Lactobacillus paracasei SX-M1559 and application thereof in preparation of drugs for reducing blood sugar and lipid and resisting inflammation

Abstract

The invention discloses a lactobacillus paracasei SX-M1559 strain and application thereof in preparation of drugs for reducing blood sugar and lipid and resisting inflammation, and belongs to the technical field of microorganisms. The invention discloses a lactobacillus paracasei SX-M1559, and the preservation number of the lactobacillus paracasei SX-M1559 is CCTCC (China Center For Type Culture Collection) NO: M 2025030. According to the lactobacillus paracasei SX-M1559 and the application thereof in preparation of the medicine for reducing blood sugar, blood fat and inflammation, the SX-M1559 is separated from fresh feces of long-life old people, teai county, Zhanjiang city, Guangdong province, the glucose content in a zebra fish hyperglycemia model body and the fat content in a zebra fish hyperlipidemia model body can be remarkably reduced, and the effect of reducing blood sugar, blood fat and inflammation is achieved. The invention provides a theoretical reference and a guidance basis for developing a probiotic preparation for preventing or treating hyperlipidemia, diabetes mellitus and inflammatory bowel disease by utilizing the new strain SX-M1559 of the casei paracasei.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and more specifically to a strain of Lactobacillus paracasei SX-M1559 and application thereof in preparing blood sugar-lowering, blood lipid-lowering and anti-inflammatory drugs. Background Art

[0002] Probiotics are a type of active microorganisms that are beneficial to the human body. They mainly live in the human intestines and reproductive tracts, and can produce definite health effects, thereby improving the host's microecological balance and playing a beneficial role. There are many types of probiotics, including lactobacilli, bifidobacteria, yeasts, Bacillus subtilis, etc. A large number of studies have shown that probiotics play an important role in improving immunity, maintaining intestinal microecological balance, lowering cholesterol, allergy relief, weight loss, and lowering blood sugar, and have been widely used in the development of functional foods or dietary supplements. Lactobacillus paracasei is a facultative anaerobic, non-motile, non-spore-forming bacillus or long bacillus, Gram-positive, catalase-negative. It is widely present in fermented dairy products, yogurt and other foods, and can also be isolated from the gastrointestinal tract of healthy humans. As a probiotic, Lactobacillus paracasei can regulate the proportion of intestinal flora, reduce the number of harmful bacteria, and improve the intestinal environment.

[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. The shortcomings of existing technologies for diabetes treatment are mainly reflected in: it is difficult to achieve high-quality blood sugar standards, and some 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] Hyperlipidemia, also known as high blood lipids or dyslipidemia, is a disease of systemic lipid metabolism disorder; it usually refers to elevated levels of cholesterol and / or triglycerides in the serum, which may be accompanied by low levels of high-density lipoprotein (HDL). Its typical symptoms include dizziness, fatigue, limb numbness, and possible xanthomas. In severe cases, it may lead to serious complications such as coronary heart disease and cerebral stroke. Treatment methods mainly include drug therapy (such as statins and fibrates) and non-drug therapy (such as improving diet, losing weight, and increasing exercise). Existing treatment technologies still have certain shortcomings. For example, drug therapy may bring side effects, non-drug treatment requires long-term persistence of patients, and has limited effect on some patients.

[0005] Reactive oxygen species (ROS) play an important physiological role in cells and are closely related to the occurrence and development of many diseases. Under normal physiological conditions, ROS participate in regulating processes such as cell growth, differentiation and signal transduction. For example, in the immune system, ROS can promote phagocytosis and inflammatory response of leukocytes, helping the body to resist the invasion of pathogens. However, when the generation and clearance of ROS are unbalanced, leading to oxidative stress, excessive ROS can damage the proteins, lipids and DNA of cells, thereby causing cell dysfunction and death. This oxidative damage is the basis for the occurrence of many diseases, including inflammatory bowel disease, cancer, cardiovascular disease, diabetes and neurodegenerative diseases. Enteritis is an inflammatory lesion of the small intestine or colon caused by infection with bacteria, viruses, fungi or parasites. The main clinical symptoms are abdominal pain, watery stools and mucus, pus and blood in stools. Some patients may have fever or a feeling of tenesmus. According to the length of the course of the disease, enteritis can be divided into acute enteritis and chronic enteritis, and chronic enteritis is more common in clinical practice. The course of chronic enteritis is generally more than two months. Common types include chronic bacterial dysentery, chronic amoebic dysentery, schistosomiasis, nonspecific ulcerative colitis and localized enteritis.

[0006] Traditional treatments for enteritis can be divided into modern medical treatments and traditional Chinese medicine treatments. Probiotics, as an endogenous and immune defense barrier in the intestine, can antagonize pathogenic bacteria. They are safe, controllable, effective, and have few side effects. They are an ideal method for treating enteritis.

[0007] Therefore, providing a strain of Lactobacillus paracasei SX-M1559 and its use in preparing blood sugar-lowering, lipid-lowering and anti-inflammatory drugs and in preparing drugs for preventing or treating intestinal inflammation are problems that need to be solved urgently by those skilled in the art. Summary of the invention

[0008] In view of this, the present invention provides a strain of Lactobacillus paracasei SX-M1559 and application thereof in the preparation of blood sugar-lowering, blood lipid-lowering and anti-inflammatory drugs.

[0009] In order to achieve the above object, the present invention adopts the following technical solution: A strain of Lactobacillus paracasei SX-M1559, with a deposit number of CCTCC NO: M 2025030, has been deposited in China Center for Type Culture Collection (hereinafter referred to as CCTCC), address: Wuhan University, Wuhan, China, deposit date is January 6, 2025, and is classified as Lactaseibacillus paracasei SX-M1559. The detailed scheme is as follows: (1) The strain of Lactobacillus paracasei SX-M1559 was isolated from the feces of a centenarian in Suixi County, Zhanjiang City, Guangdong Province.

[0010] (2) The Lactobacillus paracasei SX-M1559 is inactivated culture supernatant freeze-dried powder, bacterial freeze-dried powder and bacterial suspension.

[0011] (3) The Lactobacillus paracasei SX-M1559 has good blood sugar lowering ability in in vitro experiments.

[0012] (4) The Lactobacillus paracasei SX-M1559 has a good cholesterol-lowering ability in in vitro experiments.

[0013] (5) The Lactobacillus paracasei SX-M1559 has a good triglyceride-lowering ability in in vitro experiments.

[0014] (6) Application of the Lactobacillus paracasei SX-M1559 in DPPH clearance rate.

[0015] (7) Application of the Lactobacillus paracasei SX-M1559 in scavenging hydroxyl radicals.

[0016] (8) The freeze-dried powder and bacterial suspension of the novel Lactobacillus paracasei SX-M1559 of the present invention can significantly reduce the glucose content in zebrafish in vivo, and has a potential blood sugar-lowering effect.

[0017] (9) The lyophilized powder and bacterial suspension of the novel Lactobacillus paracasei SX-M1559 of the present invention can significantly reduce the fat content in zebrafish with high fat content in vivo, that is, it has a potential lipid-lowering effect.

[0018] (10) The use of the Lactobacillus paracasei SX-M1559 in alleviating DSS-induced intestinal inflammation in zebrafish.

[0019] Furthermore, a probiotic preparation contains the Lactobacillus paracasei SX-M1559.

[0020] Furthermore, the Lactobacillus paracasei SX-M1559 or the probiotic preparation is used in the preparation of a blood sugar-lowering drug.

[0021] Furthermore, the Lactobacillus paracasei SX-M1559 or the probiotic preparation is used in the preparation of a drug for inhibiting α-glucosidase.

[0022] Furthermore, the Lactobacillus paracasei SX-M1559 or the probiotic preparation is used in the preparation of a drug for inhibiting α-amylase.

[0023] Furthermore, the Lactobacillus paracasei SX-M1559 or the probiotic preparation is used in the preparation of a lipid-lowering drug.

[0024] Furthermore, the Lactobacillus paracasei SX-M1559 or the probiotic preparation is used in the preparation of a cholesterol-degrading drug.

[0025] Furthermore, the Lactobacillus paracasei SX-M1559 or the probiotic preparation is used in the preparation of a triglyceride-lowering drug.

[0026] Furthermore, the Lactobacillus paracasei SX-M1559 or the probiotic preparation is used in the preparation of anti-inflammatory drugs.

[0027] Furthermore, the Lactobacillus paracasei SX-M1559 or the probiotic preparation is used in the preparation of a drug for treating inflammatory bowel disease.

[0028] It can be known from the above technical scheme that, compared with the prior art, the present invention discloses a strain of Lactobacillus paracasei SX-M1559 and its application in the preparation of hypoglycemic and lipid-lowering and anti-inflammatory drugs. Lactobacillus paracasei SX-M1559 is isolated from fresh feces of centenarians in Suixi County, Zhanjiang City, Guangdong Province, and can significantly reduce the glucose content in a zebrafish hyperglycemia model and the fat content in a zebrafish hyperlipidemia model, and alleviate DSS-induced intestinal inflammation in zebrafish. This provides a theoretical reference and guiding basis for developing a probiotic preparation for preventing or treating hyperlipidemia, diabetes and inflammatory bowel disease using a new strain of Lactobacillus paracasei SX-M1559. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0030] Figure 1 This is a colony morphology diagram formed by Lactobacillus paracasei SX-M1559 of the present invention.

[0031] Figure 2 The present invention is a microscopic morphological observation of Lactobacillus paracasei SX-M1559 after Gram staining.

[0032] Figure 3 is the α-glucosidase inhibition rate of the present invention; culture supernatant: culture supernatant freeze-dried powder; bacteria: bacteria freeze-dried powder.

[0033] Figure 4 is the α-amylase inhibition rate of the present invention; culture supernatant: culture supernatant freeze-dried powder; bacteria: bacteria freeze-dried powder.

[0034] Figure 5 It is the cholesterol degradation rate of the present invention.

[0035] Figure 6 is the triglyceride degradation rate of the present invention.

[0036] Figure 7 It is the result diagram of DPPH free radical scavenging rate of the present invention.

[0037] Figure 8 This is a graph showing the hydroxyl radical scavenging rate results of the present invention.

[0038] Fig. 9 The figure is the Gram staining result of Lactobacillus paracasei SX-M1559 adhering to Caco2 cells of the present invention.

[0039] Fig.10 is the glucose concentration in the zebrafish of the present invention.

[0040] Fig.11 This is a direct view of fat staining in zebrafish of the present invention.

[0041] Fig.12 is the fat concentration in the zebrafish of the present invention.

[0042] Fig.13 These are intuitive and statistical graphs of the effects of freeze-dried powder of Lactobacillus paracasei SX-M1559 on the aggregation of neutrophils in the zebrafish intestine.

[0043] Fig.14 These are intuitive and statistical graphs of the effects of the Lactobacillus paracasei SX-M1559 bacterial suspension group on the aggregation of neutrophils in the zebrafish intestine. DETAILED DESCRIPTION

[0044] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0045] Example 1 Isolation, identification and preservation of strains (1) Isolation of strains: Fresh stool samples from centenarians in Suixi County, Zhanjiang City, Guangdong Province were diluted in gradients and inoculated into MRS solid medium. They were cultured anaerobically at 37°C for 48-72 hours. Single colonies on the plates were streaked for three generations to obtain purified strains. Single clones obtained by streaking for three generations were inoculated into MRS liquid medium, cultured anaerobically at 37°C for 12-16 hours, and 30% glycerol (containing 0.5% cysteine) was added. The bacterial liquid and glycerol were mixed in equal volumes and stored in a -80°C refrigerator.

[0046] (2) Strain plate streaking: In a biosafety cabinet / anaerobic workstation, mix the bacterial solution, dip a disposable inoculation loop into the bottom of the bacterial solution and scrape it repeatedly 3-5 times to ensure that the bacterial solution is smeared on the inoculation loop. Use the inoculation loop to streak three areas on the culture medium plate and record the label. The colony morphology formed by Lactobacillus paracasei SX-M1559 is shown in Figure 1 .

[0047] (3) Morphological identification of strains: After Gram staining of the selected strains, oil immersion microscopy was used to examine the strains. Gram-positive strains were purple and Gram-negative strains were red. The strain was a Gram-positive bacillus, named Lactobacillus paracasei SX-M1559. The microscopic morphology after Gram staining was observed as follows: Figure 2 .

[0048] (4) Molecular biological identification of the strain: The 16S rDNA full-length of the isolated strain was amplified by PCR, wherein the 16S universal primers: 1492R (5'-GGTTACCTTGTTACGACTT-3'; SEQ ID NO.1) and 27F (5'-AGAGTTTGATCCTGGCTCAG-3'; SEQ ID NO.2) were used to perform electrophoresis on the amplified product. After obtaining the corresponding band, the amplified product was sequenced, and the sequencing result was compared with the standard bacterial species sequence at NCBI for identification. The identification result was Lactobacillus paracasei. Its 16S rDNA sequence is shown in SEQ ID NO.3.

[0049]

[0050] (5) Strain preservation: Lactocasebacillus paracasei SX-M1559, with the deposit number of CCTCC NO: M 2025030, has been deposited in the China Center for Type Culture Collection (CCTCC), address: Wuhan University, Wuhan, China. The deposit date is January 6, 2025, and the classification name is Lactaseibacillusparacasei SX-M1559.

[0051] Example 2 Preparation of bacterial suspension, culture supernatant and corresponding lyophilized powder OD 600 The bacterial suspension of Lactobacillus paracasei SX-M1559 with a value of 1.6 was inoculated into the high-pressure sterilized MRS culture medium at a ratio of 2%, and cultured at 37°C for 24 h; centrifuged at 8 000 r / min for 8 min, the supernatant was collected, and passed through a 0.22 µm microporous filter membrane to obtain the culture supernatant, which was placed in a freeze dryer for freeze-drying for 72 h to obtain the culture supernatant freeze-dried powder; the collected cells were resuspended in an equal volume of PBS to obtain a bacterial suspension, which was placed in a freeze dryer for freeze-drying for 72 h to obtain the bacterial freeze-dried powder.

[0052] Example 3 In vitro functional assay - hypoglycemic ability experiment (1) α-glucosidase inhibitory activity assay 1) Mix 25 µL of 10 mg / mL sample (lyophilized powder of bacterial cells or culture supernatant) with 50 µL of glucosidase solution (0.2 U / mL), 150 µL of PBS (0.1 mol / L, pH 7.4) and 75 µL of p-nitrophenyl-α-D-glucoside (pNPG) solution (dissolved in PBS, 20 mmol / L). After incubation at 37°C for 10 minutes, the reaction was terminated by adding 1 mL of 0.1 mol / L Na2CO3 solution and the absorbance was measured at a wavelength of 405 nm.

[0053] 2) Result determination: The α-glucosidase inhibition rate of the cultured strain was calculated using the absorbance value; an α-glucosidase inhibition rate of more than 50% was considered to have good glucose-lowering ability.

[0054] 3) Calculation formula: .

[0055] A sample is the absorbance of the experimental sample (including sample and α-glucosidase), A bank represents the absorbance of blank (containing sample but no α-glucosidase), A controlis the absorbance of the control (containing α-glucosidase and without sample), and A0 represents the absorbance without α-glucosidase and sample.

[0056] Depend on Figure 3 It can be seen that compared with the blank group (PBS group), the α-glucosidase activity inhibition rates of Lactobacillus paracasei SX-M1559 bacterial freeze-dried powder and culture supernatant freeze-dried powder were 59% and 58%, respectively, both higher than 50%, indicating that they have good blood sugar lowering ability.

[0057] (2) Determination of α-amylase inhibitory activity 1) Experimental method: 500µL of 10mg / mL sample (lyophilized powder of bacterial cells or lyophilized powder of culture supernatant) was mixed with PBS (500µL, pH 7.4) and α-amylase solution (500µL, 0.5 mg / mL), incubated at 37°C for 10 minutes, then 500µL of 1% starch solution was added and incubated at 37°C for 3 minutes. Subsequently, 1 mL of DNS solution (3,5-dinitrosalicylic acid) was added and the solution was incubated in a boiling water bath for 5 minutes. After that, 5 mL of distilled water was added and cooled to room temperature, 100μL of the solution was added to a 96-well plate, and the absorbance was measured at 540 nm using a microplate reader.

[0058] 2) Result determination: The α-amylase inhibition rate of the cultured strain was calculated using the absorbance value; an α-amylase inhibition rate of more than 50% was considered to have good glucose-lowering ability.

[0059] 3) Calculation formula: .

[0060] Among them A sample It refers to the absorbance of the experimental sample (including sample and α-amylase), A blank A refers to the absorbance of the blank sample (the sample without α-amylase); control It refers to the absorbance of the control sample (containing α-amylase but not containing sample).

[0061] Depend on Figure 4 It can be seen that compared with the blank group (PBS group), the α-amylase activity inhibition rates of Lactobacillus paracasei SX-M1559 bacterial freeze-dried powder and culture supernatant freeze-dried powder were 51% and 69%, respectively, both higher than 50%, indicating that they have good blood sugar lowering ability.

[0062] Example 4 In vitro functional experiment - lipid-lowering ability experiment (1) Cholesterol-lowering ability test 1) Experimental method: 10 mg of lyophilized powder of Lactobacillus paracasei SX-M1559 was inoculated into 1 mL of MRS liquid medium containing 1 g / L cholesterol and cultured at 37°C for 48 h; the total cholesterol (T-CHO) test kit instructions (Nanjing Jiancheng Biology, A111-1-1) were used for determination: first, the culture solution was centrifuged at 10000 rpm / min for 10 minutes, and the bacterial culture supernatant was taken. 2.5 μL of distilled water was added to 250 μL of working solution as a blank well; 2.5 μL of calibrator (cholesterol, 6.55 mmol / L) was added to 250 μL of working solution as a calibration well; 2.5 μL of bacterial culture supernatant was added to 250 μL of working solution as a sample well; after thorough mixing, 100 μL of the solution was aspirated and added to a 96-well plate, and then incubated at 37°C for 10 minutes, and the OD value of each well at a wavelength of 500 nm was read by an ELISA reader.

[0063] 2) Result determination: Calculate the cholesterol degradation rate of the cultured strain after 48 hours compared with the initial time; a cholesterol degradation rate of more than 50% is considered to have good cholesterol-lowering ability.

[0064] Calculation formula: Cholesterol content C (mmol / L) = [(A 样本 -A 空白 ) / (A 标准 -A 空白 )]×C 标准 Cholesterol degradation rate % = (C0-C 48 ) / C0×100% A 样本 , A 标准 , A 空白 OD of sample well, calibration well, and blank well respectively 500 Value; C 标准 is the concentration of the calibrator, C0 is the cholesterol content in the culture medium before culture; C 48 It is the cholesterol content in the culture medium after the experimental strain was cultured for 48 hours.

[0065] Depend on Figure 5 It can be seen that compared with the blank group (distilled water), the cholesterol degradation rate of the supernatant of the bacterial culture of Lactobacillus paracasei SX-M1559 was 77.21%, which was higher than 50%, indicating that it had a good cholesterol-lowering ability.

[0066] (2) Triglyceride-lowering ability test 1) Experimental method: 10 mg of lyophilized powder of Lactobacillus paracasei SX-M1559 was inoculated into 1 mL of MRS liquid culture medium containing 1 g / L triglyceride and cultured at 37°C for 48 h; the determination was performed according to the instructions of the triglyceride (TG) test kit (Nanjing Jiancheng Biology, A110-1-1): First, the culture solution was centrifuged at 10000 rpm / min for 10 minutes, and the bacterial culture supernatant was taken. 2.5 μL of distilled water was added to 250 μL of working solution as a blank well; 2.5 μL of calibrator (triglyceride, 2.68 mmol / L) was added to 250 μL of working solution as a calibration well; 2.5 μL of bacterial culture supernatant was added to 250 μL of working solution as a sample well; after thorough mixing, 100 μL of the solution was aspirated and added to a 96-well plate, and then incubated at 37°C for 10 minutes, and the OD value of each well at a wavelength of 500 nm was read by an ELISA reader.

[0067] 2) Result determination: Calculate the triglyceride degradation rate of the cultured strain after 48 hours compared with the initial time; a triglyceride degradation rate of more than 50% is considered to be good in triglyceride lowering ability.

[0068] Calculation formula: Triglyceride content C (mmol / L) = [(A 样本 -A 空白 ) / (A 标准 -A 空白 )]×C 标准 Triglyceride degradation rate % = (C0-C 48 ) / C0×100% A 样本 , A 标准 , A 空白 OD of sample well, calibration well, and blank well respectively 500 Value; C 标准 is the concentration of the calibrator, C0 is the triglyceride content in the culture medium before culture; C 48 It is the triglyceride content in the culture medium after the experimental strain was cultured for 48 hours.

[0069] 3) Experimental results: Figure 6 It can be seen that compared with the blank group (distilled water group), the triglyceride degradation rate of the culture solution of Lactobacillus paracasei SX-M1559 was 74.92%, which was higher than 50%, indicating that it had a good ability to reduce triglycerides.

[0070] Example 5 In vitro functional experiment - determination of antioxidant capacity (1) DPPH (1,1-Diphenyl-2-picrylhydrazyl radical) free radical scavenging rate experiment 1) Experimental method: The DPPH radical scavenging ability kit (Nanjing Jiancheng Biological, A153-1-1) was used to detect the DPPH radical scavenging ability of the culture supernatant and bacterial suspension of Lactobacillus paracasei SX-M1559. 400 μL 80% methanol solution and 600 μL working solution were added to 2 mL sterile EP tube 1 as a blank tube, 400 μL 10 mg / mL sample solution and 600 μL methanol solution were added to 2 mL sterile EP tube 2 as a sample group control tube, 400 μL 10 mg / mL sample solution and 600 μL working solution were added to 2 mL sterile EP tube 3 as a sample group test tube, 400 μL 1× PBS and 600 μL working solution were added to 2 mL sterile EP tube 4 as a blank group test tube, and 400 μL 1× PBS and 600 μL methanol solution were added to 2 mL sterile EP tube 5 as a blank group control tube. Mix the solution in the centrifuge tube, keep it at room temperature 25℃ in the dark for 30 minutes, and then centrifuge it at 4000 rpm for 5 minutes. Pipette 800 μL of the supernatant into a cuvette, adjust the color to zero with 80% methanol, and read the OD value at a wavelength of 517 nm using a visible light spectrophotometer.

[0071] 2) Calculation formula: DPPH free radical scavenging rate (%) = (1-(A 测定 -A 对照 )÷A 空白 )×100% A 对照 , A 测定、 A 空白 are the OD values ​​of the control tube, the assay tube, and the blank tube, respectively. 517 value.

[0072] 3) Result determination: Calculate the DPPH free radical scavenging rate of the assay tube; if the DPPH free radical scavenging rate is above 50%, it is considered that the strain has good antioxidant capacity.

[0073] Experimental results: Figure 7 It can be seen that by comparing with the blank group, the DPPH radical scavenging rates of the freeze-dried powder of Lactobacillus paracasei SX-M1559 and the freeze-dried powder of the culture supernatant were 65% and 64%, respectively, both higher than 50%, indicating that they have good antioxidant capacity.

[0074] (2) Hydroxyl free radical scavenging rate experiment 1) Experimental method: Using hydroxyl radicals (OH -) assay kit (Nanjing Jiancheng Biology, A018-1-1) was used to detect the hydroxyl radical scavenging ability of the culture supernatant and bacterial suspension of Lactobacillus paracasei SX-M1559. 400 μL of distilled water was added to 5 mL sterile centrifuge tube 1 as a blank well, 200 μL of distilled water and 200 μL of 0.03% H2O2 standard application solution were added to 5 mL sterile centrifuge tube 2 as standard wells, 200 μL of distilled water and 200 μL of substrate application solution were added to 5 mL sterile centrifuge tube 3 as control wells, and 200 μL of substrate application solution and 200 μL of 10 mg / mL sample solution were added to 5 mL sterile centrifuge tube 4 as sample wells. 400 μL of reagent three application solution was added to the above 4 centrifuge tubes respectively, mixed quickly, and the timing was started at the same time. The reaction was carried out at 37°C for 1 minute, and then 2 mL of color developer was immediately added to terminate the reaction, mixed well, and placed at room temperature for 20 minutes. 100 μL of the solution was added to a 96-well plate, and the OD value of each well was read at a wavelength of 550 nm using an ELISA reader.

[0075] 2) Calculation formula: Hydroxyl free radical inhibition ability (U / mL) = (A 对照 -A 测定 ) / (A 标准 -A 空白 )×C 标准 ×1 / V 样 × NA 对照 , A 测定 , A 标准、 A 空白 They are the OD of the control well, sample well, standard well, and blank well. 550 Value; C 标准 is the standard concentration, 8.824mmol / L; V 样 is the sampling volume, 0.2mL; N is the dilution multiple of the sample before testing.

[0076] 3) Result determination: Calculate the hydroxyl radical scavenging rate of the sample well; if the hydroxyl radical scavenging rate is above 50%, it is considered that the strain has good antioxidant capacity.

[0077] Experimental results: Figure 8 It can be seen that by comparing with the blank group, the hydroxyl radical scavenging rates of the freeze-dried powder of Lactobacillus paracasei SX-M1559 and the freeze-dried powder of the culture supernatant were 69% and 68%, respectively, both higher than 50%, indicating that they have good antioxidant capacity.

[0078] Example 6 Experiment on bacterial strain adhesion to Caco-2 cells 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 with a structure and function similar to differentiated small intestinal epithelial cells; the Caco-2 cell monolayer can simulate the intestinal mucosal environment and test the ability of probiotics to adhere to intestinal epithelial cells.

[0079] On the first day, Caco-2 cells were cultured at 4×10 4 The cells were plated at a density of 1000 × 100 / well in a 24-well plate and cultured in DMEM complete medium containing 10% fetal bovine serum (FBS). The next day, the medium was replaced with DMEM medium without double antibodies and cultured until polarized. Then, the cells were incubated with a suspension of Lactobacillus paracasei SX-M1559 (0.5 mL, 3.00 × 10 8 CFU / mL) for 2 h. Gram staining was then used to observe whether the bacteria adhered to the Caco-2 cells. Fig. 9 This is the Gram staining result of Lactobacillus paracasei SX-M1559 adhering to Caco-2 cells. Fig. 9 It can be seen that Lactobacillus paracasei SX-M1559 has the ability to adhere to intestinal epithelial cells, and the results are consistent with the adhesion rate.

[0080] At the same time, the bacterial solution before and after co-culture was diluted in a gradient manner, and the bacterial adhesion rate was calculated by bacterial plate coating culture. The adhesion rate calculation method is as follows: Adhesion rate (%) = (bacterial concentration after adhesion / bacterial concentration before adhesion) × 100%. The bacterial concentration of Lactobacillus paracasei SX-M1559 before adhesion to Caco-2 cells is: 3.00*10 8 CFU / mL, bacterial concentration after adhesion to Caco-2 cells: 1.57*10 8 CFU / mL, calculated by the above method, the adhesion rate of Lactobacillus paracasei SX-M1559 to Caco-2 cells was 52.30%.

[0081] Example 7 In vivo functional assay (1) Preparation of freeze-dried powder and bacterial solution OD 600The bacterial suspension of Lactobacillus paracasei SX-M1559 with a value of 1.6 was inoculated into the MRS liquid medium sterilized by high pressure sterilization at a ratio of 2%, and cultured at 37°C for 24 h; centrifuged at 8 000 r / min for 8 min, the supernatant was collected, and filtered through a 0.22 µm microporous filter to obtain the culture supernatant, which was placed in a freeze dryer and freeze-dried for 72 h to obtain the culture supernatant freeze-dried powder of the strain, which was dissolved in physiological saline and adjusted to a concentration of 0.05 mg / mL; the collected cells were resuspended in physiological saline to obtain a bacterial suspension, and the concentration was adjusted to 1×10 6 CFU / mL.

[0082] (2) Zebrafish diabetes model 1) Model construction Wild-type AB zebrafish (5 dpf) were placed in a six-well plate. The experiment set up normal group, model group, positive group (metformin hydrochloride), culture supernatant freeze-dried powder group (0.05 mg / mL), bacterial suspension group (1×10 6 CFU / mL), 6 replicate wells were set up in each group, and 10 fish were placed in each well. 50 mL of E3 culture water (composition is shown in Table 1) was added to the normal group, and 10 mg of ordinary feed (purchased from Shanghai Haisheng Biological Experiment Equipment Co., Ltd.) was fed; 50 mL of 1% glucose solution was added to the model group, positive group, and test group, and 10 mg of high-fat feed (purchased from Shanghai Feixi Biotechnology Co., Ltd.) was fed. Incubate for 7 days. After the model was constructed, 50 mL of E3 culture water was added to the normal group and model group, and 10 mg of ordinary feed was fed; 50 mL of 20 μg / mL metformin hydrochloride solution was added to the positive group, and 10 mg of high-fat feed was fed; 50 mL of the corresponding concentration solution was added to the test group, and 10 mg of high-fat feed was fed. Incubate for 5 days.

[0083] Table 1 10×E3 aquaculture water formula (1L)

[0084] 2) Determination of glucose content in zebrafish After the intervention, the zebrafish were washed twice with pure water, collected into a 1.5 mL centrifuge tube, the excess liquid was discarded, 250 μL PBS was added to homogenize the zebrafish until there were no obvious tissue fragments, and then centrifuged at 12000×g, 4℃ for 10 min. The supernatant was collected by centrifugation after homogenization, and the glucose content in the supernatant was detected using a glucose determination kit (Cat. No. S03039, manufacturer Redu).

[0085] 3) Data analysis All data were statistically analyzed using GraphPad Prism 8, and experimental data were expressed as mean ± SEM. t-test analysis, compared with the normal group: #P < 0.05, ##P < 0.01, ###P < 0.001; t-test analysis, compared with the model group: &P < 0.05, &&P < 0.01, &&&P < 0.001; one-way ANOVA, compared with the model group: *P < 0.05, **P < 0.01, ***P < 0.001.

[0086] Results Fig.10 and Table 2.

[0087] Table 2 Effects of freeze-dried powder and bacterial suspension on glucose levels in zebrafish

[0088] Depend on Fig.10 As shown in Table 2, the culture supernatant freeze-dried powder of Lactobacillus paracasei SX-M1559 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 the potential to lower blood sugar levels.

[0089] (3) Zebrafish hyperlipidemia model 1) Model construction Twenty-five zebrafish larvae with normal development at 5 dpf (days-post fertilization) were randomly selected and placed in a cell culture plate. The experiment was set up as a normal group, a model group, a positive group (atorvastatin calcium solution), a culture supernatant freeze-dried powder group (0.125 mg / mL), and a bacterial suspension group (1×10 6 CFU / mL). The normal group was added with 5 mL of E3 culture water and fed with 1 mg of ordinary feed; the model group, positive group, and test group were all added with 5 mL of 1% glucose solution and fed with 1 mg of high-fat feed to construct the high-fat model for 5 days.

[0090] 2) Test article intervention After the high-fat model was established, 5 mL of E3 culture water was added to the normal group, and 1 mg of ordinary feed (Shanghai Haisheng Biological Experiment Equipment Co., Ltd.) was fed; 5 mL of E3 culture water was added to the model group, and 1 mg of high-fat feed (Shanghai Feixi Biotechnology Co., Ltd.) was fed; 5 mL of 2.5 μg / mL atorvastatin calcium solution was added to the positive group, and 1 mg of high-fat feed was fed; 5 mL of the corresponding test solution was added to the test group, and 1 mg of high-fat feed was fed. The intervention lasted for 4 days.

[0091] 3) Determination of fat content in zebrafish - Oil Red O staining After the intervention, the zebrafish were washed twice with E3 culture water, fixed with 4% paraformaldehyde solution at 4°C, rinsed twice with PBS, dehydrated with 1,2-propylene glycol gradient, and then placed in a cell culture plate and added with Oil Red O dye. After staining, the zebrafish were decolorized with 1,2-propylene glycol, placed under a stereo microscope for observation and photographed. Image J software was used to analyze the staining of zebrafish fat Oil Red O, and the corresponding gray value (S) was calculated.

[0092] The formula for calculating the relative fat level of zebrafish is as follows: Zebrafish relative fat level (%) = (S 模型组、阳性组、供试品组 / S 正常组 )× 100%.

[0093] 3) Data analysis All data were statistically analyzed using GraphPad Prism 8, and experimental data were expressed as mean ± SEM. t-test analysis, compared with the normal group: #P < 0.05, ##P < 0.01, ###P < 0.001; t-test analysis, compared with the model group: &P < 0.05, &&P < 0.01, &&&P < 0.001; one-way ANOVA, compared with the model group: *P < 0.05, **P < 0.01, ***P < 0.001.

[0094] Results Fig.11 , Fig.12 and Table 3.

[0095] Table 3 Effects of freeze-dried powder and bacterial suspension on fat content in zebrafish

[0096] Depend on Fig.11 , Fig.12 As shown in Table 3, the culture supernatant freeze-dried powder of Lactobacillus paracasei SX-M1559 at a concentration of 0.125 mg / mL and the bacterial suspension at a concentration of 1×10 6 CFU / mL concentration, it can reduce the fat content in zebrafish and has a potential lipid-lowering effect.

[0097] (4) DSS (dextran sulfate sodium) induced zebrafish intestinal inflammation model - freeze-dried powder group 1) Model construction Twenty-five 3 dpf healthy Tg (mpx:EGFP) transgenic fluorescent zebrafish were randomly selected and placed in a 6-well cell culture plate; the experiment set up a normal group, a model group, a positive group (mesalazine solution), and a culture supernatant freeze-dried powder group (0.125 mg / mL). Except for the normal group, 4 mL of E3 culture water was added, and the other groups were added with 4 mL of 0.5% DSS (Shanghai McLean Biochemical Technology Co., Ltd., D808272) to construct an intestinal inflammation model, which was placed in a 28.5℃ incubator for 3 days. After the model was constructed, E3 culture water was added to the normal group and the model group, 4 mL of 50 μg / mL mesalazine solution was added to the positive group, and 4 mL of 0.125 mg / mL culture supernatant freeze-dried powder solution of Lactobacillus paracasei SX-M1559 was added to the bacterial liquid group, and placed in a 28.5℃ incubator for intervention for 2 days.

[0098] 2) Determination of neutrophil content in zebrafish After the incubation, the solution in each well was discarded, and the wells were washed twice with E3 culture water. Then, the zebrafish were placed under an inverted fluorescence microscope for observation and photography.

[0099] 3) Data statistics: The experimental data were expressed as mean ± SEM and analyzed by t-test. Compared with the normal group: ###P<0.001; compared with the model group: *P<0.05, **P<0.01, ***P<0.001.

[0100] Results Fig.13 and Table 4.

[0101] Table 4 Effect of freeze-dried powder on the number of neutrophils in the intestine of zebrafish

[0102] Depend on Fig.13 As shown in Table 4, the culture supernatant freeze-dried powder of Lactobacillus paracasei SX-M1559 at a concentration of 0.125 mg / mL can significantly inhibit the aggregation of neutrophils in the zebrafish intestine, and has the potential to alleviate intestinal inflammation.

[0103] (5) DSS-induced zebrafish intestinal inflammation model - bacterial suspension group 1) Model construction Twenty-five 3 dpf transgenic Tg (mpx:EGFP) zebrafish were randomly selected and placed in a 6-well cell culture plate. The experiment set up a normal group, a model group, a positive group (mesalazine solution), a bacterial suspension group (1×10 6CFU / mL). Except for the normal group, 4 mL of E3 culture water was added, and the other groups were added with 4 mL of 0.5% DSS (Shanghai McLean Biochemical Technology Co., Ltd., D808272) to construct the intestinal inflammation model, which was placed in a 28.5°C incubator for 3 days. After the model was constructed, E3 culture water was added to the normal group and the model group, 4 mL of 50 μg / mL mesalazine solution was added to the positive group, and 4 mL of the corresponding bacterial solution was added to the bacterial solution group, and they were placed in a 28.5°C incubator for intervention for 2 days.

[0104] 2) Determination of neutrophil content in zebrafish After the incubation, the solution in each well was discarded, and the wells were washed twice with E3 culture water. Then, the zebrafish were placed under an inverted fluorescence microscope for observation and photography.

[0105] 3) Data statistics: The experimental data were expressed as mean ± SEM and analyzed by t-test. Compared with the normal group: ###P<0.001; compared with the model group: *P<0.05, **P<0.01, ***P<0.001.

[0106] Results Fig.14 and Table 5.

[0107] Table 5 Effects of bacterial suspension on the number of neutrophils in the zebrafish intestine

[0108] Depend on Fig.14 As shown in Table 5, the bacterial suspension of Lactobacillus paracasei SX-M1559 was 1×10 6 CFU / mL concentration can significantly inhibit the aggregation of neutrophils in the zebrafish intestine, and has the potential to alleviate intestinal inflammation.

[0109] Example 8 Strain Safety Assessment Experiment The bacterial suspension with an OD600 value of 1.6 was inoculated into the sterilized MRS liquid medium at a ratio of 2% and cultured at 37°C for 24 h; centrifuged at 8 000 r / min for 8 min, the supernatant was discarded, the bacterial cells were collected and resuspended in physiological saline to obtain a bacterial suspension, and the concentration was adjusted to 1×10 8 CFU / mL concentration.

[0110] Twelve male mice and 12 female mice were divided into a saline group (control group) and a Lactobacillus paracasei SX-M1559 group (experimental group), with 6 mice in each group. The mice were given 0.1 mL / 10 g of saline (saline group) and 0.1 mL / 10 g of 1×10 8After intragastric administration of CFU / mL SX-M1559 bacterial solution (SX-M1559 group), samples (heart, liver, spleen, lung, and kidney) were collected after 3 weeks of conventional feeding to calculate the organ index. Organ index calculation formula: organ weight / mouse body weight. Tables 6 and 7 show the results of the safety assessment of Lactobacillus paracasei SX-M1559 strain.

[0111] Table 6

[0112] Table 7

[0113] The results in Tables 6 and 7 show that there was no significant difference between the normal saline group and the Lactobacillus paracasei SX-M1559 group, suggesting that this strain had no significant effect on organs.

[0114] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A strain of Lactobacillus paracasei SX-M1559, characterized in that: Its deposit number is CCTCC NO: M 2025030.

2. A probiotic preparation, characterized in that: The method comprises the Lactobacillus paracasei SX-M1559 according to claim 1.

3. Application of the Lactobacillus paracasei SX-M1559 according to claim 1 or the probiotic preparation according to claim 2 in preparing a hypoglycemic drug.

4. Application of the Lactobacillus paracasei SX-M1559 according to claim 1 or the probiotic preparation according to claim 2 in preparing a drug for inhibiting alpha-glucosidase.

5. Use of the Lactobacillus paracasei SX-M1559 according to claim 1 or the probiotic preparation according to claim 2 in preparing a drug for inhibiting alpha-amylase.

6. Use of the Lactobacillus paracasei SX-M1559 according to claim 1 or the probiotic preparation according to claim 2 in preparing a lipid-lowering drug.

7. Use of the Lactobacillus paracasei SX-M1559 according to claim 1 or the probiotic preparation according to claim 2 in preparing a cholesterol-degrading medicament.

8. Use of the Lactobacillus paracasei SX-M1559 according to claim 1 or the probiotic preparation according to claim 2 in preparing a triglyceride-lowering drug.

9. Use of the Lactobacillus paracasei SX-M1559 according to claim 1 or the probiotic preparation according to claim 2 in the preparation of anti-inflammatory drugs.

10. Use of the Lactobacillus paracasei SX-M1559 according to claim 1 or the probiotic preparation according to claim 2 in the preparation of a medicament for treating inflammatory bowel disease.

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