Lactobacillus acidophilus strain la-pg-06 with blood pressure-lowering activity and preparation method thereof

Abstract

The present application belongs to the field of microbial technology, and relates to a lactobacillus acidophilus strain La-PG-06 with blood pressure lowering ability and a preparation method thereof. The strain provided by the present application is lactobacillus acidophilus La-PG-06, which is named Lactobacillus acidophilus in Latin, has a preservation number of CGMCC No. 31864, a preservation time of September 4, 2024, and a preservation unit of China General Microbiological Culture Collection Center, located at No. 3, Beichen West Road, Chaoyang District, Beijing. The strain is isolated and purified from pickled Chinese cabbage, has the ability to inhibit angiotensin converting enzyme (ACE) activity, thereby exerting the function of lowering blood pressure; the strain has strong acid tolerance, salt tolerance and tolerance to artificial simulated gastrointestinal fluid, and has high antioxidant activity and antibacterial ability, and has strong adhesion ability.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a strain of Lactobacillus acidophilus La-PG-06 with blood pressure-lowering ability and its preparation method. Background Technology

[0002] Hypertension is a common disease worldwide, a clinical syndrome characterized by elevated systolic or diastolic blood pressure. It can lead to coronary heart disease, arteriosclerosis, myocardial infarction, stroke, and other conditions. The global prevalence of hypertension is as high as 10% to 20%, and it is widely distributed across the population, with high rates not only among the elderly and middle-aged but also among children. It is a significant threat to human health. Therefore, the prevention and treatment of hypertension is a hot topic of research in today's society.

[0003] Angiotensin-converting enzyme (ACE) is a enzyme containing Zn 2+ Dipeptide carboxypeptidase (ACE) is mainly found in endothelial cells of various tissues such as the lungs, brain, and kidneys, and is also present in epithelial cells, plasma, and urine. The main function of ACE is to catalyze the conversion of angiotensin I to angiotensin II, which can trigger strong vasoconstriction and promote the synthesis and release of the adrenal cortex hormone aldosterone. Therefore, ACE is a key enzyme in blood pressure regulation; inhibiting its activity can achieve the effect of lowering blood pressure.

[0004] Probiotics are a class of live microorganisms that have beneficial effects on the body's health, regulating the balance of the intestinal microecology and the metabolic activities of intestinal microorganisms. Studies have shown that during fermentation, probiotics release peptide fragments with antihypertensive activity from food proteins through the hydrolytic action of their extracellular proteases and peptidases (carboxypeptidases and aminopeptidases). These peptides can bind to the ACE active site, competitively inhibiting ACE activity, preventing the conversion of angiotensin I to angiotensin II, and catalyzing the hydrolysis of bradykinin into inactive fragments, thereby lowering blood pressure. Therefore, the screening and research of probiotics with antihypertensive functions has become an increasingly important focus. Summary of the Invention

[0005] Therefore, the present invention aims to provide a strain of Lactobacillus acidophilus that is resistant to acid, bile salts, and simulated gastrointestinal fluid, and has a high blood pressure lowering ability.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a strain of Lactobacillus acidophilus with the ability to lower blood pressure, the strain being Lactobacillus acidophilus La-PG-06, with the Latin name Lactobacillus acidophilus, the accession number being CGMCC No. 31864, the accession date being September 4, 2024, the depositary being the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] A method for preparing a strain of Lactobacillus acidophilus with antihypertensive ability, applicable to a strain of Lactobacillus acidophilus with antihypertensive ability, comprising the following steps:

[0008] S1. Dilute the fecal sample with physiological saline, spread it on MRS agar solid medium, and incubate at 37°C for 48 h. Pick single colonies with different morphology and size on the plate and streak them on MRS agar medium multiple times for purification until they are identified as single colonies. Pick single colonies for microscopic examination, physiological and biochemical analysis and 16S rDNA homology comparison, and freeze-preserve the isolated strains with glycerol.

[0009] S2. Fully activate and expand the Lactobacillus acidophilus La-PG-06 that has been frozen in glycerol;

[0010] S3. After activating the indicator bacteria, inoculate them into LB liquid medium. After activating Lactobacillus acidophilus La-PG-06, inoculate it into MRS liquid medium. After culturing at 37℃ for 24 h, conduct the Lactobacillus acidophilus La-PG-06 antibacterial test.

[0011] S4. The activated Lactobacillus acidophilus La-PG-06 bacterial suspension was streaked onto Columbia blood agar plates, inverted and incubated in an incubator for 48 h to perform a hemolysis test.

[0012] S5. Inoculate the fully activated and expanded Lactobacillus acidophilus La-PG-06 bacterial culture onto MRS agar medium, incubate at 37°C upside down for 24 h, and conduct antibiotic tests on the strain.

[0013] S6. The fully activated and expanded Lactobacillus acidophilus La-PG-06 bacterial culture was inoculated into MRS medium with different pH values ​​and cultured at 37℃ for 24 h. Then, the acid and alkali resistance of the strain was tested.

[0014] S7. The fully activated and expanded Lactobacillus acidophilus La-PG-06 bacterial culture was inoculated into MRS medium with different bile salt concentrations and cultured at 37℃ for 24 h. Then, the salt tolerance and bile salt tolerance of the strain were tested.

[0015] S8. Centrifuge the fully activated and expanded Lactobacillus acidophilus La-PG-06 bacterial solution, collect the bacterial sludge, and prepare a bacterial suspension with physiological saline for naked bacterial simulated gastrointestinal test.

[0016] S9. The in vitro antioxidant capacity of fully activated and expanded Lactobacillus acidophilus La-PG-06 was determined.

[0017] S10. Perform a cell adhesion test on the fully activated and expanded Lactobacillus acidophilus La-PG-06;

[0018] S11. The ability of fully activated and expanded Lactobacillus acidophilus La-PG-06 to inhibit ACE was determined.

[0019] Compared with the prior art, the beneficial effects of the present invention are that the strain described in the present invention is isolated and purified from sauerkraut. This strain has the ability to inhibit angiotensin-converting enzyme (ACE) activity, thereby exerting the function of lowering blood pressure. This strain also has strong acid resistance, salt resistance, and tolerance to artificial simulated gastrointestinal fluid. The strain has high antioxidant activity and antibacterial ability, and strong adhesion ability. Attached Figure Description

[0020] Figure 1 Image showing the colony morphology and Gram staining results of Lactobacillus acidophilus La-PG-06 in a petri dish;

[0021] Figure 2 The graph shows the results of the test on the inhibition of pathogenic bacteria by the supernatant of Lactobacillus acidophilus La-PG-06 fermentation broth;

[0022] Figure 3 The image shows the results of the hemolysis test for Lactobacillus acidophilus La-PG-06.

[0023] Figure 4 A graph showing the acid resistance of Lactobacillus acidophilus La-PG-06;

[0024] Figure 5 A graph showing the alkali resistance of Lactobacillus acidophilus La-PG-06;

[0025] Figure 6 A graph showing the salt tolerance of Lactobacillus acidophilus La-PG-06;

[0026] Figure 7 A graph showing the bile salt tolerance of Lactobacillus acidophilus La-PG-06;

[0027] Figure 8 A graph showing the ability of Lactobacillus acidophilus La-PG-06 to tolerate simulated gastrointestinal fluid;

[0028] Figure 9 The graph shows the results of the test on the DPPH free radical scavenging ability of Lactobacillus acidophilus La-PG-06 strain;

[0029] Figure 10 The graph shows the test results of the hydroxyl radical scavenging ability of Lactobacillus acidophilus La-PG-06 strain;

[0030] Figure 11 Lactobacillus acidophilus strain La-PG-06 against ABTS + Figure showing the test results of free radical scavenging ability;

[0031] Figure 12 The results of the test on the superoxide anion free radical scavenging ability of Lactobacillus acidophilus strain La-PG-06;

[0032] Figure 13 This is a diagram showing the self-aggregation ability of Lactobacillus acidophilus strain La-PG-06.

[0033] Figure 14 This is a graph showing the co-aggregation ability of Lactobacillus acidophilus strain La-PG-06.

[0034] Figure 15 The figure shows the results of the assay for inhibiting angiotensin-converting enzyme activity by Lactobacillus acidophilus La-PG-06. Detailed Implementation

[0035] Example 1: Screening and identification of Lactobacillus acidophilus La-PG-06

[0036] 1. Strains Isolation and Purification

[0037] Take 1 mL of sauerkraut fermentation broth, dilute it with physiological saline, and then take 100 μL of the diluted broth at different dilution gradients of 10 μL and 10 μL respectively. -2 ~10 -7 The fermentation broth was evenly spread onto MRS medium and incubated at 37°C for 48 h. Single colonies of different morphologies and sizes were picked from the plates and streaked multiple times on MRS agar for purification until a pure strain was identified.

[0038] 2. Microscopic examination

[0039] Single colonies were picked and inoculated into MRS broth medium. After culturing for 24 h, Gram staining was performed. The bacterial culture was smeared, Gram-stained, and the cell morphology and arrangement were observed under a light microscope. The observations were recorded and photographed for later use. Finally, Gram-positive cultures were identified as suspected lactic acid bacteria. 1.0 mL of the pure culture was transferred to a storage tube containing 50% glycerol for preservation and future use.

[0040] 3. Physiological and biochemical identification

[0041] The hydrogen peroxide test, indole test, and carbohydrate utilization test were used to identify and screen the bacterial strains. The isolated and purified strain was milky white, with a raised center, a smooth and moist surface, and a diameter of 1.15 mm; the strain was Gram-positive, and the cells were spherical, growing singly or in clusters. Figure 1 Its physicochemical characteristics include a negative hydrogen peroxide test; a positive indole reaction; a positive aescin hydrolysis test; and the ability to ferment cellobiose, maltose, mannitol, salicin, sorbitol, sucrose, raffinose, inulin, and lactose.

[0042] 4. Molecular biological identification

[0043] The bacterial strain was expanded and cultured, and the bacterial culture was centrifuged to obtain bacterial cells, which were then sequenced. Sequencing was performed by Nanjing Paisennong Gene Technology Co., Ltd. The 16S sequence of strain La-PG-06 was submitted after sequencing was completed.The rDNA sequence (CGACAGGTGCTATACTGCAAGTCGAGCGAGCTGAACCAACAGATTCACTTCGGTGATGACGTTGGGAACGCGAGCGGCGGATGGGTGAGTAACACGTGGGGAACCTGCCCCATAGTCTGGGATACCACTTGGAAACAGGTGCTAATACCGGATAAGAAAGCAGATCGCATGATCAGCTTATAAAAGGCGGCGTAAGCTGTCGCTATGGGATGGCCCCGCGGTGCATTAGCTAGTTGGTAGGGTAACGGCCTACCAAGGCAATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTGGTGAAGAAGGATAGAGGTAGTAACTGGCCTTTATTTGACGGTAATCAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGAAGAATAAGTCTGATGTGAAAGCCCTCGGCTTAACCGAGGAACTGCATCGGAAACTGTTTTTCTTGAGTGCAGAAGAGGAGAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAAGTGTTGGGAGGTTTCCGCCTCTCAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCTAGGGC) is deposited in NCBIA comparison with the BLAST database revealed that the gene sequence of strain La-PG-06 was 100% homologous to Lactobacillus acidophilus. Combined with Gram staining results and physiological and biochemical characteristics, strain La-PG-06 was identified as Lactobacillus acidophilus.

[0044] Example 2: Antibacterial activity test of Lactobacillus acidophilus La-PG-06

[0045] After activating the indicator bacteria *Escherichia coli*, *Listeria monocytogenes*, and *Staphylococcus aureus*, they were inoculated into LB liquid medium and incubated at 37°C for 24 h. Bacterial suspensions were then prepared for use. *Lactobacillus acidophilus* La-PG-06 was activated and inoculated into liquid MRS medium and incubated at 37°C for 24 h. The supernatant was collected after centrifugation at 8000×g for 5 min. Using the Oxford cup agar diffusion method, *Staphylococcus aureus*, *Bacillus subtilis*, *Escherichia coli*, *Pseudomonas aeruginosa*, *Listeria monocytogenes*, and *Pseudomonas aeruginosa* were spread on plates. Sterile physiological saline served as a blank control. Four Oxford cups were evenly placed on each plate. Three Oxford cups were filled with 200 μL of fermentation supernatant, and one Oxford cup was filled with 200 μL of sterile physiological saline. The plates were incubated at 37°C for 24 h. The presence of inhibition zones around the bottom of the Oxford cups was observed, and the diameter of the inhibition zones was measured and averaged.

[0046] From Table 1 and Figure 2 As can be seen, inhibition zones appeared in all the petri dishes, and the diameter of the inhibition zones was greater than 14.0 mm. This indicates that Lactobacillus acidophilus La-PG-06 has an inhibitory effect on the growth of Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Listeria monocytogenes, and Salmonella. The diameters of the inhibition zones were 19.13±0.61 mm, 18.93±0.50 mm, 20.47±0.42 mm, 24.67±0.61 mm, 21.00±0.20 mm, and 22.73±0.31 mm, respectively, showing that Lactobacillus acidophilus La-PG-06 has a significant antibacterial effect and strong antibacterial ability.

[0047] Table 1. Experimental data on the antibacterial activity of Lactobacillus acidophilus La-PG-06

[0048] Staphylococcus aureus 19.13±0.61 Bacillus subtilis 18.93±0.50 E. coli 20.47±0.42 Pseudomonas aeruginosa 24.67±0.61 Listeria monocytogenes 21.00±0.20 Salmonella serovar Typhimurium 22.73±0.31

[0049] Note: The diameter of the Oxford cup is 7.833 ± 0.172.

[0050] Example 3 Hemolysis test of Lactobacillus acidophilus strain La-PG-06

[0051] Lactobacillus acidophilus La-PG-06 bacterial suspension was inoculated onto Columbia blood agar medium and incubated at 37°C for 24 h. Hemolysis was then observed around the colonies. A semi-transparent hemolytic zone on the plate indicated α-hemolysis, a clearly defined, completely transparent hemolytic zone indicated β-hemolysis, and the absence of a hemolytic zone indicated γ-hemolysis (i.e., no hemolysis).

[0052] The test results for Lactobacillus acidophilus La-PG-06 are as follows: Figure 3 As shown, with Staphylococcus aureus as the control strain (right figure), Lactobacillus acidophilus La-PG-06 (left figure) is not hemolytic and is a safe strain.

[0053] Example 4 Antibiotic susceptibility test of Lactobacillus acidophilus strain La-PG-06

[0054] The fully activated and expanded Lactobacillus acidophilus La-PG-06 bacterial suspension was plated on MRS agar medium. Antibiotic susceptibility test strips were placed on the surface of the petri dish using sterile forceps. The dish was incubated upside down at 37°C for 24 h. Three strips of the same drug were placed on each petri dish, with a spacing of at least 24 mm between each strip and a center distance of at least 15 mm from the edge of the petri dish. The dish was left to stand for 5 min and then incubated upside down at 37°C for 24 h. The diameter of the inhibition zone was measured and recorded.

[0055] The susceptibility of strains to various drugs was determined according to the Clinical Laboratory Standards Institute (CLSI) Antimicrobial Susceptibility Testing Standards (CLSI-M100-S19). The following criteria were used to determine susceptibility: inhibition zone ≥ 18 mm was considered sensitive; inhibition zone ≥ 12 mm was considered moderately sensitive; and inhibition zone ≤ 12 mm was considered resistant. The test results were reported as resistant (R), moderately sensitive (I), or sensitive (S).

[0056] The results of the antibiotic susceptibility test of Lactobacillus acidophilus La-PG-06 are shown in Table 2. As can be seen from Table 2, Lactobacillus acidophilus La-PG-06 showed an R susceptibility to ampicillin, streptomycin, vancomycin, kanamycin, and gentamicin. The inhibition zone diameter (mm) for ampicillin was 12.13±0.11, while no inhibition zone was formed for the other antibiotics. The susceptibility to erythromycin and clindamycin was I, with inhibition zone diameters (mm) of 20.73±0.06 and 17.53±0.08, respectively. The susceptibility to tetracycline and chloramphenicol was S, with inhibition zone diameters (mm) of 15.53±0.13 and 19.47±0.22, respectively. Lactobacillus acidophilus La-PG-06 was most sensitive to chloramphenicol.

[0057] Table 2. Results of antibiotic susceptibility testing of Lactobacillus acidophilus strain La-PG-06

[0058] Ampicillin 12.13±0.11 I tetracycline 13.53±0.13 I Streptomycin 0 R Vancomycin 0 R Kanamycin 0 R Erythromycin 20.73±0.06 S clindamycin 17.53±0.08 I Gentamicin 0 R Chloramphenicol 19.47±0.22 S

[0059] Note: R indicates drug resistance, I indicates moderate sensitivity, and S indicates sensitivity.

[0060] Example 5: Tolerance test of Lactobacillus acidophilus La-PG-06

[0061] 1. Results of acid resistance test

[0062] After the strain was activated and passaged twice in MRS medium at 37℃, a bacterial suspension was prepared and resuspended in MRS medium with pH values ​​adjusted to 5.0, 4.0, 3.0, 2.0 and 1.0 respectively using 4.0 mol / L HCl. After incubation at 37℃ for 24 h, the bacterial suspension was collected. The absorbance of the bacterial suspension was measured at 600 nm wavelength, with 3 replicates. The absorbance of pH 6.0 MRS medium at 600 nm wavelength was used as a control. The survival rate was calculated.

[0063] Survival rate = N t / N 0； Where: N t The absorbance values ​​are for MRS culture media with pH values ​​ranging from 1.0 to 5.0. N0 represents the absorbance of the MRS medium at pH 6.0.

[0064] In healthy individuals, the pH of gastric juice on an empty stomach ranges from 0.9 to 1.8. After ingestion, the pH of gastric juice changes, fluctuating between approximately 1.8 and 5.0. Therefore, this experiment selected pH values ​​of 1.5, 2, 2.5, 3, 4, and 5 to test the acid tolerance of the bacterial strains. The results are as follows: Figure 4 As shown. By Figure 4 It can be seen that Lactobacillus acidophilus La-PG-06 exhibits poor tolerance at pH 1.5, with a survival rate of only 14.87% after 24 hours of treatment; however, it demonstrates strong tolerance at pH 5.0, with a survival rate of 91.38% after 24 hours of treatment. This indicates that the bacterium has a strong ability to tolerate acidic conditions.

[0065] 2. Alkali resistance test results In the alkali resistance test of the strain, the pH of the MRS medium was adjusted with 4.0 mol / L NaOH, and MRS media with different pH values ​​were prepared: 6.0, 7.0, 8.0, 9.0, 10.0, and 11.0. The fully activated and expanded *Lactobacillus acidophilus* strain La-PG-06 was inoculated into the media with different pH values. After culturing at 37℃ for 24 h, the OD of the bacterial suspension was measured. 600The nm value was calculated in triplicate, with the OD value of MRS medium (pH 6.0) as a control. The survival rate was calculated to determine the ability of Lactobacillus acidophilus La-PG-06 to tolerate alkali.

[0066] That is, survival rate = N t / N0; Where: N t The values ​​represent the OD values ​​of MRS media with pH values ​​ranging from 7.0 to 11.0. N0 is the OD value of the MRS medium at pH 6.0.

[0067] The results of the alkali resistance test of Lactobacillus acidophilus strain La-PG-06 are as follows: Figure 5 As shown. From Figure 5 It can be seen that *Lactobacillus acidophilus* La-PG-06 exhibits the best tolerance at pH 7.0, with a survival rate as high as 94.23% after 24 hours of treatment. At pH 11.0, the strain also shows high tolerance, with a survival rate of 73.94% after 24 hours of treatment. This indicates that *Lactobacillus acidophilus* La-PG-06 has a strong ability to tolerate alkaline conditions and maintains a high survival rate even in strongly alkaline environments.

[0068] 3. Salt tolerance test results For the salt tolerance test of the strain, fully activated and expanded Lactobacillus acidophilus La-PG-06 bacterial suspension was inoculated at a 3% inoculum into MRS medium containing 1.0, 2.0, 3.0, 4.0, and 5.0 g / 100 mL NaCl concentrations, respectively. After incubation at 37°C for 24 h, the OD of the bacterial suspension was measured. 600 The nm value was used as a control, with the OD value of the strain in MRS medium without NaCl added. Three replicates were set up to calculate the survival rate and determine the effect of different NaCl concentrations on the activity of Lactobacillus acidophilus La-PG-06.

[0069] That is, survival rate = N t / N0; Where: N t The values ​​are OD values ​​of MRS media with NaCl concentrations ranging from 1.0 to 5.0 g / 100 mL. N0 is the OD value of the MRS medium when the NaCl concentration is 0 g / 100 mL.

[0070] The results of the salt tolerance test for Lactobacillus acidophilus strain La-PG-06 are as follows: Figure 6 As shown. The tolerance of the strain to NaCl was tested by selecting NaCl concentrations ranging from 1.0 to 5.0 g / 100 mL. Figure 6It can be seen that the survival rate of Lactobacillus acidophilus La-PG-06 decreased slightly with the increase of NaCl concentration, but after culturing for 24 h under NaCl concentration of 5.0 g / 100 mL, its survival rate was still 99.00%, indicating that the bacterium has a strong ability to tolerate salt conditions.

[0071] 4. Results of the bile salt tolerance test After two generations of activation and subculturing, Lactobacillus acidophilus La-PG-06 strain was inoculated at a 3% inoculum into MRS medium containing 0.1%, 0.3%, 0.5%, 0.7%, and 0.9% bile salts, respectively. After incubation at 37°C for 24 h, the bacterial suspension was collected, and the absorbance of the bacterial suspension was measured at a wavelength of 600 nm. Three replicates were set up, with the absorbance of MRS medium without bile salts at a wavelength of 600 nm as a control. The survival rate of the strain was calculated.

[0072] Survival rate = N t / N 0； Where: N t The number of viable bacteria in MRS culture media containing different concentrations of bile salts; N0 represents the number of viable bacteria in MRS medium without bile salts.

[0073] The concentration of bile salts in the small intestine of most humans is around 0.3% (w / v). Probiotics need to tolerate the bile salts in the intestine to exert their beneficial effects. In this study, *Lactobacillus acidophilus* strain La-PG-06 was treated with five different mass fractions of bile salts (0.1%, 0.3%, 0.5%, 0.7%, and 0.9%) for 24 h (with strains cultured without bile salts serving as a control). The results are as follows: Figure 7 As shown. From Figure 7 It can be seen that the survival rate of the strain gradually decreases with the increase of bile salt concentration. After 24 h of treatment with 0.3% bile salt, the survival rate was still 40.13%, indicating that Lactobacillus acidophilus La-PG-06 has good bile salt tolerance.

[0074] 5. Results of simulated gastrointestinal fluid tolerance test Lactobacillus acidophilus strain La-PG-06 was activated and passaged twice in MRS medium at 37℃. A bacterial suspension was prepared, resuspended in simulated gastric fluid, and treated at 37℃ for 3 h. After centrifugation and discarding the supernatant, the bacterial sludge was collected and washed three times with sterile physiological saline. The sludge precipitate was collected aseptically and mixed with physiological saline at a ratio of 1:5. The mixture was then serially diluted before colony counting.

[0075] Take 1.0 g of bacterial sludge after 3 h of gastric fluid treatment, add simulated bile, and treat at 37℃ for 20 min. Centrifuge and discard the supernatant, collect the bacterial sludge, and wash it three times with sterile physiological saline. Collect the precipitate under aseptic conditions, add physiological saline at a ratio of bacterial sludge: physiological saline of 1:5, mix well, perform serial dilution, and count the colonies.

[0076] Take 1.0 g of bacterial sludge after treating the bile for 20 min, add simulated intestinal fluid, and treat at 37℃ for 4 h. Centrifuge and discard the supernatant, collect the bacterial sludge, and wash it three times with sterile physiological saline. Collect the precipitate under aseptic conditions, add physiological saline at a ratio of bacterial sludge: physiological saline of 1:5, mix well, perform serial dilution, and count the colonies.

[0077] Survival rate of artificial gastrointestinal fluid and bile = N t / N 0； Where: N t To simulate the number of viable bacteria after treatment with gastric juice, bile, and intestinal fluid; N0 represents the number of viable bacteria before treatment.

[0078] Gastric juice contains mucus, gastric acid, pepsin, etc. For probiotics to exert their beneficial effects, they must survive in the stomach, meaning they need to be able to tolerate the acidic environment and resist pepsin. Food typically stays in the stomach for 3 hours; therefore, this study used simulated gastric juice to treat Lactobacillus acidophilus La-PG-06 bacterial culture for 3 hours. After digestion in the stomach, food enters the intestines. The small intestine environment is weakly alkaline and contains trypsin and bile. The viable count of probiotics in the intestines reaches 10-1. 6 Probiotics need to have a concentration of at least CFU / mL to exert their probiotic function; therefore, they must be able to tolerate the weakly alkaline environment of the intestine and resist trypsin and bile. Food typically stays in the small intestine for 3–8 hours. Therefore, this experiment used simulated intestinal fluid to treat Lactobacillus acidophilus La-PG-06 bacterial culture for 4 hours. The results are as follows: Figure 8 As shown. By Figure 8 It can be seen that after 3 hours of treatment with gastric juice, the survival rate of Lactobacillus acidophilus La-PG-06 was 90.73%, with a viable count of 12.79 ± 0.03 lg (cfu / g). After 20 minutes of treatment with bile, the survival rate was 86.22%, with a viable count of 12.15 ± 0.02 lg (cfu / g). After 4 hours of treatment with intestinal juice, the survival rate was still 77.90%, with a viable count of 10.98 ± 0.09 lg (cfu / g). This demonstrates that Lactobacillus acidophilus La-PG-06 has a strong ability to tolerate simulated gastric and intestinal juices and bile.

[0079] Example 6: In vitro antioxidant capacity test of Lactobacillus acidophilus La-PG-06 1. Results of DPPH free radical scavenging ability Prepare a 0.1 mmol / L DPPH solution using anhydrous ethanol, store it protected from light, and adjust the absorbance at 517 nm to 1.25 ± 0.05 with anhydrous ethanol before use. Take 2.0 mL of bacterial culture into a 10 mL test tube, add 2.0 mL of DPPH solution, and incubate at room temperature in the dark for 20 min. Measure the absorbance at 517 nm and record it as A1. Record the absorbance when ethanol is used instead of DPPH as A2. Record the absorbance when ethanol is used instead of the sample as A0. Calculate the DPPH free radical scavenging rate using the formula.

[0080] ; like Figure 9 As shown, the DPPH free radical scavenging rate of Lactobacillus acidophilus La-PG-06 was 92.37±4.22%.

[0081] 2. Results of scavenging ability against hydroxyl radicals Take 1 mL of bacterial culture and place it in a 10 mL test tube. Then, add 1 mL of 9 mmol / L ferrous sulfate solution, 1 mL of 9 mmol / L salicylic acid ethanol solution, and 1 mL of 8.8 mmol / L H₂O₂ solution sequentially. Shake well and incubate the test tube at 37℃ for 30 min. After cooling to room temperature, measure the absorbance at 510 nm (A1). The absorbance of H₂O instead of H₂O₂ is A2. The absorbance of H₂O instead of the sample is A0. Calculate the hydroxyl radical scavenging rate using the formula.

[0082] ; like Figure 10 The hydroxyl radical scavenging rate of Lactobacillus acidophilus La-PG-06 was 96.72 ± 1.90%.

[0083] 3. Regarding ABTS + Free radical scavenging ability results First, prepare a 7 mmol / L ABTS solution and a 2.45 mmol / L K₂S₂O₈ solution, mix them in equal proportions, and react at 4°C in the dark for 12–16 h. Before use, dilute with anhydrous ethanol to a wavelength of 0.7 ± 0.02 at 734 nm. Take 0.5 mL of bacterial culture in a test tube, add 4.5 mL of the diluted ABTS stock solution, react at 30°C for 10 min, and measure the absorbance at 734 nm as A1. The absorbance at 734 nm using H₂O instead of ABTS is A2. The absorbance at 734 nm using H₂O instead of the sample is A0. Calculate the absorbance of ABTS using the formula. + Free radical scavenging rate.

[0084] ; Figure 11 shows the ABTS of Lactobacillus acidophilus La-PG-06. + The free radical scavenging rate was 76.94 ± 9.01%.

[0085] 4. Results of superoxide anion radical scavenging ability Take 1 mL of bacterial culture in a test tube, add 1 mL of 10 mmol / L pyrogallol solution and 5 mL of 50 mmol / L Tris-HCl (pH 8.2), react at 25℃ for 4 min, then add 1 mL of 8 mol / L HCl to terminate the reaction. Measure the absorbance at 320 nm as A1. The absorbance at 320 nm is A1 when H2O replaces pyrogallol. 2， H₂O was used to replace the sample, and the absorbance at wavelength 320 nm was A₀. The superoxide anion radical scavenging rate was calculated according to the formula.

[0086] ; like Figure 12 The superoxide anion radical scavenging rate of Lactobacillus acidophilus La-PG-06 was 58.83±0.57%.

[0087] In summary, the results above indicate that Lactobacillus acidophilus La-PG-06 has a high antioxidant capacity.

[0088] Example 7 Hydrophobicity test of Lactobacillus acidophilus strain La-PG-06 For the surface hydrophobicity test of the strain, centrifuge the fully activated and expanded *Lactobacillus acidophilus* La-PG-06 strain for 10 min and discard the supernatant. Add an appropriate amount of PBS buffer and wash 2-3 times. Adjust the concentration of the test strain to 0.4 at 600 nm using PBS buffer. After adjustment, take 3 ml of the bacterial solution and add 1 ml of chloroform. No chloroform is added to the control group. Shake for 30 s and let stand for 30 min to separate the organic and aqueous phases. Remove the organic phase. Using the buffer as a blank control, measure the OD value at 560 nm. Repeat the experiment three times and calculate the results.

[0089] Hydrophobicity determination = [1-(A)] x / A0)]×100% In the formula: A x A is the measured value; A0 is the initial value of 0.4.

[0090] The surface hydrophobicity of Lactobacillus acidophilus strain La-PG-06 is shown in Table 3. As can be seen from Table 3, after standing for 30 min, the surface hydrophobicity of Lactobacillus acidophilus strain La-PG-06 is 30.25% ± 2.13%, indicating strong hydrophobicity.

[0091] Table 3 Results of hydrophobicity assay of bacterial strains Lactobacillus acidophilus La-PG-06 30.25%±2.13% Example 8 Self-aggregation test of Lactobacillus acidophilus strain La-PG-06 For the bacterial strain self-aggregation assay, the fully activated and expanded *Lactobacillus acidophilus* strain La-PG-06 was centrifuged for 10 min, and the supernatant was discarded. An appropriate amount of PBS buffer was added, and the strain was washed 2-3 times. Finally, it was resuspended in PBS. The total OD value of the bacterial suspension was measured at 600 nm. The bacterial suspension was incubated at 37℃ for 2, 4, 6, 8, and 24 h. 0.1 mL of the supernatant was transferred to another EP tube, 3.9 mL of PBS was added, and the suspension was shaken before measuring the OD value. The experiment was repeated three times and the results were calculated.

[0092] Self-aggregation ability = [1-(A)] t / A0)]×100%.

[0093] In the formula: A0 is the absorbance at 0h; A t The absorbance values ​​were measured at 2, 4, 6, 8, and 24 hours.

[0094] The self-aggregation results of Lactobacillus acidophilus strain La-PG-06 are as follows: Figure 13 As shown. By Figure 13 It can be seen that the self-aggregation ability of Lactobacillus acidophilus La-PG-06 strain was above 90% from 2 to 24 hours, indicating strong self-aggregation ability and strong adhesion to intestinal epithelial cells.

[0095] Example 9: Co-aggregation test of Lactobacillus acidophilus strain La-PG-06 For the co-aggregation assay, *Staphylococcus aureus*, *Escherichia coli*, *Salmonella typhimurium*, and *Bacillus subtilis* were used as indicator bacteria. *Lactobacillus acidophilus* La-PG-06 and the indicator bacteria were activated and inoculated into MRS and LB liquid media, respectively, and cultured at 37°C for 24 h. Equal volumes of *Lactobacillus acidophilus* La-PG-06 and the indicator bacteria were mixed, centrifuged to collect the bacteria, washed twice with PBS, and finally resuspended in PBS. The total OD value of the bacterial suspension was measured at 600 nm. 4 mL of bacterial suspension was incubated at 37°C for 2, 4, 6, 8, and 24 h. 0.1 mL of the supernatant was transferred to another EP tube, 3.9 mL of PBS was added, and the suspension was shaken before measuring the OD value. The experiment was repeated three times and the results were calculated.

[0096] Co-aggregation ability = [1-(A)] t / A0)]×100% In the formula: A0 is the absorbance at 0h; A t The absorbance values ​​were measured at 2, 4, 6, 8, and 24 hours.

[0097] The co-aggregation results of Lactobacillus acidophilus strain La-PG-06 are as follows: Figure 14 As shown. Lactic acid bacteria can co-aggregate with pathogens, interfering with the retention and colonization of pathogens in the gastrointestinal tract. Figure 14 It can be seen that, using Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Listeria monocytogenes, and Salmonella typhimurium as indicator bacteria, the co-aggregation ability of Lactobacillus acidophilus La-PG-06 strain with each indicator bacteria was weak at 2 h, at 92.08%, 92.84%, 90.77%, 78.08%, and 87.25%, respectively; from 4 to 24 h, the co-aggregation ability increased, and the co-aggregation ability of Lactobacillus acidophilus La-PG-06 strain with each indicator bacteria was above 85%.

[0098] Example 10 ACE Activity Detection Test of Lactobacillus acidophilus La-PG-06 The inhibition rate of ACE of Lactobacillus acidophilus strain La-PG-06 was detected using the Solarbio ACE inhibitor activity assay kit.

[0099] The bacterial culture was inoculated into 100 mL of modified MRS liquid medium and cultured at 37 °C for 17 h to obtain activated third-generation bacterial culture, which was then used. The obtained third-generation bacterial culture was inoculated at a 3% inoculum into 11% (w / w) skim milk and cultured at 37 °C for 20 h. The pH was measured and adjusted to 6.0 ± 0.02. An appropriate amount of culture was taken and centrifuged at 4 °C and 8000 r / min for 10 min. The supernatant was collected, and the pH was adjusted to 6.0 ± 0.02 again. 1 mL of the supernatant was taken and centrifuged at 4 °C and 10000 r / min for 10 min. The supernatant after centrifugation was used as the test sample.

[0100] The activated third-generation test strain was centrifuged at 8000 r / min for 10 min, the supernatant was discarded, and the bacterial pellet was collected. The pellet was washed with sterile PBS buffer, and this process was repeated three times. The pellet was then resuspended in PBS buffer, and the bacterial suspension concentration was adjusted to 1×10⁻⁶. 4 CFU / mL. After culturing the bacterial culture at 37℃ for 12 h, centrifuge at 12,000 r / min for 10 min and collect the supernatant. Filter through a 0.22 μm filter membrane to obtain the extracellular supernatant. According to the cell number (10... 4Add reagent one to the bacterial suspension at a ratio of 500-1000 mL to reagent one of the Solarbio ACE inhibitor activity assay kit. The bacterial suspension is then sonicated (200 W, 5 s operation, 5 s interval, 6 min). The lysate is centrifuged at 8000 r / min for 10 min, filtered through a 0.22 μm filter membrane, and the supernatant is collected to obtain the intracellular extract of the bacteria. The test strain is then heat-inactivated at 121℃ for 20 min to obtain an inactivated intact bacterial suspension. The extracellular supernatant and intracellular extract of the inactivated test strain are prepared according to the above method.

[0101] Preheat the UV spectrophotometer for 30 min, adjust the wavelength to 340 nm, zero the instrument with distilled water, and add the sample to a 5 mL quartz cuvette according to the steps in Table 4. Mix thoroughly, and measure the absorbance A1 at 340 nm after 10 s. Immediately place the instrument at 37℃ for an accurate reaction time of 30 min, and measure the absorbance A2 at 30 min and 10 s. ΔA 测定 =A1 测定 -A2 测定 , △A 空白 =(A1 空白1 -A2 空白1 )-(A1 空白2 -A2 空白2 ).

[0102] ACE inhibition rate (%) = (△A) 空白 -△A 测定 )÷△A 空白 ×100%; Table 4. Reagent Addition Amount Sample supernatant - - 135 - Reagent 1 135 1485 - - Reagent 2 1350 1350 1350 1350 Reagent 3 working solution 1350 - 1350 1350 Reagent 4 - - - 135 The results of the ACE inhibition rate test of Lactobacillus acidophilus strain La-PG-06 are as follows: Figure 15 As shown. By Figure 15 It can be seen that the live bacterial suspension, extracellular supernatant, and intracellular substances of *Lactobacillus acidophilus* La-PG-06 showed strong ACE inhibition abilities, at 75.95%, 72.15%, and 55.70%, respectively. Furthermore, the ACE inhibition rates of all live bacterial treatment groups were higher than those of all dead bacterial treatment groups, indicating that this strain has a strong antihypertensive function. However, the inhibition rates were all lower than the control group (captopril group). However, captopril in the control group has certain side effects on humans, such as dry cough, hyperkalemia, transient renal dysfunction, and rash.

[0103] Table 6 Comparison of ACE inhibition ability with other probiotic strains Lactobacillus rhamnosus HCS01-013 61.54 Wang Yali, Li Xuelong, Yu Ping, et al. Screening of lactic acid bacteria with antihypertensive function and their probiotic effects [J]. China Brewing, 2022, 41(10):130-133. Lactobacillus reuteri ZJUIDS09 70.13 Song Xiaoling, Gao Jiting, Cao Feiwei, et al. Screening, probiotic characteristics evaluation and application of lactic acid bacteria producing ACE inhibitory peptides [J]. Food Industry Technology, 2022, 43(10):149-157. Lactobacillus helveticus casein hydrolysate (C4) 48.12 Ruiqi Ren, Lu Liu et al, Identification and in-silico screening of ACE-inhibitory peptides fromcasein hydrolysate via fermentation with different probiotics.[J] International Dairy Journal, 2026, 172: 106425. Lactobacillus helveticus H11+ commercial starter culture PYS-010 67.34 Tian Chunle. Development of Probiotic Brown Fermented Milk with ACE Inhibitory Activity [D]. Inner Mongolia Agricultural University, 2024.000487. As shown in Table 6, the inhibition rate of *Lactobacillus acidophilus* La-PG-06 was higher than that of other strains in the literature. Furthermore, this strain exhibited strong tolerance in a simulated human gastrointestinal environment, with a high survival rate in tolerance tests. This demonstrates that *Lactobacillus acidophilus* La-PG-06 possesses significant value and development potential in the field of blood pressure reduction.

[0104] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A strain of Lactobacillus acidophilus with antihypertensive ability, characterized in that: The strain is Lactobacillus acidophilus La-PG-06, with the Latin name Lactobacillus acidophilus. Its accession number is CGMCC No. 31864, the deposit date is September 4, 2024, and the depositary institution is the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

2. A method for preparing a strain of Lactobacillus acidophilus with antihypertensive ability, applicable to the Lactobacillus acidophilus strain with antihypertensive ability described in claim 1, characterized in that, Includes the following steps: S1. Dilute the fecal sample with physiological saline, spread it on MRS agar solid medium, and incubate at 37°C for 48 hours. Pick single colonies with different morphologies and sizes from the plate and streak them on MRS agar medium multiple times for purification until they are identified as single colonies. Pick single colonies for microscopic examination, physiological and biochemical analysis and 16S rDNA homology comparison, and freeze-preserve the isolated strains with glycerol. S2. Fully activate and expand the Lactobacillus acidophilus La-PG-06 that has been frozen in glycerol; S3. After activating the indicator bacteria, inoculate them into LB liquid medium. After activating Lactobacillus acidophilus La-PG-06, inoculate it into MRS liquid medium. After culturing at 37℃ for 24 h, conduct the Lactobacillus acidophilus La-PG-06 antibacterial test. S4. The activated Lactobacillus acidophilus La-PG-06 bacterial suspension was streaked onto Columbia blood agar plates, inverted and incubated in an incubator for 48 h to perform a hemolysis test. S5. Inoculate the fully activated and expanded Lactobacillus acidophilus La-PG-06 bacterial culture onto MRS agar medium, incubate at 37°C upside down for 24 h, and conduct antibiotic tests on the strain. S6. The fully activated and expanded Lactobacillus acidophilus La-PG-06 bacterial culture was inoculated into MRS medium with different pH values ​​and cultured at 37℃ for 24 h. Then, the acid and alkali resistance of the strain was tested. S7. The fully activated and expanded Lactobacillus acidophilus La-PG-06 bacterial culture was inoculated into MRS medium with different bile salt concentrations and cultured at 37℃ for 24 h. Then, the salt tolerance and bile salt tolerance of the strain were tested. S8. Centrifuge the fully activated and expanded Lactobacillus acidophilus La-PG-06 bacterial solution, collect the bacterial sludge, and prepare a bacterial suspension with physiological saline for naked bacterial simulated gastrointestinal test. S9. The in vitro antioxidant capacity of fully activated and expanded Lactobacillus acidophilus La-PG-06 was determined. S10. Perform a cell adhesion test on the fully activated and expanded Lactobacillus acidophilus La-PG-06; S11. The ability of fully activated and expanded Lactobacillus acidophilus La-PG-06 to inhibit ACE was determined.

3. The method for preparing a strain of Lactobacillus acidophilus with antihypertensive activity according to claim 2, characterized in that: When activating and expanding the Lactobacillus acidophilus La-PG-06 in S2, the Lactobacillus acidophilus La-PG-06 strain preserved at -20℃ glycerol is first thawed and then inoculated into liquid MRS medium at an inoculation rate of 2% (V / V). After incubation at 37℃ for 24 h, the revived bacterial solution is inoculated into MRS liquid medium and incubated at 37℃ for another 24 h to ensure that the strain is fully activated and expanded.

4. The method for preparing a strain of Lactobacillus acidophilus with antihypertensive activity according to claim 2, characterized in that: When performing the antibacterial test on Lactobacillus acidophilus La-PG-06 in S3, the Oxford cup agar diffusion method was used. Staphylococcus aureus, Escherichia coli, and Listeria monocytogenes were used as indicator bacteria for plate spreading, and physiological saline was used as a blank control. Four Oxford cups were evenly placed on each plate. 200 μL of Lactobacillus acidophilus La-PG-06 bacterial suspension was added to three Oxford cups, and physiological saline was added to one Oxford cup. After incubation at 37°C for 24 h, the size of the inhibition zone was measured with vernier calipers.

5. The method for preparing a strain of Lactobacillus acidophilus with antihypertensive activity according to claim 2, characterized in that: When performing a hemolytic test on Lactobacillus acidophilus La-PG-06 in S4, the strain was streaked on a blood agar plate and cultured at 37°C for 48 h. The plate was then removed for observation. Hemolysis was indicated by the presence of a transparent area, a green area, or no area around the colony. The transparent area represented β-hemolysis, the green area represented α-hemolysis, and the absence of the area represented γ-hemolysis.

6. The method for preparing a strain of Lactobacillus acidophilus with antihypertensive activity according to claim 2, characterized in that: When performing antibiotic susceptibility testing on Lactobacillus acidophilus La-PG-06 in S5, the fully activated and expanded Lactobacillus acidophilus La-PG-06 bacterial suspension is plated on MRS agar medium. Antibiotic susceptibility test discs are placed on the surface of the culture dish using sterile forceps. The dish is then incubated upside down at 37°C for 24 h to perform antibiotic susceptibility testing. Three discs of the same drug are placed on each culture dish, with a spacing of at least 24 mm between each disc and a center distance of at least 15 mm from the edge of the culture dish. The dish is left to stand for 5 min, then incubated upside down at 37°C for 24 h. The diameter of the inhibition zone is measured and recorded.

7. The method for preparing a strain of Lactobacillus acidophilus with antihypertensive activity according to claim 2, characterized in that: When conducting acid and alkali resistance tests on Lactobacillus acidophilus La-PG-06 in S6, the pH values ​​of multiple MRS liquid culture media with a pH of 6.0 were adjusted to 5.0, 4.0, 3.0, 2.0, and 1.0 respectively using 4.0 mol / L HCl, and the pH values ​​of multiple MRS liquid cultures with a pH of 6.0 were adjusted to 11.0, 10.0, 9.0, 8.0, and 7.0 respectively using NaOH. The strain was inoculated into culture media with different pH values, and after being cultured at 37℃ for 24 h, the absorbance of the bacterial suspension was measured at a wavelength of 600 nm to calculate its survival rate and test the acid and alkali resistance of Lactobacillus acidophilus La-PG-06.

8. The method for preparing a strain of Lactobacillus acidophilus with antihypertensive activity according to claim 2, characterized in that: When conducting salt and bile salt tolerance tests on *Lactobacillus acidophilus* La-PG-06 in S7, fully activated and expanded *Lactobacillus acidophilus* La-PG-06 bacterial suspension was inoculated at a 3% inoculum into MRS medium containing 1.0, 2.0, 3.0, 4.0, and 5.0 g / 100 mL NaCl concentrations, respectively; the strain was also inoculated at a 3% inoculum into MRS medium containing 0.1%, 0.3%, 0.5%, 0.7%, and 0.9% bile salt concentrations, respectively. After incubation at 37°C for 24 h, the absorbance of the bacterial suspension was measured at 600 nm, with the absorbance at 600 nm of the strain in MRS medium without bile salts as a control. The survival rate was calculated to evaluate the effects of salt and bile salts on the activity of *Lactobacillus acidophilus* La-PG-06.

9. The method for preparing a strain of Lactobacillus acidophilus with antihypertensive activity according to claim 2, characterized in that: When performing simulated gastrointestinal assays on *Lactobacillus acidophilus* La-PG-06 in S8, the strain was activated and passaged twice in MRS medium at 37°C. 1.0 g of bacterial sludge was centrifuged and added to 5.0 mL of simulated gastric fluid. After treatment at 37°C for 3 h, the precipitate was collected under aseptic conditions. Sterile physiological saline was added at a ratio of 1.0 g bacterial sludge to 5.0 mL physiological saline, mixed thoroughly, diluted, and spread for colony counting. Then, 1.0 g of bacterial sludge treated with simulated gastric fluid was added to 5.0 mL of simulated bile. After treatment at 37°C for 20 min, the mixture was centrifuged at 8000 r / min for 10 min. The centrifuged bacterial sludge was then collected for colony counting. Finally, 1.0 g of bacterial sludge treated with simulated bile was added to 5.0 mL of simulated intestinal fluid. After treatment at 37°C for 4 h and 8 h respectively, the mixture was centrifuged at 8000 r / min for 10 min. After 1 minute, the centrifuged bacterial sludge was collected for colony counting. The colony count in MRS medium that had not undergone simulated gastrointestinal treatment was used as a control to calculate the survival rate.

10. The method for preparing a strain of Lactobacillus acidophilus with antihypertensive activity according to claim 2, characterized in that: When conducting in vitro antioxidant capacity tests on the *Lactobacillus acidophilus* La-PG-06 strain in S9, the strain was activated at 37°C in MRS medium for two generations, and then its DPPH free radical scavenging capacity, hydroxyl free radical scavenging capacity, and ABTS were evaluated. + The free radical scavenging capacity and superoxide anion free radical scavenging capacity were determined by comparing the antioxidant capacity of VC and LGG bacterial culture treated in the same way. When performing the cell adhesion ability test on Lactobacillus acidophilus La-PG-06 in S10, the fully activated and expanded Lactobacillus acidophilus La-PG-06 strain was centrifuged for 10 min, and the supernatant was discarded. An appropriate amount of PBS buffer was added, and the sample was washed 2-3 times. The concentration of the test strain was adjusted to 0.4 at 600 nm using PBS buffer. After adjustment, 3 mL of the bacterial solution was taken, and 1 mL of chloroform was added. No chloroform was added to the control group. After shaking for 30 s, the mixture was allowed to stand for 30 min to separate the organic and aqueous phases. The organic phase was removed, and the OD value was measured at 560 nm using the buffer as a blank control. The experiment was repeated three times, and the hydrophobicity value of the strain was calculated. In the bacterial self-aggregation test, the treated bacteria were suspended in PBS, and the total OD value of the bacterial suspension was measured at 600 nm. 4 mL of bacterial suspension was incubated at 37℃ for 2, 4, 6, 8, and 24 h. 0.1 mL of the supernatant was then placed in another EP tube, 3.9 mL of PBS was added, and the suspension was shaken before measuring the OD value. The experiment was repeated three times, and the bacterial self-aggregation ability was calculated. For the co-aggregation assay, Staphylococcus aureus, Escherichia coli, Salmonella typhimurium, and Bacillus subtilis were used as indicator bacteria. Lactobacillus acidophilus La-PG-06 and the indicator bacteria were activated and inoculated into MRS and LB liquid media, respectively, and cultured at 37°C for 24 h. Equal volumes of Lactobacillus acidophilus La-PG-06 and the indicator bacteria were mixed, centrifuged to collect the bacteria, washed twice with PBS, and finally resuspended in PBS. The total OD value of the bacterial suspension was measured at 600 nm. 4 mL of bacterial suspension was allowed to stand at 37°C for 2, 4, 6, 8, and 24 h. 0.1 mL of the supernatant was transferred to another EP tube, 3.9 mL of PBS was added, and the suspension was shaken before measuring the OD value. When testing the ACE inhibition ability of Lactobacillus acidophilus La-PG-06 in S11, the bacterial culture was inoculated into 100 mL of modified MRS liquid medium and cultured at 37 °C for 17 h to obtain activated third-generation bacterial culture, which was then ready for use. The obtained third-generation bacterial culture was inoculated at a 3% inoculum into 11% skim milk and cultured at 37 °C for 20 h. The pH was measured and adjusted to 6.0 ± 0.

02. An appropriate amount of culture was taken and centrifuged at 4 °C and 8000 r / min for 10 min. The supernatant was collected, and the pH was adjusted to 6.0 ± 0.02 again. 1 mL of the supernatant was taken and centrifuged at 4 °C and 10000 r / min for 10 min. The supernatant after centrifugation was used as the test sample. The activated third-generation test strain was centrifuged at 8000 r / min for 10 min, the supernatant was discarded, and the bacterial pellet was collected. The pellet was washed with sterile PBS buffer, and the process was repeated three times. The pellet was then resuspended in PBS buffer, and the bacterial suspension concentration was adjusted to 1×10⁻⁶. 4 CFU / mL. After culturing the bacterial culture at 37℃ for 12 h, centrifuge at 12000 r / min for 10 min and collect the supernatant. Filter through a 0.22 μm filter membrane to obtain the extracellular supernatant. According to the cell number (10... 4 Add reagent 1 to the bacterial suspension at a volume ratio (mL) of 500-1000:1 to that of reagent 1 in the Solarbio ACE Inhibitor Activity Assay Kit. Sonicate the mixture (200 W, 5 s, 5 s interval, 6 min). Centrifuge the mixture at 8000 r / min for 10 min, filter through a 0.22 μm filter membrane, and collect the supernatant to obtain the intracellular extract of the bacteria. Heat-inactivate the test strain at 121℃ for 20 min to obtain an inactivated intact bacterial suspension. Prepare the extracellular supernatant and intracellular extract of the inactivated test strain using the method described above. Preheat the UV spectrophotometer for 30 min, adjust the wavelength to 340 nm, zero the instrument with distilled water, and add the sample to a 5 mL quartz cuvette according to the steps in the kit. Mix thoroughly, and measure the absorbance A1 at 340 nm after 10 s. Immediately place the instrument at 37℃ for an accurate reaction time of 30 min, and measure the absorbance A2 after 30 min and 10 s.

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