, and then the culture supernatant of Lactobacillus crispatus SX-E770 was placed in a freeze dryer for freeze drying for 72 h to obtain the culture supernatant freeze-dried powder of Lactobacillus crispatus SX-E770; the bacterial precipitate after centrifugation was collected and resuspended in PBS of the same volume as the bacterial solution before centrifugation to obtain a bacterial suspension of Lactobacillus crispatus SX-E770 (containing 1×10 9 CFU / mL), and then put it into a freeze dryer for freeze drying for 72 hours to obtain freeze-dried powder of Lactobacillus crispatus SX-E770.

[0055] 2. Blood sugar lowering ability In this example, an in vitro α-amylase activity inhibition experiment was used to evaluate the glucose-lowering ability of Lactobacillus crispatus SX-E770.

[0056] (1) Experimental methods The culture supernatant freeze-dried powder or bacterial freeze-dried powder of Lactobacillus crispatus SX-E770 obtained in this example was fully dissolved in sterile water to obtain a sample to be tested with a concentration of 10 mg / mL. 500 µL of the sample to be tested was mixed with 500 µL PBS (pH 7.4) and 500 µL 0.5 mg / mL α-amylase solution, and incubated at 37°C for 10 minutes; then 500 µL 1wt% starch solution was added, mixed and incubated at 37°C for 3 minutes; then 1 mL DNS solution (3,5-dinitrosalicylic acid) was added, mixed and incubated in a boiling water bath for 5 minutes; then 5 mL distilled water was added, mixed and cooled to room temperature, and then 100 μL was taken from the obtained reaction solution and added to a 96-well plate, and the absorbance was measured at 540 nm using an ELISA reader.

[0057] (2) Result determination The α-amylase inhibition rate of the sample to be tested is calculated based on the measured absorbance. An α-amylase inhibition rate of more than 50% is considered to be good in hypoglycemic ability. The calculation formula for the α-amylase inhibition rate is: α-amylase inhibition rate (%) = [1−(A sample −A blank ) / A control ]×100%; among which A sample It refers to the absorbance of the experimental sample (i.e. the reaction solution containing the sample to be tested and α-amylase), A blank It refers to the absorbance of the blank group (i.e. the reaction solution does not contain α-amylase but contains the sample to be tested); A control It refers to the absorbance of the control sample (i.e. the reaction solution contains α-amylase but not the sample to be tested).

[0058] (3) Experimental results like Figure 4 As shown, compared with the blank group (PBS group), the α-amylase activity inhibition rates of the freeze-dried powder of Lactobacillus crispatus SX-E770 and the freeze-dried powder of the culture supernatant were 61.43% and -8.09%, respectively, among which the α-amylase activity inhibition rate of the freeze-dried powder of the bacteria was higher than 50%, indicating that the freeze-dried powder of Lactobacillus crispatus SX-E770 had a good blood sugar lowering ability.

[0059] 3. Antioxidant Capacity (1) Total antioxidant capacity test - equivalent to Fe 2+ Antioxidant capacity concentration Under acidic conditions, antioxidants can reduce Fe 3+ -TPTZ produces blue Fe 2+ -TPTZ, Fe can be calculated based on the absorbance reading at 593 nm 2+ The antioxidant concentration was regarded as the total antioxidant capacity of the sample.

[0060] 1) Experimental methods The total antioxidant capacity (T-AOC) assay kit (FRAP method) (Legen Bio, TO1005-100T) was used to detect the antioxidant properties of Lactobacillus crispatus SX-E770 obtained in this example. The specific method is as follows: First, prepare a FRAP working solution with a volume ratio of FRAP Assay Buffer: TPTZ solution: ferric chloride solution = 10:1:1. 2+ The standard solution is specifically prepared by diluting the ferrous sulfate standard solution (10 mM) with distilled water to a concentration of 0.05, 0.1, 0.3, 0.5, 0.7, 0.9, 1.2 or 1.5 mM.

[0061] Add 30 μL of distilled water to 264 μL of FRAP working solution as a blank well; add 30 μL of 8 different concentrations of FeSO4 standard solutions to 264 μL of FRAP working solution as standard wells; fully dissolve the culture supernatant freeze-dried powder or bacterial freeze-dried powder of Lactobacillus crispatus SX-E770 obtained in this example with sterile water to obtain a sample to be tested with a concentration of 10 mg / mL, and add 30 μL of the sample to be tested to 264 μL of FRAP working solution as a measurement well.

[0062] After the solutions in each well were thoroughly mixed, 100 μL of the solution was pipetted into each 96-well plate, which was then placed in a 37°C water bath for 30 min, and the OD value of each well at a wavelength of 593 nm was read using an enzyme reader.

[0063] 2) Result determination After subtracting the OD value of the blank well from each well, the OD calibration value of each well is obtained. The OD calibration value of the standard well is used as the horizontal axis, and the concentration of the standard substance corresponding to the OD calibration value of the standard well is used as the vertical axis. Use the graphing software (or EXCEL table) to construct the standard curve and the corresponding linear regression equation. Substitute the OD calibration value of the measurement well into the linear regression equation to obtain the Fe concentration corresponding to the sample concentration of 10 mg / mL. 2+ Antioxidant capacity of Fe 2+ Concentrations greater than 1 nM are considered to have good overall antioxidant capacity.

[0064] 3) Experimental results The linear regression equation constructed in this embodiment is y=1.5927x+0.2777 (R 2 =0.9759). After substituting into the calculation, Figure 5 As shown in the figure, compared with the blank group, the Fe 2+ The concentrations were 0.712 nM and 1.183 nM, respectively. The Fe 2+The concentration was higher than 1 nM, indicating that the culture supernatant of Lactobacillus crispatus SX-E770 had good antioxidant capacity.

[0065] (2) DPPH (1,1-Diphenyl-2-picrylhydrazyl radical) free radical scavenging rate experiment 1) Experimental methods The DPPH free radical scavenging ability kit (Nanjing Jiancheng Biology, A153-1-1) was used to detect the DPPH free radical scavenging ability of Lactobacillus crispatus SX-E770 obtained in this example, and the specific method is as follows: Add 400 μL of 80 v / v% methanol solution and 600 μL of working solution to 2 mL sterile EP tube 1 as a blank tube; fully dissolve the culture supernatant freeze-dried powder or bacterial freeze-dried powder of Lactobacillus crispatus SX-E770 obtained in this example with sterile water to obtain a sample to be tested with a concentration of 10 mg / mL, add 400 μL of the sample to be tested and 600 μL of 80 v / v% methanol solution to 2 mL sterile EP tube 2 as a control tube for the sample group; add 400 μL of the sample to be tested and 600 μL of working solution to 2 mL sterile EP tube 3 as a measuring tube for the sample group; add 400 μL of 1×PBS and 600 μL of working solution to 2 mL sterile EP tube 4 as a measuring tube for the blank group; add 400 μL of 1×PBS and 600 μL of 80 v / v% methanol solution to 2 mL sterile EP tube 5 as a control tube for the blank group.

[0066] Mix the solutions in the above sterile EP tubes 1 to 5, keep them at room temperature 25°C in the dark for 30 min, centrifuge them at 4000 rpm for 5 min, pipette 800 μL of the supernatant after centrifugation into a cuvette, adjust to zero with 80% methanol, and read the OD value at a wavelength of 517 nm using a visible light spectrophotometer.

[0067] 2) Result determination The DPPH free radical scavenging rate was calculated according to the following formula: DPPH free radical scavenging rate (%) = (1-(A 测定 -A 对照 )÷A 空白 )×100%; where A 对照 , A 测定 and A 空白 are the OD values ​​of the control tube, test tube and blank tube in this experiment. 517 The DPPH free radical scavenging rate was above 50%, indicating that the strain had good antioxidant capacity.

[0068] 3) Experimental results like Figure 6As shown, compared with the blank group, the DPPH free radical scavenging rates of the freeze-dried powder of Lactobacillus crispatus SX-E770 and the freeze-dried powder of the culture supernatant were 36.25% and 78.02%, respectively, indicating that the culture supernatant had good antioxidant capacity.

[0069] (3) Hydroxyl free radical scavenging rate experiment 1) Experimental methods Using hydroxyl radicals (OH - ) assay kit (Nanjing Jiancheng Biology, A018-1-1) was used to detect the hydroxyl radical scavenging ability of Lactobacillus crispatus SX-E770. The specific method is as follows: Add 400 μL of distilled water to a 5mL sterile centrifuge tube 1 as a blank well; add 200 μL of distilled water and 200 μL of 0.03% H2O2 standard application solution to a 5mL sterile centrifuge tube 2 as a standard well; add 200 μL of distilled water and 200 μL of substrate application solution to a 5mL sterile centrifuge tube 3 as a control well; fully dissolve the culture supernatant freeze-dried powder or bacterial freeze-dried powder of Lactobacillus crispatus SX-E770 obtained in this example with sterile water to obtain a sample to be tested with a concentration of 10 mg / mL, and add 200 μL of substrate application solution and 200 μL of the sample to be tested to a 5mL sterile centrifuge tube 4 as a sample well.

[0070] Add 400 μL of reagent three application solution to each of the above sterile centrifuge tubes 1 to 4, mix quickly, start timing at the same time, react at 37°C for 1 minute, then immediately add 2 mL of color developer to terminate the reaction, mix well, let stand at room temperature for 20 minutes, then pipette 100 μL of the solution into a 96-well plate, and read the OD value of each well at a wavelength of 550 nm using an enzyme reader.

[0071] 2) Result determination The hydroxyl radical scavenging rate (%) was calculated according to the following formula: (A' 对照 -A' 测定 ) / (A' 标准 -A' 空白 )×100%; where A' 对照 , A' 测定 , A' 标准 and A' 空白 are the OD values ​​of the control well, sample well, standard well, and blank well in this experiment. 550 A hydroxyl radical scavenging rate of more than 50% is considered to be a good antioxidant capacity of the strain.

[0072] 3) Experimental results like Figure 7 As shown, compared with the blank group, the hydroxyl radical scavenging rates of the freeze-dried powder of Lactobacillus crispatus SX-E770 and the freeze-dried powder of the culture supernatant were 56.65% and 74.04%, respectively, both higher than 50%, indicating that they have good antioxidant capacity.

[0073] 4. Adhesion ability Adhesion ability is the first step for probiotics to colonize in the intestine. In probiotic research, adhesion ability is one of the important indicators for evaluating the potential of probiotics. Probiotics can competitively inhibit the adhesion of pathogens by adhering to intestinal epithelial cells, thereby exerting a probiotic effect. The Caco-2 cell model is a human cloned colon adenocarcinoma cell, which is similar in structure and function to differentiated small intestinal epithelial cells; the intestinal mucosal environment can be simulated by the Caco-2 cell monolayer to test the ability of probiotics to adhere to intestinal epithelial cells. This example uses a strain adhesion Caco-2 cell experiment to determine the adhesion ability of Lactobacillus crispatus SX-E770.

[0074] (1) Experimental methods On the first day of the experiment, Caco-2 cells were cultured at 4×10 4 The cells were plated at a density of 100 μg / well in a 24-well plate and cultured in DMEM complete medium containing 10 v / v% fetal bovine serum (FBS). The next day, the culture medium was replaced with DMEM medium without double antibodies and cultured until polarized state. Then, the cells were incubated with Lactobacillus crispatus SX-E770 suspension (0.5 mL, 10 8 CFU / mL) for 2 h. Gram staining was then used to observe whether the bacteria adhered to the Caco-2 cells.

[0075] At the same time, the bacterial solution before and after co-culture was diluted in a gradient manner and then plated on a bacterial plate to calculate the bacterial adhesion rate. The adhesion rate calculation method is as follows: Adhesion rate (%) = (bacterial concentration after adhesion / bacterial concentration before adhesion) × 100%.

[0076] (2) Experimental results According to statistics, the bacterial concentration of Lactobacillus crispatus SX-E770 before adhering to Caco-2 cells was 9.23×10 9 CFU / mL, the bacterial concentration after adhering to Caco-2 cells was 3.23×10 9 CFU / mL, so the adhesion rate of Lactobacillus crispatus SX-E770 to Caco-2 cells was 34.99%.

[0077] Gram staining results of Lactobacillus crispatus SX-E770 adhering to Caco-2 cells Figure 8 As shown, Lactobacillus crispatus SX-E770 can be seen on the edge and surface of Caco-2 cells, indicating that Lactobacillus crispatus SX-E770 has the ability to adhere to intestinal epithelial cells, which is consistent with the adhesion rate results.

[0078] The above results show that the Lactobacillus crispatus SX-E770 of the present invention has excellent blood sugar lowering, antioxidant and adhesion abilities.

[0079] Example 3 In vivo functional assay of Lactobacillus crispatus SX-E770 1. Preparation of culture supernatant freeze-dried powder and bacterial solution of Lactobacillus crispatus SX-E770 The Lactobacillus crispatus SX-E770 obtained in Example 1 was resuspended in MRS medium and cultured anaerobically at 37°C until the OD value of the bacterial solution was measured by an ultraviolet spectrophotometer. 600 The value was 1.6, and then the bacterial liquid was inoculated into the MRS liquid culture medium sterilized by high pressure at a ratio of 2v / v%, and anaerobically cultured at 37℃ for 24h; the cultured bacterial liquid was collected and centrifuged at 8000 r / min for 8 min, and the supernatant after centrifugation was collected and filtered through a 0.22 µm microporous filter membrane to obtain the culture supernatant of Lactobacillus crispatus SX-E770, and then the culture supernatant of Lactobacillus crispatus SX-E770 was placed in a freeze dryer and freeze-dried for 72h to obtain the culture supernatant freeze-dried powder of Lactobacillus crispatus SX-E770, and then dissolved in physiological saline at a final concentration of 0.05mg / mL to obtain the culture supernatant freeze-dried powder solution; the bacterial precipitate after centrifugation was collected and the final concentration was 1×10 6 CFU / mL was resuspended in physiological saline to obtain a bacterial suspension.

[0080] 2. Construction, treatment and detection of zebrafish diabetes model (1) Construction of zebrafish diabetes model Wild-type AB zebrafish (5 dpf) were placed in a six-well plate and randomly divided into 5 groups, namely normal group, model group, positive group, culture supernatant freeze-dried powder group and bacterial suspension group. Each group was set up with 6 replicate wells, and 10 fish were placed in each well.

[0081] E3 culture water was prepared according to the formula shown in Table 1. 50 mL of E3 culture water was added to the normal group, and 10 mg of ordinary feed (purchased from Shanghai Haisheng Biological Laboratory Equipment Co., Ltd.) was fed; 50 mL of glucose with a final concentration of 1wt% was added to the model group, positive group, culture supernatant freeze-dried powder group and bacterial suspension group, and 10 mg of high-fat feed (purchased from Shanghai Feixi Biotechnology Co., Ltd.) was fed. Incubate for 7 days.

[0082] Table 1E3 Aquaculture water formula (1L)

[0083] (2) Treatment of zebrafish diabetes model After the incubation, 50 mL of E3 culture water was added to the normal group and the model group, and 10 mg of ordinary feed was fed; 50 mL of E3 culture water containing 20 μg / mL metformin hydrochloride was added to the positive group, and 10 mg of high-fat feed was fed; 50 mL of the culture supernatant freeze-dried powder solution prepared in this example was added to the culture supernatant freeze-dried powder group, and 10 mg of high-fat feed was fed; 50 mL of the bacterial suspension prepared in this example was added to the bacterial suspension group, and 10 mg of high-fat feed was fed. Incubation was continued for 5 days.

[0084] (3) Determination of glucose content in zebrafish After the treatment, the zebrafish in each group were washed twice with pure water, collected into 1.5 mL centrifuge tubes, and the excess liquid was discarded. 250 μL PBS was added for homogenization and disruption until there were no obvious tissue fragments. The tubes were centrifuged at 12000×g and 4°C for 10 min. The supernatant after centrifugation was taken and the glucose content was detected using a glucose assay kit (Cat. No. S03039, manufacturer Redu).

[0085] (4) Data analysis All data were statistically analyzed using GraphPad Prism 8, and experimental data were expressed as mean ± SEM. The significant differences compared with the normal group were expressed as #P<0.05, ##P<0.01, ###P<0.001 by t-test analysis; the significant differences compared with the model group were expressed as &P<0.05, &&P<0.01, &&&P<0.001 by t-test analysis; the significant differences compared with the model group were expressed as *P<0.05, **P<0.01, ***P<0.001 by one-way ANOVA analysis.

[0086] like Figure 9 As shown in the figure, the glucose concentration in zebrafish of the model group was significantly higher than that in the normal group, indicating that the zebrafish diabetes model was successfully established; the glucose concentration in zebrafish of the positive group was significantly lower than that in the model group, indicating that metformin hydrochloride effectively lowers blood sugar; the glucose concentration in zebrafish of the culture supernatant freeze-dried powder group and the bacterial suspension group was significantly lower than that in the model group, indicating that the culture supernatant freeze-dried powder of Lactobacillus crispatus SX-E770 at a concentration of 0.05 mg / mL and the bacterial suspension at a concentration of 1×10 6 CFU / mL concentration, it can reduce the glucose content in zebrafish and has excellent blood sugar lowering effect.

[0087] Example 4 Safety evaluation of Lactobacillus crispatus SX-E770 1. Experimental methods Twelve male mice (strain ICR) were randomly divided into a saline group and a SX-E770 group, with 6 mice in each group. The mice in the saline group were gavaged with 0.1 mL / 10 g of saline every day, and the mice in the SX-E770 group were gavaged with 0.1 mL / 10 g of Lactobacillus crispatus SX-E770 bacterial solution (1×10 8 CFU / mL), intragastrically for 3 consecutive days. After 3 weeks of conventional feeding, the visceral organs (heart, liver, spleen, lung and kidney) of mice in each group were collected to calculate the organ index. The calculation formula was: organ index = weight of visceral organ / mouse body weight.

[0088] Twelve female mice were treated and their organ indexes were calculated using the above method.

[0089] 2. Experimental results Table 2 Statistical results of mouse organ index

[0090] As shown in Table 2, there was no significant difference in organ index between the saline group and the SX-E770 group for both male and female mice, indicating that the Lactobacillus crispatus SX-E770 of the present invention has substantially no negative impact on organs and has good safety.

[0091] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A strain of Lactobacillus crispatus ( Lactobacillus crispatus )SX-E770, characterized by, The preservation number of the Lactobacillus crispatus SX-E770 is CCTCC NO: M 2025033, and it was deposited in the China Center for Type Culture Collection on January 6, 2025.

2. The Lactobacillus crispatus according to claim 1 ( Lactobacillus crispatus )Application of SX-E770 in the preparation of hypoglycemic products.

3. The Lactobacillus crispatus according to claim 1 ( Lactobacillus crispatus )Application of SX-E770 in the preparation of antioxidant products.

4. The Lactobacillus crispatus according to claim 1 ( Lactobacillus crispatus )Application of SX-E770 in the preparation of hypoglycemic and antioxidant products.

5. The Lactobacillus crispatus according to claim 1 ( Lactobacillus crispatus )Application of SX-E770 in the preparation of products for inhibiting amylase activity.

6. The Lactobacillus crispatus according to claim 1 ( Lactobacillus crispatus )Application of SX-E770 in the preparation of free radical scavenging products.

7. The Lactobacillus crispatus according to claim 1 ( Lactobacillus crispatus ) Use of SX-E770 in the preparation of products for preventing and / or treating diabetes.

8. The use according to any one of claims 2 to 7, characterized in that: The Lactobacillus crispatus ( Lactobacillus crispatus ) The 16S rDNA sequence of SX-E770 is shown in SEQ ID NO:

3.

9. The use according to any one of claims 2 to 7, characterized in that: The Lactobacillus crispatus ( Lactobacillus crispatus )SX-E770 includes the Lactobacillus crispatus ( Lactobacillus crispatus ) at least one of live bacteria, culture solution or culture supernatant of SX-E770.

10. A microbial agent, characterized in that: Containing the Lactobacillus crispatus according to claim 1 ( Lactobacillus crispatus )SX-E770.

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