Bifidobacterium longum subsp. infantis o-161 and postbiotic, antihypertensive peptide and application thereof

Abstract

This invention belongs to the field of microbial technology, providing a strain of *Bifidobacterium longum* subsp. infantis O-161 derived from breast milk, along with its postbiotic, antihypertensive peptides, and applications. *Bifidobacterium longum* subsp. infantis O-161 was isolated and identified from fresh, mature breast milk samples taken at 43 days of age using a modified anaerobic screening method (CCTC NO: M 20232203). After a comprehensive evaluation of the strain's biological characteristics, probiotic properties, and safety, its supernatant postbiotic was prepared, confirming its ability to inhibit angiotensin-converting enzyme (ACE), protect against angiotensin II (AngII)-induced endothelial dysfunction, and exert a vasodilatory effect on constricted mesenteric artery vascular rings in animals, with this vasodilatory effect exhibiting a certain endothelial dependence. Five potential antihypertensive peptides with high ACE inhibition rates, including GGWPLP, were screened using mass spectrometry and other methods for their use in assisting in lowering blood pressure and improving vascular function.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to the preparation of a postbiotic of a human milk-derived Bifidobacterium longum subsp. infantis O-161 and the application and products of its antihypertensive peptides that assist in lowering blood pressure and improving vascular function. Background Technology

[0002] Hypertension is a chronic cardiovascular disease characterized by persistently high systemic arterial blood pressure (≥140 / 90 mmHg). Long-term hypertension can lead to stroke, ischemic heart disease, chronic kidney disease, and other complications. More than ten million people die from hypertension or its complications each year. Currently, the main medications for treating hypertension include renin inhibitors, ACE inhibitors, AT1R antagonists, dihydropyridine calcium channel blockers that block calcium ion channels, and thiazide diuretics that reduce water and sodium reabsorption. Most of these medications have adverse reactions such as abdominal pain, diarrhea, and edema. Therefore, exploring safe and green food-derived biological resources and their active ingredients to help improve vascular function and thus control blood pressure has received widespread attention.

[0003] Bifidobacteria are among the earliest microorganisms to naturally colonize the human gut. Their abundance and diversity in the colon are closely linked to host health. Some members of the Bifidobacteria genus have been widely used in infant formula, other dairy products (yogurt, cheese), beverages, confectionery, dietary supplements (such as powders, capsules, and drops), and oral care products, with dietary supplements and fermented milk accounting for a significant portion. Their main claimed functions include increasing the diversity and abundance of beneficial bacteria in the human gut, balancing the gut microbiota, enhancing the immune system, alleviating gut-related problems, restoring healthy skin, and improving mental well-being. As microorganisms with probiotic potential, the functionality and probiotic properties of Bifidobacteria are strictly dependent on specific strains. Therefore, to fully realize their probiotic effects, it is necessary to continuously isolate, culture, and characterize new strains in detail.

[0004] Epibiotics are rich in probiotic metabolites, which are non-living cells. After entering the body, they can exert their biological functions without colonization or competitive survival, thus offering higher safety and greater bioavailability. Furthermore, epibiotics possess anti-inflammatory, antioxidant, and antibacterial properties, making them highly promising for developing biological functions. The blood pressure-lowering effect and mechanism of epibiotics have become a research hotspot in the field of cardiovascular health in recent years. On the one hand, epibiotics contain abundant bioactive peptides, which can utilize their peptide functions to inhibit renin, angiotensin-converting enzyme (ACE), and act as angiotensin receptor blockers. On the other hand, epibiotics can regulate the gut microbiota, increasing the number of beneficial bacteria such as Lactobacillus and Bifidobacterium, reducing the number of opportunistic pathogens such as Escherichia coli, and significantly increasing the content of short-chain fatty acids such as lactic acid and acetic acid, thus improving the intestinal microenvironment and positively impacting blood pressure. Currently, there is limited research on the effects of postbiotics on the body's vascular and endothelial functions, as they are natural active factors used for the prevention and treatment of hypertension, either alone or as an adjunct. Therefore, screening and evaluating the antihypertensive potential and related mechanisms of postbiotics produced by probiotic strains is of great significance for the development of the probiotic functions of postbiotics and also provides new ideas and methods for the prevention and treatment of hypertension. Summary of the Invention

[0005] Given the shortcomings of existing antihypertensive drugs, such as significant side effects, low activity of dietary ACE inhibitory peptides, and a single mechanism of action, the purpose of this invention is to provide a human milk-derived Bifidobacterium infantis subspecies O-161, to prepare a post-genetic agent that can help lower blood pressure and improve vascular function, and to clarify the application of this post-genetic agent and its active functional peptides in the preparation of antihypertensive related products, thereby providing a new strategy for hypertension intervention.

[0006] To achieve the above objectives, the present invention employs the following technical solution: This invention discloses a human milk-derived *Bifidobacterium longum* subspecies *infant* O-161 strain, which belongs to *Bifidobacterium longum* subspecies *infant*, and is named *Bifidobacterium longum* subspecies *infant*. Bifidobacterium longum subsp. infantis The isolate O-161 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20232203 and deposit date November 13, 2023.

[0007] This invention discloses a metabiotic, which is obtained by fermentation of the above-mentioned Bifidobacterium longum subsp. infantis O-161 and is a supernatant type metabiotic.

[0008] This invention discloses the application of the above-mentioned metagenes in the preparation of products with ACE enzyme inhibitory function.

[0009] This invention discloses the application of the above-mentioned metagenes in the preparation of products that alleviate AngII-induced endothelial dysfunction in human umbilical vein endothelial cells.

[0010] Preferably, alleviating AngII-induced endothelial dysfunction in human umbilical vein endothelial cells includes promoting the secretion of nitric oxide and endothelial nitric oxide synthase, inhibiting the production of reactive oxygen species, inhibiting excessive cell migration, and reducing the transcription and expression of inflammatory factors IL-6 and TNF-α.

[0011] This invention discloses the above-mentioned application in the preparation of products with peripheral mesenteric artery dilation function.

[0012] This invention also discloses a class of antihypertensive peptides derived from the aforementioned metagenic peptides, including short peptides with amino acid sequences as shown in any of SEQ ID NO. 1-5. The specific sequences are: GGWPLP, WPANPPL, GLCPLP, PAMPPL, and VCAFLPLP.

[0013] Preferably, the antihypertensive peptide has an inhibition rate of ≥75% against ACE, and a half-maximal inhibitory concentration (IC50) of ≥75%. 50 The values ​​range from 14.07 μM to 148 μM.

[0014] This invention also discloses the application of the above-mentioned antihypertensive peptide in the preparation of products with ACE enzyme inhibition function.

[0015] This invention also discloses the application of the above-mentioned antihypertensive peptide in the preparation of products with antihypertensive function.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention utilizes a modified anaerobic screening method to isolate and identify a strain of *Bifidobacterium longum* subsp. *infantii* O-161 from a 43-day-old fresh, mature breast milk sample. This strain is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 20232203. Based on previous research, this application comprehensively evaluates the strain's basic biological characteristics, potential probiotic properties, and safety. Furthermore, it prepares a metabiotic from the supernatant of the O-161 fermentation broth, determines the inhibitory rate of the metabiotic on angiotensin-converting enzyme (ACE), explores the alleviating effect of the metabiotic on AngII-induced endothelial dysfunction in human umbilical vein endothelial cells (HUVECs), and examines the vasodilatory effect of the metabiotic on the peripheral mesenteric arteries of experimental rats. Finally, the sequence of the metabiotic-derived antihypertensive peptide is isolated and identified, and the ACE inhibition rate and IC50 of the synthesized antihypertensive peptide are verified.

[0017] The specific advantages are summarized as follows: 1. The supernatant metagenic component of the fermentation broth of O-161 exhibits antihypertensive activity. It showed a 75.12% inhibition rate against angiotensin-converting enzyme (ACE). 2. O-161 postbiotic can preventively protect against AngII-induced endothelial dysfunction. After establishing an endothelial dysfunction model of human umbilical vein endothelial cells (HUVECs) induced by angiotensin II (AngII), intervention with 3% O-161 postbiotic can effectively alleviate endothelial dysfunction, including significantly increasing NO secretion, inhibiting ROS production, inhibiting excessive cell migration, reducing cellular inflammatory responses (decreased transcription and expression of IL-6 and TNF-α), and promoting eNOS expression. 3. O-161 postbiotic can inhibit AngII-induced endothelial dysfunction. AngII and 3% O-161 postbiotic acted together on human umbilical vein endothelial cells (HUVECs) to significantly inhibit endothelial dysfunction, including reducing ROS production, inhibiting excessive cell migration, and reducing cellular inflammatory responses (decreased transcription and expression of IL-6 and TNF-α). 4. O-161 postbiotics have a significant endothelium-dependent vasodilatory effect on the peripheral mesenteric artery ring of experimental rats in a contracted state, with a vasodilation rate of 31.12%.

[0018] 5. The identification, synthesis, and pressure-lowering function verification of bioactive peptides in the supernatant of O-161 fermentation broth metagenic peptides were completed. Nano LC-MS / MS was used to comprehensively identify the peptide components in the metagenic samples and obtain their detailed peptide profiles. Bioinformatics databases and analytical prediction tools such as PeptideRanker, BIOPEP, and ToxinPred were used to systematically search and predict the bioactivity potential of the identified peptides, and target peptides with potential ACE-inhibiting activity, namely GGWPLP, WPANPPL, GLCPLP, PAMPPL, and VCAFLPLP, were screened.

[0019] 6. Based on the above sequence, antihypertensive peptides were synthesized, with an ACE inhibition rate of 87.23±6.73% and an IC50 value between 14.07μM and 148μM. The peptide VCAFPLP showed the highest inhibition rate at 91.87%, while the peptide PAMPPL showed the lowest IC50 at 41.61μM. Attached Figure Description

[0020] Figure 1 This is a schematic diagram illustrating the working mechanism of the present invention.

[0021] Figure 2 CFS pretreatment with Bifidobacterium O-161 improves AngII-induced excessive cell migration; A shows the scratch map of AngII-induced cell migration after CFS with O-161, magnified ×4; B shows the scratch area of ​​cells at 0h, 24h, and 48h, as quantitatively analyzed by ImageJ, reflecting the degree of cell migration.

[0022] Figure 3 Pretreatment with Bifidobacterium O-161 supernatant-type postbiotic CFS enhanced the inhibition of NO production by AngII in HUVEC cells; *** indicates P <0.001.

[0023] Figure 4 The effect of Bifidobacterium O-161 supernatant-type postbiotic CFS pretreatment on AngII-induced eNOS production in HUVEC cells; where A is a schematic diagram of the relative expression of eNOS detected by Western Blot; B is the quantitative analysis result; C is the ELISA detection of eNOS production; * and ** represent P < 0.05 and P < 0.01, respectively.

[0024] Figure 5 This study investigated the effect of Bifidobacterium O-161 supernatant-type postbiotic CFS pretreatment on improving AngII-induced oxidative stress in HUVEC cells. A shows the fluorescence image of ROS production in AngII-induced cells induced by O161 CFS (×10); B shows the amount of ROS produced in cells measured by Image J, with the average fluorescence intensity being the ratio of the experimental group to the control group.* P <0.05.

[0025] Figure 6 The effect of Bifidobacterium O-161 supernatant postbiotic CFS pretreatment on AngII-induced production of inflammatory factors in HUVEC cells; where A and C represent the transcription and expression of TNF-α, and B and D represent the transcription and expression of IL-6, respectively; * and ** indicate P < 0.05 and P < 0.01, respectively.

[0026] Figure 7 The study investigated the effects of co-treatment with Bifidobacterium O-161 supernatant-type postbiotic CFS on improving AngII-induced excessive cell migration. A shows the scratch map of AngII-induced cell migration induced by CFS, with a migration magnification of ×4. B shows the scratch area of ​​cells at 0h, 24h, and 48h, as quantitatively analyzed using ImageJ, reflecting the degree of cell migration.

[0027] Figure 8 Co-treatment with Bifidobacterium O-161 supernatant-type postbiotic CFS improved AngII-induced NO reduction;* P <0.05.

[0028] Figure 9This study investigated the effects of co-treatment with Bifidobacterium O-161 supernatant-type postbiotic CFS on improving AngII-induced oxidative stress. A shows the fluorescence image of ROS production in cells induced by CFS under AngII-induced stress, magnification: ×10; B shows the amount of ROS produced in cells measured using ImageJ, with the average fluorescence intensity reflecting the ratio of fluorescence intensity between the experimental and control groups. P <0.01.

[0029] Figure 10 The effect of co-treatment intervention on the production of inflammatory factors in HUVEC cells induced by AngII; where A and C represent the transcription and expression of the inflammatory factor TNF-α, respectively, and B and D represent the transcription and expression of the inflammatory factor IL-6, respectively; * and ** indicate P < 0.05 and P < 0.01, respectively.

[0030] Figure 11 To analyze and identify the peptide sequences and amino acid composition of Bifidobacterium O-161 postbiotic using nano-HPLC-MS / MS; where A is the distribution map of short peptides; and B is the site distribution map of amino acids in short peptides.

[0031] Figure 12 This is a molecular docking simulation of five peptides derived from postgenes with angiotensin-converting enzyme (ACE, PDB: 1O8A).

[0032] Figure 13 The ACE inhibition rate of the five peptides derived from post-genetics is shown; the positive control Captopril is the ACE inhibitor captopril; ** indicates P < 0.01.

[0033] Figure 14 The IC50 values ​​for ACE inhibition of five target peptides are shown; where A is WPANPPL; B is PAMPPL; C is GLCPLP; D is GGWPLP; and E is VCAFPLP.

[0034] Preservation Instructions The strain O-161, belonging to the subspecies of Bifidobacterium longum (breast milk-derived), is named *Bifidobacterium longum* subspecies *infant*. Bifidobacterium longum subsp infantis The isolate O-161 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20232203 and deposit date November 13, 2023. Detailed Implementation

[0035] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0036] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of the invention described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0037] The present invention will now be described in further detail with reference to the accompanying drawings: The following examples involve information on the experimental strains, cells, and primers used: Bifidobacterium longum infant subspecies Bifidobacterium longum subsp. infantis O-161 was a strain isolated and preserved in our laboratory; human umbilical vein endothelial cells (HUVE) were commercially available cells. Adult male SD rats were purchased from the Experimental Animal Center of Xi'an Jiaotong University. Primers used were synthesized by Sangon Biotech (Shanghai) Co., Ltd. Gene names and sequences are shown in Table 1.

[0038] Table 1. Gene names and sequences of primers used in the experiment.

[0039] Example 1: Preparation of postbiotic supernatant from fermentation broth of Bifidobacterium lactis strain O-161 (from human milk) Bifidobacterium longum infantis subspecies ( Bifidobacterium longum subsp infantis The isolate O-161 was a strain isolated and identified in our laboratory and is currently deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 20232203. The frozen strain was removed from -80℃, inoculated into MRSc medium, and anaerobically cultured at 37℃ for 24-48 hours. After activation, it was used for subsequent experiments.

[0040] This study prepared cell-free supernatant (CFS) using a filtration sterilization method. The preparation method was as follows: After 24 hours of growth of Bifidobacterium O-161, the accumulation of secondary metabolites reached its peak. The 24-hour fermentation broth was used to prepare the CFS. The bacterial suspension was centrifuged at 10,000 rpm at 4°C for 15 min, the bacterial cells were discarded, and the supernatant was filtered through a 0.22 μm syringe filter to obtain the fermentation broth supernatant CFS. The CFS was aliquoted into 1 ml containers and stored at -20°C for later use.

[0041] Example 2: Assay of the inhibitory activity of Bifidobacterium O-161 postbiotic on angiotensin-converting enzyme (ACE) First, 10 μL of 0.1 U ACE enzyme was mixed with 40 μL of CFS sample, sodium borate buffer (0.1 M boric acid and 0.3 M NaCl, pH 8.2), and 2 mg / mL captopril. The mixture was incubated at 37°C for 5 min. Then, 80 μL of 5 mM hippuric acid-histidine-leucine (HHL) was added as the reaction substrate, and the mixture was incubated at 37°C for 60 min. The reaction was terminated with 200 μL of 1 M HCl. Captopril served as a positive control.

[0042] High-performance liquid chromatography (HPLC) was used to detect hippuric acid (HA) after the reaction. A ZORBAX Eclipse XDB-C18 column (250 × 4.5 mm, 5 μm particle size) was used to detect the absorbance of HA at 228 nm. Detection conditions: mobile phase A (aqueous phase containing 0.05% TFA), mobile phase B (acetonitrile), 80% phase A and 20% phase B, flow rate of 1 mL / min, column temperature 35℃, injection volume 10 μL.

[0043]

[0044] Among them, A blank A represents the peak area of ​​HA produced after the reaction in the blank group (sodium borate buffer). sample This represents the peak area of ​​HA after the reaction of post-biotic CSF with ACE.

[0045] The experimental results showed that the supernatant of Bifidobacterium longum subsp. infantis isolate O-161 inhibited ACE enzyme by 75.12%, while captopril, as a control, had an inhibition rate of 97.65%.

[0046] Example 3: Protective effect of Bifidobacterium O-161 postbiotic on AngII-induced endothelial dysfunction (postbiotic treatment followed by induction of endothelial dysfunction). 10 5HUVEC cells were seeded in 24-well plates and cultured overnight. When the cell confluence reached approximately 90%, the medium was changed to basal medium. 3% O-161 was added as a preventative intervention. After 12 hours, the cells were washed with PBS, and AngII was added to a final concentration of 1×10⁻⁶. -6 M, continue culturing for 24 hours to construct an endothelial dysfunction model. The blank control group received no O-161 extrinsic agent or AngII; the model group received only AngII, with all other treatments the same. After treatment, cell supernatant and cells were collected according to experimental requirements for subsequent experiments.

[0047] (1) O-161 post-biotic inhibits AngII-induced excessive migration of HUVEC cells - scratch assay Excessive cell migration is one of the core pathological manifestations of endothelial dysfunction. Its essence lies in the abnormally enhanced cell migration ability caused by an imbalance in the regulatory network of cytoskeleton rearrangement, adhesion molecule expression, and extracellular matrix degradation. Cells were cultured in basal medium containing 3% O-161 post-biotic for 12 hours. Three scratches were made in each well. The scratched cells were washed with PBS and then treated with 1 μM AngII for 48 hours. The scratches were photographed using an inverted microscope at 0 h, 24 h, and 48 h, and the scratch area was analyzed and calculated using ImageJ software. Cells that underwent scratching but did not receive treatment served as a control.

[0048]

[0049] This represents the scratch area at 0h. Indicates the scratch area after 24 hours or 48 hours. The results showed that AngII stimulation caused excessive cell proliferation, with cell migration rates of 41.87±6.95% and 52.26±9.11% at 24h and 48h, respectively. After metagenic intervention, excessive cell migration was significantly improved (24h: 35.64±1.73% vs 41.87±6.95%). P =0.0159; 48h: 41.60±3.11%%vs 52.26±9.11%, P =0.0223). (See also: Figure 2 ) (2) O-161 postbiotic promotes AngII-induced NO expression and enhances eNOS activity in HUVEC cells. In HUVEC cells, the core function of endothelial nitric oxide synthase (eNOS) is to catalyze the production of nitric oxide (NO). NO is an endothelial-dependent relaxing factor, and measuring NO can immediately reflect the eNOS activity and overall functional integrity of HUVEC cells. The expression level of eNOS can indicate whether endothelial cells are in a protective or damaged phenotype and is widely used in the initial screening and mechanism research of cardiovascular drugs. By comparing NO levels and eNOS expression before and after drug administration, it is possible to determine whether metagenic CFS has the potential to promote NO release, improve vasodilation, or have antihypertensive effects.

[0050] 10 5 HUVEC cells were seeded in 24-well plates overnight, pretreated with 3% O-161 postbiotic for 12 h, and then stimulated with 1 µM Ang II for 24 h. After treatment, the NO content in the cell culture medium was detected by a NO kit, and the amount of eNOS produced by the cells was detected by an eNOS ELISA kit. At the same time, cells were collected, proteins were extracted, and Western blotting of eNOS expression was performed. The protein bands were saved and analyzed by ImageJ.

[0051] NO, as an important endothelial relaxant, promotes the synthesis of cyclic guanosine monophosphate (cGMP) by activating guanylate cyclase (sGC) in smooth muscle cells, ultimately leading to vascular smooth muscle relaxation. This, in turn, regulates vascular tone, improves local blood perfusion, and plays an irreplaceable role in maintaining vascular homeostasis. Metagenes were first used to prophylactically treat cells for 12 hours, followed by endothelial dysfunction induced by AngII. The results showed... Figure 3 After prophylactic intervention with O-161, the NO content produced by HUVEC cells was significantly increased compared with the model group (1.78±0.56µM vs 4.9±0.77µM, P<0.0001), with no significant difference compared with the negative control group.

[0052] like Figure 4 As shown in Figures A and B, compared with the control group, the relative expression level of eNOS in the AngII model group was significantly decreased (0.722 vs 1.103, P = 0.003). Compared with the AngII model group, the relative expression level of eNOS in endothelial cells of the metagenic pretreatment group showed an increasing trend (0.875 vs 0.722, P > 0.05). Figure 4As shown in Figure C, compared with the control group, the secretion of eNOS in the AngII model group was significantly decreased (26.15 ng / ml vs 62.20 ng / ml, P < 0.001). Compared with the AngII model group, the production of eNOS in endothelial cells in the post-genetic pretreatment group was significantly increased (51.48 vs 26.15, P = 0.003), and the production was basically the same as that in the control group, with no significant difference (P > 0.05), consistent with the results of Western Blot.

[0053] (3) O-161 postbiotic can reduce ROS production in AngII-induced HUVEC cells. Oxidative stress is one of the main manifestations of endothelial dysfunction. Its essence lies in the imbalance between the body's ROS production and the antioxidant system's ability to clear them, leading to excessive ROS accumulation and endothelial damage that prevents the endothelium from fulfilling its normal biological functions. The degree of increase in reactive oxygen species (ROS) in the body is negatively correlated with endothelial cell survival, NO production, and inflammatory factor secretion. By comparing the changes in cellular ROS before and after post-biotic CFS treatment, drugs with cardiovascular protective effects can be rapidly screened.

[0054] 10 5 HUVEC cells were seeded in 24-well plates overnight, pretreated with 3% O-161 postbiotic for 12 h, and then stimulated with 1 µM AngII for 24 h. The original culture medium was discarded, the cells were washed twice with PBS, and then incubated with DCFH-DA fluorescent probe at 37°C in the dark for 30 min. After washing away the fluorescent probe that did not enter the cells with PBS, the intracellular fluorescence intensity was detected by fluorescence microscopy and fluorescence microplate reader. The intracellular fluorescence intensity after capture was quantified using ImageJ software and finally normalized to the fluorescence intensity relative to the control group.

[0055] The results are as follows Figure 5 As shown, the amount of ROS produced by endothelial dysfunction cells increased to 1.47 times that of the negative control group, while the amount of ROS produced decreased significantly after metabiotic intervention, indicating that metabiotic has a significant protective effect against excessive ROS production induced by AngII in HUVEC cells.

[0056] (4) O-161 postbiotic can alleviate the AngII-induced inflammatory response in HUVEC cells. Overactivation of the RAAS system leads to excessive accumulation of AngII in blood vessels, causing vascular remodeling and inflammatory responses, resulting in damage to endothelial cells and vascular smooth muscle cells and metabolic disorders.

[0057] 10 5HUVEC cells were seeded in 24-well plates overnight, pretreated with 3% O-161 for 12 h as a prebiotic, and then stimulated with 1 µM Ang II for 24 h. Supernatant and cells were collected separately. RNA was extracted from the collected cells and reverse transcribed into cDNA. IL-6 and TNF-α were selected as marker inflammatory factors, with β-actin as an internal reference gene. A two-step qPCR amplification method was used, and the relative expression level of the target gene was calculated using 2^(-ΔΔCt). The expression levels of the inflammatory factors IL-6 and TNF-α in the collected cell culture supernatant were detected using an ELISA kit.

[0058] The effects of post-biotic treatment on the transcription and expression of inflammatory factors IL-6 and TNF-α in HUVEC cells under AngII-induced endothelial dysfunction were detected by RT-qPCR and ELISA. Figure 6 As shown, post-genetic pretreatment can reduce / alleviate cellular inflammation in AngII-induced endothelial dysfunction.

[0059] Compared with the control group, the relative expression levels of IL-6 (1.024 vs 0.781, P<0.001) and TNF-α in the AngII model group were increased (1.0 vs 0.705, P=0.068). Compared with the AngII model group, the transcription of IL-6 in the postbiotic pretreatment group was significantly decreased (0.474 vs 1.024, P<0.001), while the transcription of TNF-α was increased but not significantly different (1.203 vs 1.0, P>0.05).

[0060] Compared with the control group, the expression levels of inflammatory factors showed that the secretion of IL-6 (13611.75 vs 8543, P=0.0033) and TNF-α in the AngII model group were significantly increased (70.58 vs 42.35, P=0.0316); compared with the AngII model group, the secretion of IL-6 (5953.8 vs 136111.75, P<0.001) and TNF-α (16.41 vs 70.58, P<0.001) in the post-biotic pretreatment group were significantly decreased.

[0061] Example 4: Bifidobacterium O-161 postbiotic inhibits AngII-induced endothelial dysfunction (postbiotic treatment and endothelial dysfunction induction were performed simultaneously). 10 5 HUVEC cells were seeded in 24-well plates and cultured overnight. When the cell confluence reached approximately 90%, the complete culture medium was discarded and replaced with basal medium, with the addition of 3% O-161 postbiotic and AngII (final concentration 1×10⁻⁶). -6M) The cells were treated for a total of 24 hours. After completion, the cells were washed twice with PBS, and AngII was added to a final concentration of 1×10⁻⁶ in the wells. -6 M, continue culturing for 24 hours. The blank control group received no O-161 post-biotic and no AngII; the model group received only AngII without O-161 post-biotic intervention, with all other treatment steps being the same. After treatment, cell supernatant and cells were collected according to experimental requirements for subsequent experiments.

[0062] (1) O-161 postbiotic can inhibit AngII-induced excessive migration of HUVEC cells. Cells were treated with 3% O-161 post-genetic agent and 1 μM AngII for 48 h; three scratches were made in each well. Observation continued for 48 h, and the scratches were photographed using an inverted microscope at 0 h, 24 h, and 48 h. The scratch area was analyzed and calculated using ImageJ software. Cells that underwent scratching but did not receive treatment served as controls.

[0063]

[0064] This represents the scratch area at 0h. This indicates the scratch area after 24 hours or 48 hours.

[0065] The experimental results are as follows: During the co-treatment intervention, AngII stimulation of cells led to excessive cell migration, with migration rates reaching 30.96±1.68% and 54.78±6.92% at 24h and 48h, respectively, equivalent to 1.28 times and 1.35 times that of the control group. After subsequent co-treatment with biotics, excessive cell migration was inhibited, with migration rates decreasing to 25.09±1.82% (24h) and 40.43±2.58% (48h), respectively. Figure 7 As shown.

[0066] (2) O-161 post-biotic did not improve the reduction of NO secretion in HUVEC cells induced by AngII. 10 5 HUVEC cells were seeded in 24-well plates overnight. After treating the cells with 3% O-161 postbiotic and 1 µM AngII for 24 h, the NO content in the cell culture medium was detected using a NO detection kit.

[0067] Experimental results showed that co-treatment with post-genetic agents did not improve the reduction in NO secretion induced by AngII in HUVEC cells (see [link to experimental results]). Figure 8 ).

[0068] (3) O-161 postbiotic can reduce ROS production in AngII-induced HUVEC cells. The degree of ROS elevation is negatively correlated with endothelial cell survival rate, NO production, and inflammatory factor secretion. By comparing the changes in cell ROS before and after post-biotic CFS treatment, drugs with cardiovascular protective effects can be quickly screened.

[0069] 10 5 HUVEC cells were seeded in 24-well plates overnight, and treated with 3% O-161 postbiotic and 1µM AngII for 24 h. The original culture medium was discarded, the cells were washed twice with PBS, and then incubated with DCFH-DA fluorescent probe at 37°C in the dark for 30 min. After washing away the fluorescent probe that did not enter the cells with PBS, the intracellular fluorescence intensity was detected using a fluorescence microplate reader. The intracellular fluorescence intensity after capture was quantified using ImageJ software and finally normalized to the fluorescence intensity relative to the control group.

[0070] Experimental results: The amount of ROS generated in the model group increased to 1.54 times that of the control group, while the amount of ROS generated after metabiotic intervention was significantly lower than that in the AngII group (1.13±0.11µM vs 1.54±0.07µM, P<0.001), indicating that metabiotics have a good protective effect against AngII-induced endothelial dysfunction (see...). Figure 9 ).

[0071] (4) O-161 postbiotic can inhibit AngII-induced inflammatory response in HUVEC cells. 10 5 HUVEC cells were seeded in 24-well plates overnight. Cells were then treated with 3% O-161 postbiotic and 1 µM AngII for 24 h; the supernatant and cells were collected separately.

[0072] RNA was extracted from collected cells and reverse transcribed into cDNA. IL-6 and TNF-α were selected as marker inflammatory factors, and β-actin was used as an internal reference gene. A two-step qPCR amplification method was employed, and the relative expression levels of the target genes were calculated using 2^(-ΔΔCt). The expression levels of the inflammatory factors IL-6 and TNF-α in the collected cell culture supernatant were detected using an ELISA kit.

[0073] Experimental results showed that, compared with the control group, the transcriptional levels of inflammatory factors, particularly the relative expression levels of IL-6 and TNF-α, were increased in the AngII model group (IL-6: 1.003 vs 1.610). P =0.042; TNF-α: 1.018 vs 1.856, P <0.001); compared with the AngII model group, the post-biogenetic co-treatment group had IL-6 (0.817 vs 1.003, P =0.002) and TNF-α (1.411 vs 1.856,P The relative expression level of IL-6 in the co-treatment group was significantly decreased (=0.077). Compared with the AngII model group, the expression levels of inflammatory factors in the post-biotic co-treatment group were significantly lower (8230 vs 13611.8). P =0.0086) and TNF-α (50.17 vs 69.45, P The expression of (=0.0184) decreased significantly (see [reference needed]). Figure 10 This indicates that O-161 postbiotic has a positive effect on inhibiting AngII-induced inflammatory responses in HUVEC cells.

[0074] Example 5: The vasodilatory effect of Bifidobacterium O-161 postbiotic on isolated rat mesenteric arterial rings Animal husbandry and preparation of isolated rat mesenteric artery rings: Male SD rats were used in the experiment. The animals were purchased from the Experimental Animal Center of Xi'an Jiaotong University School of Medicine, were 12 weeks old, and weighed between 180-220g. They were housed in a well-ventilated environment at 25℃ with alternating light and dark cycles for 12 hours, and had free access to food and water. The experimental ethics were approved by the Biomedical Ethics Committee of Xi'an Jiaotong University School of Medicine (XJTUAE2023-1677).

[0075] Rats were sacrificed, and the abdominal cavity was quickly opened to separate the mesenteric artery. The artery was placed in a prepared glass dish containing 4°C KH solution, fixed, and the excess tissue surrounding the artery was slowly dissected. The separated mesenteric artery was cut into approximately 2mm ring segments. KH buffer was added to a tissue bath beforehand, and the vascular rings were placed in the bath and gently hung on a stress-bearing plate. The system was then connected to a BL420S physiological signal acquisition system. The system was equilibrated at 37°C for 1 hour, with the KH buffer changed every 10 minutes. During this period, 95% O2 and 5% CO2 were continuously and stably introduced, adjusting the basal tension to 0.5g before equilibration to a stable state.

[0076] Functional integrity check of isolated mesenteric artery ring: Before the start of each experiment, the functional integrity of the isolated mesenteric artery ring was checked. When the difference in contraction amplitude between two experiments was within 10%, it indicated that the stability of the vessel met the experimental requirements. The vasomotor response was tested using 1µM phenylephrine (PE) and 1mM acetylcholine (ACh). A contraction amplitude >50% and a vasodilation effect >50% indicated good vasomotor function and intact vascular endothelium.

[0077] Mesenteric artery endothelial removal: To examine whether the vasodilatory effect of post-genes on the vascular ring depends on the endothelial layer of the mesenteric artery, an aortic ring with the endothelium removed was used to test the vasodilatory effect in the absence of endothelium. First, Triton X-100 was injected into the separated vessel and left for 10 seconds. The vessel was immediately flushed with KH solution, and ET-1 was added to constrict the vessel. After reaching a plateau, ACh was added. Vasodilation of less than 20% indicated successful endothelial removal.

[0078] Post-adrenergic vasodilatory function test: After testing vasoactivity and reactivity, 1 ml of KH buffer was added to the bath, and the mesenteric artery was pre-constricted to a stable plateau with 1 µM endothelin ET-1. Then, 10 μL of different concentrations (1 × 10⁻⁶) of the drug were cumulatively added to the bath, starting with low concentrations. -3 2×10 -3 7×10 -3 2×10 -2 2×10 -1 7×10 -1 The post-epigenetic derivatives were used. The percentage of vasodilation in the post-epigenetic derivatives was calculated, with the contraction amplitude at which the 1 μmol / L ET-1 reached its plateau defined as 100%.

[0079]

[0080] Experimental results showed that the direct regulatory effect of metabiotics on vascular endothelial function could be systematically evaluated by detecting the vasodilatory response of the mesenteric artery. After acetylcholine acted on the contractile vascular ring caused by the proconstrictor endothelin-1 (ET-1), the vasodilatory rate reached 57.23%, indicating that the endothelial function of this vascular ring was intact, with good contractile and vasodilatory functions. Metabiotics, after acting on the contractile vascular ring, showed a good vasodilatory effect, with a vasodilatory rate of 31.12%. After endothelial removal, the vasodilatory ability of metabiotics decreased to 13.64%, indicating that the vasodilatory effect of Bifidobacterium O-161 is not entirely dependent on endothelial integrity and may be related to other multiple regulatory mechanisms.

[0081] Example 6: Isolation and Identification of Antihypertensive Peptides from Bifidobacterium O-161 Postbiotic (1) Isolation of antihypertensive peptides Peptide sequences were identified using nano-HPLC-MS / MS. First, an appropriate amount of post-biotic CFS was passed through a C18 desalting column, and then detected using a Q Exactive™ Plus ultra-high performance liquid chromatography-mass spectrometry system. Data were processed using Thermo ProteomeDiscoverer version 2.3 software, with the Bifidobacterium database downloaded and digestion set to none. The search parameters were set to a fragment ion mass tolerance of 10 ppm, a precursor ion mass tolerance of 5 ppm, no dynamic modification, and static modification consisting of aminomethylation of cysteine ​​(c) with a modification mass of +57.021 Da.

[0082] The experimental results are as follows: Based on ultra-fast high-resolution mass spectrometry analysis and interpretation, a total of 2761 peptides were identified from Bifidobacterium O161CFS, of which 1450 were small peptides. 910 peptides were no more than octapeptides, accounting for 62.77% of the short peptides. Proline (P), alanine (A), glycine (G), threonine (T), and serine (S) were the most abundant in the small peptides. Figure 11 The data shows the amino acid composition at each site. After data homogenization, the amino acids with a high proportion at the N-terminus of the CFS peptide are proline (P), alanine (A), lysine (K), and threonine (T), while those at the C-terminus are proline (P), glycine (G), and glutamine (Q).

[0083] (2) Screening and identification of antihypertensive peptides Computer simulation analysis was used to screen and identify antihypertensive peptides.

[0084] The first step involved predicting the bioactivity of the retrieved peptide sequences using PeptideRanker software (0-1 points, with higher scores indicating higher bioactivity potential). Peptides with PeptideRanker scores > 0.9 were selected for further analysis. The AllerTOP v2.1 database was used to analyze the biosensitivity of the target peptide sequences; Toxinpred was used to analyze the potential toxicity, hydrophobicity, amphiphilicity, isoelectric point, and molecular weight of the target peptides; the Expasy-pI / Mw tool was used to analyze the isoelectric point (pI); and PepDraw was used to assess the hydrophobicity and net charge of the peptides. This process screened out non-toxic, non-sensitizing, and highly bioactive peptides.

[0085] The second step involves using the BIOPEP database to predict whether the peptide is a potential precursor to a bioactive peptide. This is achieved by calculating the frequency of bioactive fragments in the protein sequence (A) and the potential bioactivity of the protein fragment (B), to preliminarily determine its potential as an ACE inhibitory peptide. PepSite 2 analysis is then used to analyze the interaction between the peptide and ACE (PDB: 108A). A P < 0.05 indicates a strong binding affinity between the peptide and the ACE molecule, suggesting that the peptide is a potential ACE inhibitory peptide. The third step involved using the BIOPEP database to simulate the breakdown of peptides by pepsin 13 (EC 3.4.23.1), trypsin 12 (EC 3.4.21.4), and chymotrypsin 11 (EC 3.4.21.1) to screen for peptides that still possess potential ACE inhibitory activity after gastrointestinal digestion.

[0086] After combining the above three steps, potential ACE inhibitory peptides with high biological activity, non-toxicity, and non-sensitizing properties were initially screened.

[0087] Experimental results showed the predicted evaluation of the bioactivity, sensitization, toxicity, and physicochemical properties of antihypertensive peptides in Bifidobacterium O161CFS postbiotics. 1450 peptides from the Bifidobacterium O161CFS postbiotic were scored using PeptideRanker, with a PeptideRanker score >0.8 as the threshold. 115 peptides with high bioactivity potential were selected; among them, 112 short peptides showed no biotoxicity, and 76 showed no sensitization. These 76 short peptides possess high bioactivity, are non-toxic and non-sensitizing, and have good application potential. Potential biofunctional prediction of these 76 potential antihypertensive peptides revealed that all of them exhibited ACE inhibitory properties. Some short peptides also showed inhibitory activity against dipeptidyl peptidase IV and α-glucosidase, indicating that these short peptides have great potential in lowering blood pressure and blood sugar.

[0088] The interaction between the 76 short peptides with high bioactivity, non-toxicity, and potential ACE inhibitory activity obtained from the above screening was analyzed using the PepSite 2 program. The results showed that 63 of the 76 screened peptides in O161CFS had significant interactions with ACE (P < 0.05). After computer-simulated gastrointestinal digestion, 23 peptides retained ACE inhibitory activity after enzymatic digestion. Most of these peptides also possessed antioxidant, dipeptidyl peptidase IV inhibitor, and Xaa-Pro inhibitor properties, indicating that these 23 peptides showed good stress resistance and are potential ACE inhibitors, antioxidants, and dipeptidyl peptidase-IV inhibitors. (Table 2)

[0089] Table 2. Summary of 23 bioactive, non-toxic, non-allergenic peptides with ACE inhibitory activity from Bifidobacterium O161CFS that retain bioactivity after computer-simulated gastrointestinal digestion.

[0090] Remarks: a: ACE inhibitor; b: dipeptidyl peptidase IV inhibitor; c: xaa-proinhibitor; d: lactocepin inhibitor; e: inhibitor of tripeptidyl peptidase II; f: neprilysin inhibitor; g: antioxidant; h: renin inhibitor; i: dipeptidyl peptidase III inhibitor Finally, based on the PeptideRanker scores from highest to lowest, and combined with the predicted ACE inhibitory activity, α-glucosidase inhibitory activity, and dipeptidyl peptidase IV inhibitory activity, the top five peptides were selected for further molecular docking evaluation, synthesis, and verification of their ACE inhibitory function.

[0091] (3) Molecular docking verification of the interaction between five antihypertensive peptides and ACE protein Molecular docking technology was used to systematically evaluate the interaction and binding affinity between target peptides and ACE. The docking strategy employed a dual-platform complementary validation: precise docking based on the active site was performed using AutoDock Vina 1.2.2, supplemented by blind docking using the CB-DOCK2 online platform (https: / / cadd.labshare.cn / cb-dock2 / ), comprehensively analyzing the peptide-ACE binding mode, binding site, and binding free energy. The peptide's two-dimensional structure was plotted using ChemDraw 22.0.2, and its three-dimensional conformation was constructed and optimized using Chem3D 22.0.0. The receptor was a human ACE crystal structure (PDB ID: 1O8A), which was converted to PDBQT format after removing water of crystallization and adding hydrogen. Vina docking used a 40 Å × 40 Å × 40 Å box to cover the ACE active pocket and surrounding region, and the binding energy (kcal / mol) and optimal conformation were output through conformation search. Further validation was performed using CB-DOCK2 blind docking, where the platform automatically completed preprocessing, protein cavity detection, and batch docking to obtain Vina scores and visualization results.

[0092] Experimental results show that, in the CB-Dock 2 web server, cavities in the ACE molecule (PDB: 1O8A) were first searched, and the top five cavities (C1-C5) in terms of size were selected as candidates for blind docking. The docking results showed that these peptides could dock with all cavities, but cavity C1 showed the best binding results. C1 cavity has the largest volume, with a calculated volume of 13554 Å. 3 It binds to different peptides, and the binding affinity of the peptide to the ACE protein can be predicted based on the docking score. A lower binding score indicates a stronger binding affinity.

[0093] The docking results are shown in Figure 12. The minimum binding fractions of these eight peptides with ACE protein are: GGWPLP (-10.1 Kcal / mol), WPANPPL (-9.7 Kcal / mol), PAMPPL (-10.2 Kcal / mol), VCAFLPLP (-8.7 Kcal / mol), and GLCPLP (-9.1 Kcal / mol), indicating that these peptides have good interactions with ACE protein. As shown in Table 3, the calculated binding fractions obtained in AutodockVina show that these five peptides all have strong binding energies with ACE molecules.

[0094] Table 3. Molecular docking results between CB-Dock2 and AutodockVina

[0095] The results indicate that these five peptides have a strong affinity for ACE molecules and bind spontaneously, which is consistent with the above-mentioned inhibitory ability of the peptides on ACE. Therefore, the inhibitory effect of Bifidobacterium O-161 postbiotic on ACE can be partly attributed to the binding of these peptides to the active site of ACE, thereby exerting an effective inhibitory effect.

[0096] Example 7: Synthesis and activity detection of active antihypertensive peptides in Bifidobacterium O-161 postbiotics Five pure peptides, Gly-Gly-Trp-Pro-Leu-Pro (GGWPLP), Trp-Pro-Ala-Asn-Pro-Pro-Leu (WPANPPL), Gly-Leu-Cys-Pro-Leu-Pro (GLCPLP), Pro-Ala-Met-Pro-Pro-Leu (PAMPPL), and Val-Cys-Ala-Phe-Leu-Pro-Leu-Pro (VCAFLPLP), were synthesized by Shanghai Sangon Biotech Co., Ltd. The purity of the peptides was confirmed by liquid chromatography-mass spectrometry, and the purity was >98%.

[0097] The ACE inhibition rate of the synthesized antihypertensive peptide was detected using the method described in Example 2, and its IC50 was calculated.

[0098] The experimental results are as follows: 1. ACE inhibition rate of peptides Further verification using in vitro ACE activity inhibition confirmed that the five target peptides competitively block the hydrolytic effect of angiotensin I by binding to the active pocket of ACE. Results are as follows: Figure 13 The results showed that the ACE inhibition rates of the target peptides were all above 75%, with the following order from highest to lowest: VCAFPLP (91.87%), PAMPPL (89.87%), WPANPPL (89.63%), GGWPLP (89.45%), and GLCPLP (75.31%). The inhibition rate of captopril, used as a positive control, was 93.48%. Except for GLCPLP, the ACE inhibition rates of the other four peptides showed no statistically significant difference compared to captopril (P>0.05).

[0099] 2. IC50 of peptide inhibition rate against ACE The ACE inhibition IC50 values ​​of five peptides were measured, and the results are shown below. Figure 14The IC50 values ​​of the target peptides ranged from 40.61 μM to 148 μM, with the values ​​increasing from low to high as follows: PAMPPL (IC50 = 41.61 μM), GGWPLP (IC50 = 47.61 μM), GLCPLP (IC50 = 70.76 μM), WPANPPL (IC50 = 109.6 μM), and VCAFPLP (IC50 = 148.0 μM). These results are largely consistent with those obtained from computer simulations and molecular docking analyses.

[0100] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A strain of Bifidobacterium longum subsp. infantis O-161 of human milk origin, characterized in that, The strain belongs to Bifidobacterium longum subsp. infantis, and is named as Bifidobacterium longum subsp. infantis strain Bifidobacterium longum subsp. infantis The isolated strain O-161 is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M 20232203, and the preservation date is November 13, 2023.

2. A postbiotic, characterized in that, It is prepared by fermentation of Bifidobacterium longum subsp. infantis O-161 as described in claim 1, and is a supernatant type metabiotic.

3. The use of the post-genetic agent according to claim 2 in the preparation of products with ACE enzyme inhibition function.

4. The use of the metagenin according to claim 2 in the preparation of products that alleviate AngII-induced endothelial dysfunction in human umbilical vein endothelial cells.

5. The use according to claim 4, wherein the compound is ###0002### Alleviating AngII-induced endothelial dysfunction in human umbilical vein endothelial cells involves promoting the secretion of nitric oxide and endothelial nitric oxide synthase, inhibiting the production of reactive oxygen species, suppressing excessive cell migration, and reducing the transcription and expression of inflammatory factors IL-6 and TNF-α.

6. The use of the post-genetic agent according to claim 2 in the preparation of products with peripheral mesenteric artery dilation function.

7. A class of antihypertensive peptides characterized in that, Derived from the metagenic agent of claim 2, including short peptides with amino acid sequences as shown in any of SEQ ID NO. 1 to 5.

8. The blood pressure lowering peptide according to claim 7, wherein, The blood pressure lowering peptide has an ACE inhibition rate of ≥75%, and a half-inhibition concentration IC 50 value is 14.07 μM-148 μM.

9. The use of the antihypertensive peptide according to claim 7 or 8 in the preparation of a product having ACE enzyme inhibitory function.

10. The use of the antihypertensive peptide according to claim 7 or 8 in the preparation of products with antihypertensive function.

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