Lactobacillus rhamnosus post-fermentation biogen, preparation method thereof and application of lactobacillus rhamnosus post-fermentation biogen in improvement of atherosclerosis, reduction of thrombosis risk and regulation of ceramide metabolism

Abstract

The invention belongs to the technical field of preparation of fermented biogen, and particularly relates to lactobacillus rhamnosus fermented biogen, a preparation method of the lactobacillus rhamnosus fermented biogen and application of the lactobacillus rhamnosus fermented biogen to improvement of atherosclerosis, reduction of thrombosis risk and regulation of ceramide metabolism. In the preparation process of the lactobacillus fermented biogen, yeast protein releases polypeptide with vasodilation and inflammation inhibition functions, and after the polypeptide is matched with anthocyanin and araboxylan oligosaccharide which have the functions of regulating ceramide metabolism and protecting the cardiovascular system, the activity of the polypeptide is improved; under the fermentation action of the lactobacillus rhamnosus NKU ML1-2, lactobacillus fermentation postbiotics with the function of protecting heart and cerebral vessels are formed, and the lactobacillus fermentation postbiotics as a dietary nutrition intervention means can directly act on vascular cells through multiple ways to promote the function repair of endothelial cells; apoptosis and oxidative damage of endothelial cells are inhibited, generation of ceramide substances is regulated, and an innovative nutrition intervention strategy is provided for prevention and treatment of atherosclerosis related diseases and prevention of thrombosis.

Description

Technical Field

[0001] This invention belongs to the field of post-fermentation biogenic preparation technology, specifically relating to post-fermentation biogenics of Lactobacillus rhamnosus, its preparation method, and its application in improving atherosclerosis, reducing the risk of thrombosis, and regulating ceramide metabolism. Background Technology

[0002] Atherosclerosis (AS) is a chronic inflammation of the blood vessel wall. Ceramides and tumor necrosis factor-α (TNF-α) can enhance vascular inflammation and influence the development of AS. AS is also a major pathogenic mechanism of coronary heart disease and has become one of the important causes of death from non-communicable diseases. Studies known to the inventors have shown that endothelial dysfunction is a core element in the occurrence and progression of AS, and the activity and oxidative stress levels of endothelial cells (EPCs) are closely related to the AS process. Furthermore, ceramides show significant accumulation in the development of AS, promoting necrotic core formation by inducing macrophage pyroptosis and exacerbating plaque instability.

[0003] Currently, existing medications for treating atherosclerosis mainly include statins, antiplatelet drugs, and antihypertensive drugs. However, statins may cause adverse reactions such as muscle pain, liver damage, and elevated blood sugar; antiplatelet drugs such as aspirin increase the risk of bleeding; and long-term use may lead to cumulative toxicity.

[0004] Compared to existing probiotics and their compositions known to the inventors, postbiotics exhibit higher stability, are unaffected by biological activity, and have greater safety, specifically in the following four aspects: 1) Significantly superior stability compared to existing probiotics and their compositions: Existing probiotics and their compositions rely on the activity of live bacteria. Live bacteria are sensitive to environmental factors such as temperature, acidity / alkalinity, processing, and storage, and their survival rate is easily reduced. Cold chain and special encapsulation are required to maintain activity, increasing production, storage, and transportation costs and difficulties. Postbiotics are non-living microorganisms and active ingredient complexes, do not rely on live bacteria, have extremely strong environmental tolerance, and their active ingredients remain stable under room temperature storage and high-temperature processing. They require no special protection, are compatible with various products, reduce storage and transportation costs, and facilitate large-scale promotion. 2) Unaffected by biological activity, with more stable and controllable efficacy: The efficacy of existing probiotics depends on the colonization of live bacteria in the gut, which is greatly affected by the environment and individual host differences. Insufficient live bacteria or colonization failure can lead to unstable efficacy or even ineffectiveness. Postbiotics do not contain live bacteria, and their active ingredients can be directly absorbed by the human body, unaffected by individual differences or the activity of live bacteria. Their efficacy is stable and controllable, and can be precisely adjusted through ingredient content. Verification has shown that some postbiotics are no less effective than live probiotics. 3) Safety is higher than existing probiotics and their combinations: Existing probiotics contain live bacteria, which pose risks of infection and bacterial translocation to immunocompromised individuals and other special populations. Some strains may carry drug-resistant genes, rendering them ineffective and increasing health risks when used concurrently with antibiotics. 4) Postbiotics undergo inactivation treatment to eliminate live bacteria, thus avoiding related safety risks and making them suitable for special populations. Their ingredients are clearly defined and purified, eliminating the risk of drug-resistant genes, allowing for concurrent use with antibiotics. They are applicable to a wide range of populations, and no significant adverse reactions have been observed clinically, demonstrating a significant safety advantage.

[0005] It is evident that postbiotics have significant advantages in stability, bioactivity, and safety compared to probiotics and their combined products. However, postbiotic products that can improve symptoms related to atherosclerosis still require further development and research. Summary of the Invention

[0006] The purpose of this invention is to provide a post-fermentation biotic of Lactobacillus rhamnosus, its preparation method, and its application in improving atherosclerosis, reducing the risk of thrombosis, and regulating ceramide metabolism. The post-fermentation biotic of Lactobacillus rhamnosus can directly act on vascular cells, promote endothelial cell function repair, inhibit endothelial cell apoptosis and oxidative damage, and regulate the production of ceramides, providing an innovative nutritional intervention strategy for the prevention and treatment of atherosclerosis-related diseases and the prevention of thrombosis.

[0007] This invention provides a method for preparing lactobacillus post-fermentation biogenics, comprising the following steps: fermenting *Lactobacillus rhamnosus* (… Lacticaseibacillus rhamnosus NKU ML1-2 was inoculated into a fermentation system for liquid fermentation to obtain the liquid fermentation product; the biopreservation name of the Lactobacillus rhamnosus NKU ML1-2 is... Lacticaseibacillus rhamnosusNKU ML1-2, taxonomic name is Lacticaseibacillus rhamnosus The deposit date is August 13, 2025. The depositary institution is Guangdong Provincial Center for Microbial Culture Collection (GDMCC). The depositary address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, Institute of Microbiology. The deposit number is GDMCC No:66837. The bacterial cells in the liquid fermentation product are removed to obtain the post-fermentation biogener of Lactobacillus; The fermentation system uses water as a solvent and includes yeast protein, arabinoxylan, and blueberry powder. The amount of yeast protein added is 10% to 15% of the mass of the fermentation system, the amount of arabinoxylan added is 2% to 10% of the mass of the fermentation system, and the amount of blueberry powder added is 0.2% to 1% of the mass of the fermentation system. The anthocyanin content in the blueberry powder is 20% to 40%.

[0008] Preferably, the inoculum size of *Lactobacillus rhamnosus* NKU ML1-2 is 10. 7 ~10 9 CFU / mL.

[0009] Preferably, the liquid fermentation is a shaking fermentation, the shaking fermentation speed is 180~220 rpm, the shaking fermentation temperature is 35~40℃, and the shaking fermentation time is 22~26 h.

[0010] The present invention also provides a post-fermentation biogenic agent of Lactobacillus, which is prepared by the preparation method described in the above technical solution; the post-fermentation biogenic agent of Lactobacillus contains short-chain fatty acids and / or polypeptides.

[0011] Preferably, the short-chain fatty acids include one or more of butyric acid, propionic acid, acetic acid, nonanoic acid, hexanoic acid, heptanoic acid, isobutyric acid, isovaleric acid, valeric acid, decanoic acid, and caprylic acid.

[0012] This invention also provides one or both of the following applications of the lactobacillus fermentation precursor described in the above technical solution: 1) Application in the preparation of products for the prevention and / or improvement of atherosclerosis; 2) Application in the preparation of products for preventing thrombosis.

[0013] Preferably, the product includes products that improve vascular endothelial dysfunction.

[0014] Preferably, the improvement of vascular endothelial dysfunction includes one or more of the following: improving inflammatory damage to vascular cells, improving oxidative damage to vascular cells, and regulating ceramide metabolism in vascular cells.

[0015] The present invention also provides a product for preventing and / or improving atherosclerosis, the product comprising the lactobacillus fermentation precursor described in the above technical solution.

[0016] Preferably, the lactobacillus fermentation precursor is the only effective component.

[0017] Beneficial effects: This invention provides a method for preparing post-fermentation biogenics from Lactobacillus, comprising the following steps: inoculating Lactobacillus rhamnosus NKU ML1-2 into a fermentation system for liquid fermentation to obtain a liquid fermentation product; removing the bacterial cells from the liquid fermentation product to obtain the post-fermentation biogenics from Lactobacillus; the fermentation system uses water as a solvent and includes yeast protein, arabinoxylan, and blueberry powder, wherein the amount of yeast protein added is 10%~15% of the mass of the fermentation system, the amount of arabinoxylan added is 2%~10% of the mass of the fermentation system, and the amount of blueberry powder added is 0.2%~1% of the mass of the fermentation system; the anthocyanin content in the blueberry powder is 20%~40%; the biopreservation name of Lactobacillus rhamnosus NKU ML1-2 is... Lacticaseibacillus rhamnosus NKU ML1-2, with accession number GDMCC No: 66837. In the preparation of the lactobacillus fermentation post-biotic described in this invention, the fermentation product of the probiotic *Lactobacillus rhamnosus* NKU ML1-2 is rich in functional factors such as vasodilatory peptides and short-chain fatty acids; yeast protein releases polypeptides with vasodilatory and anti-inflammatory functions, which, after being combined with anthocyanins and arabinoxylan oligosaccharides that regulate ceramide metabolism and protect cardiovascular function, form a lactic acid bacteria fermentation post-biotic with cardiovascular and cerebrovascular protective functions under the fermentation action of *Lactobacillus rhamnosus* NKU ML1-2. As a dietary nutritional intervention, this lactic acid bacteria fermentation post-biotic can directly act on vascular cells through multiple pathways to promote endothelial cell function repair, inhibit endothelial cell apoptosis and oxidative damage, and regulate the production of ceramide substances, providing an innovative nutritional intervention strategy for the prevention and treatment of atherosclerosis-related diseases and the prevention of thrombosis. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0019] Figure 1 This is a standard curve of polypeptide content in the probiotics after fermentation with Lactobacillus rhamnosus in Example 1; Figure 2 The graph shows the results of determining the polypeptide content in the probiotics after fermentation with Lactobacillus rhamnosus in Example 1. Figure 3 The graph shows the results of determining the polypeptide content in the probiotics after fermentation with Lactobacillus rhamnosus in Example 2. Figure 4 The results of the angiotensin-converting enzyme ACE inhibitor activity assay of the progenitor cells after fermentation with Lactobacillus rhamnosus in Example 2; Figure 5 The results of the DPPH free radical scavenging rate determination of the biogenic agent after fermentation of Lactobacillus rhamnosus in Example 2; Figure 6 The content of short-chain fatty acids in the biogenic elements after fermentation of Lactobacillus rhamnosus in Example 5; Figure 7 The graph shows the results of the determination of angiotensin-converting enzyme ACE inhibitor activity and DPPH free radical scavenging rate of the progenitor cells after fermentation with Lactobacillus rhamnosus in Example 5. Figure 8 This is a verification result of the biogenic efficacy of Lactobacillus rhamnosus fermentation in enhancing the activity of vascular endothelial cells (HUVECs) in Example 6; Figure 9 The results of the assay in Example 7 show that the biogenics of Lactobacillus rhamnosus fermentation alleviated the inflammatory damage of HUVECs induced by oxidized low-density lipoprotein. Figure 10 The results of the assay in Example 8 show that the biogenics of Lactobacillus rhamnosus fermentation alleviated oxidative damage to HUVECs induced by oxidized low-density lipoprotein. Figure 11 The results of the assay for the effect of Lactobacillus rhamnosus fermentation on the oxidized low-density lipoprotein-induced apoptosis of HUVECs cells were obtained in Example 9. Figure 12 The results of the assay for ceramide production in HUVECs induced by oxidized low-density lipoprotein after fermentation of Lactobacillus rhamnosus in Example 10 are as follows.

[0020] Biological Preservation Information: Lacticaseibacillus rhamnosus NKU ML1-2, taxonomic name is Lacticaseibacillus rhamnosus Latin name Lacticaseibacillus rhamnosus It was deposited on August 13, 2025 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, with accession number GDMCC No: 66837. Detailed Implementation

[0021] This invention provides a method for preparing post-fermentation biogenics from Lactobacillus, comprising the following steps: inoculating Lactobacillus rhamnosus NKU ML1-2 into a fermentation system for liquid fermentation to obtain a liquid fermentation product; the biopreservation name of Lactobacillus rhamnosus NKUML1-2 is... Lacticaseibacillus rhamnosus NKU ML1-2, accession number GDMCCNo: 66837; The bacterial cells in the liquid fermentation product are removed to obtain the post-fermentation biogener of Lactobacillus; The fermentation system uses water as a solvent and includes yeast protein, arabinoxylan, and blueberry powder. The amount of yeast protein added is 10% to 15% of the mass of the fermentation system, the amount of arabinoxylan added is 2% to 10% of the mass of the fermentation system, and the amount of blueberry powder added is 0.2% to 1% of the mass of the fermentation system. The anthocyanin content in the blueberry powder is 20% to 40%.

[0022] This invention involves inoculating *Lactobacillus rhamnosus* NKU ML1-2 into a fermentation system for liquid fermentation to obtain a liquid fermentation product. The fermentation system uses water as a solvent and includes yeast protein, arabinoxylan, and blueberry powder. The amount of yeast protein added is 10%–15% of the fermentation system mass, the amount of arabinoxylan added is 2%–10% of the fermentation system mass, and the amount of blueberry powder added is 0.2%–1% of the fermentation system mass. As one embodiment, the amount of yeast protein added can be 10%–12%; the protein content of the yeast protein is ≥70.0 g / 100 g; and the yeast protein can release polypeptides with vasodilatory and anti-inflammatory functions in the fermentation system. This invention does not have a specific limitation on the source of the yeast protein; it can be purchased from conventional commercial channels in the art. As one embodiment, the amount of arabinoxylan added is 5%–8%; the CAS number of the arabinoxylan is 9040-27-1; and the arabinoxylan has cardiovascular protective effects. This invention does not specifically limit the source of the arabinoxylan; it can be purchased from conventional commercial channels in the art. As one embodiment, the amount of blueberry powder added is 0.2%~0.5%; the anthocyanin content in the blueberry powder is 20%~40%, further preferably 25%~40%; the anthocyanins have cardiovascular protective effects and can promote the utilization of arabinoxylan by *Lactobacillus rhamnosus*; this invention does not specifically limit the source of the blueberry powder; it can be conventionally purchased or prepared by the manufacturer, as long as it meets the 20%~40% anthocyanin content requirement. As one embodiment, the inoculum size of *Lactobacillus rhamnosus* NKU ML1-2 is 10... 7 ~10 9 CFU / mL, further to 10 8CFU / mL; compared with other Lactobacillus rhamnosus, the Lactobacillus rhamnosus NKU ML1-2 has an anti-inflammatory advantage. As one embodiment, the liquid fermentation is a shaking fermentation, with a shaking speed of 180-220 rpm, more preferably 220 rpm; the shaking fermentation temperature is 35-40℃, more preferably 37-38℃; and the shaking fermentation time is 22-26 h, more preferably 23-24 h. In this invention, the liquid fermentation has the advantage of promoting the fermentation and transformation of substrate active substances by Lactobacillus rhamnosus and reducing fermentation time.

[0023] After obtaining the liquid fermentation product, the present invention removes the bacterial cells from the liquid fermentation product to obtain the post-fermentation probiotic of Lactobacillus. As one embodiment, the bacterial cells can be removed by microfiltration, using a membrane with a pore size of 0.22 μm.

[0024] This invention also provides a post-fermentation biogenic agent from *Lactobacillus*, prepared using the method described above; the post-fermentation biogenic agent from *Lactobacillus* contains short-chain fatty acids and / or polypeptides. As one embodiment, the short-chain fatty acids include, but are not limited to, one or more of butyric acid, propionic acid, acetic acid, nonanoic acid, hexanoic acid, heptanoic acid, isobutyric acid, isovaleric acid, valeric acid, decanoic acid, and octanoic acid, and may further be one or more of acetic acid, propionic acid, butyric acid, isobutyric acid, and isovaleric acid.

[0025] The present invention also provides the application of one or two of the following biogenic elements derived from Lactobacillus fermentation described in the above technical solutions: 1) application in the preparation of products for preventing and / or improving atherosclerosis; 2) application in the preparation of products for preventing thrombosis. As one embodiment, the product includes a product for improving vascular endothelial dysfunction; further, the improvement of vascular endothelial dysfunction may be one or more of improving inflammatory damage to vascular cells, improving oxidative damage to vascular cells, and regulating ceramide metabolism in vascular cells.

[0026] This invention also provides a product for preventing and / or improving atherosclerosis, the product comprising the lactobacillus fermentation precursor described in the above-described technical solution. As one embodiment, in the product, the lactobacillus fermentation precursor is the sole active ingredient. This invention does not impose any special limitations on the amount and type of excipients in the product; conventional selection or limitation is acceptable as needed.

[0027] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0028] Example 1 The optimal amount of yeast protein was selected for the fermentation substrate formulation scheme. The steps are as follows: 1. The fermentation substrates were set up into the following groups, and the optimal yeast protein fermentation concentration was screened based on the amount of peptides released: Take cryopreserved *Lactobacillus rhamnosus* NKU ML1-2 and add 24 mL of MRS medium to a 50 mL centrifuge tube to fully activate the 1 mL of bacterial culture. Incubate at 37°C on a shaker for 24 hours, then subculture. For subculture, mix 1 mL of bacterial culture with 30 mL of MRS medium and incubate at 37°C on a shaker for 20-24 hours. Subculture three times to obtain the subcultured bacterial culture. Take 40 mL of the subcultured bacterial culture, centrifuge at 6000g for 10 minutes to obtain bacterial sludge, and resuspend the sludge in 20 mL of sterile water.

[0029] Set up a 20 mL fermentation system, using water as the solvent, with an inoculum size of 10. 8 The amounts of yeast protein (Angel Yeast Protein F80EC, hereinafter the same) added were 2%, 5%, 10%, and 15% of the fermentation system mass, respectively, and labeled as JM2%, JM5%, JM10%, and JM15%. Liquid fermentation was adopted, and the mixture was fermented in a shaker at 37℃ for 24 hours, followed by centrifugation at 8000 rpm for 10 minutes. The supernatant was then filtered through a 0.22 μm filter membrane to effectively remove bacterial cells, yielding the post-fermentation biogenic agent of *Lactobacillus rhamnosus*.

[0030] 2. Determination of the content and activity of biotic components in Lactobacillus rhamnosus after fermentation The peptide content in the JM2%, JM5%, JM10%, and JM15% groups was determined using the o-phthalaldehyde (OPA) method: 20 μL of the sample prepared in step 1 was mixed with 200 μL of OPA solution. The mixture was incubated at room temperature for 2 min, and the absorbance was read at 340 nm using a microplate reader. A standard curve was constructed using L-glutathione to calculate the peptide content. The glutathione standard curve concentrations (mg / mL) were set as follows: 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, and 1.5. Figure 1 As shown in the figure. The OPA mixture is prepared as follows: Weigh 7.620g of sodium tetraborate decahydrate, 200mg of sodium dodecyl sulfate (SDS), and 176mg of dithiothreitol (DTT) reagent and dissolve them in 150mL of water to obtain a mixed solution; dissolve 160mg of OPA in 4mL of ethanol and add it to the prepared mixed solution above, then dilute to 200mL with deionized water to obtain the OPA mixture.

[0031] The results of the polypeptide content determination are as follows: Figure 2As shown, inoculation with *Lactobacillus rhamnosus* NKU ML1-2 resulted in significant hydrolysis of yeast protein and a significant increase in peptide release. The JM2% group had the lowest peptide content (0.888±0.05), followed by the JM5% group (1.538±0.03). While there was no significant difference between the JM10% and JM15% groups, both were significantly higher than the JM2% and JM5% groups. This indicates that peptide release increases with increasing yeast protein content, but stops increasing above 10%. Therefore, 10% is considered the optimal yeast protein content.

[0032] Example 2 The steps for screening the optimal addition amount of bacterial strains in the fermentation substrate compounding scheme are as follows: 1. Yeast protein peptides have been shown to have cardiovascular protective effects; ACE can constrict blood vessels and disrupt endothelial cell function; oxidative stress is one of the core driving forces of endothelial dysfunction and atherosclerosis. Therefore, the fermentation substrate was divided into the following groups, and the optimal strain addition was screened based on peptide release, angiotensin-converting enzyme (ACE) inhibitory activity, and product antioxidant activity: Take cryopreserved *Lactobacillus rhamnosus* NKU ML1-2 and add 24 mL of MRS medium to a 50 mL centrifuge tube to fully activate the 1 mL of bacterial culture. Incubate at 37°C on a shaker for 24 hours, then subculture. During subculturing, mix 1 mL of bacterial culture with 30 mL of MRS medium and incubate at 37°C on a shaker for 20-24 hours to obtain the subcultured bacterial culture. Take 40 mL of the subcultured bacterial culture, centrifuge at 6000g for 10 minutes to obtain bacterial sludge, and resuspend the sludge in 20 mL of sterile water.

[0033] A 20 mL fermentation system was set up, with the yeast protein added at the optimal amount shown in Example 1, and the inoculum size was 10. 6 10 7 10 8 10 9 The CFU / mL samples were labeled LJM6, LJM7, LJM8, and LJM9, respectively. A liquid fermentation method was used, with the samples fermented on a shaker at 37℃ for 24 hours, followed by centrifugation at 8000 rpm for 10 minutes. The supernatant was then filtered through a 0.22 μm filter membrane to effectively remove bacterial cells, yielding the post-fermentation biogenic strain of *Lactobacillus rhamnosus*.

[0034] 2. The polypeptide content in groups LJM6, LJM7, LJM8, and LJM9 was determined using the o-phthalaldehyde (OPA) method, the same as in Example 1. The results are as follows: Figure 3 As shown, based on a 10% yeast protein addition, changing the inoculum amount of the strains did not result in significant differences in peptide levels among the LJM7, LJM8, and LJM9 groups, but all were significantly higher than those in the LJM6 group.

[0035] 3. The ACE inhibitory activity of the LJM6, LJM7, LJM8, and LJM9 groups was determined using an angiotensin-converting enzyme (ACE) inhibitor activity assay kit (purchased from Beijing Solarbio Science & Technology Co., Ltd., catalog number: BC5570). The results are as follows: Figure 4 As shown, it can be seen that, based on the addition of 10% yeast protein, changing the inoculum amount of the strain yielded results similar to those obtained from the peptide level assay. That is, there was no significant difference in the ACE inhibition rate among the LJM7, LJM8, and LJM9 groups, but all of them were significantly higher than that of the LJM6 group.

[0036] 4. The antioxidant activity of LJM6, LJM7, LJM8, and LJM9 groups was determined using a DPPH free radical scavenging experiment. The specific procedure was as follows: 0.5 mL of post-biotic solution was mixed with an equal volume of 0.2 Mm DPPH anhydrous ethanol solution, allowed to stand in the dark for 30 min, centrifuged at 8000g for 10 min, and the supernatant was measured at 517 nm for absorbance. ABTS free radical scavenging ability: ABTS stock solution (7.4 mmol / L) and K2S2O8 stock solution (2.6 mmol / L) were mixed and allowed to stand at room temperature in the dark for 12 h, then diluted 50 times with anhydrous ethanol. A0 value detection: 1.6 mL of the ABTS solution and 0.6 mL of anhydrous ethanol were thoroughly mixed, and the absorbance was measured at 734 nm. A value detection: 1.6 mL of ABTS was mixed with 0.6 mL of fermentation broth, and the absorbance was measured at 734 nm. Scavenging rate = (A0 - A) / A0 × 100%. Results are as follows. Figure 5 As shown in the figure, it can be seen that, based on a 10% yeast protein addition, changing the inoculum amount of the strains did not result in significant differences in ACE inhibition rates among the LJM7, LJM8, and LJM9 groups, but all were significantly higher than those of the LJM6 group. This result is similar to the results of peptide level and ACE inhibition activity assays.

[0037] Based on the detection results in steps 2-4, with 10 8 The optimal addition amount is determined by the strain concentration of CFU / mL.

[0038] Example 3 The optimal addition ratio of arabinoxylan and blueberry powder was determined through the following steps: 1. The fermentation substrates were set into the following groups, using angiotensin-converting enzyme (ACE) inhibitory activity and product antioxidant activity as standards to screen the optimal blending ratio of arabinoxylan and blueberry powder (purchased from Taobao, store name: Peptide Beauty Bio-Raw Material Manufacturer, product name: Blueberry Anthocyanin 40% Blueberry Powder Anthocyanin Powder, the same applies to the following examples): Take cryopreserved *Lactobacillus rhamnosus* NKU ML1-2 and add 24 mL of MRS medium to a 50 mL centrifuge tube to fully activate the 1 mL of bacterial culture. Incubate at 37°C on a shaker for 24 hours, then subculture. During subculturing, mix 1 mL of bacterial culture with 30 mL of MRS medium and incubate at 37°C on a shaker for 20-24 hours to obtain the subcultured bacterial culture. Take 40 mL of the subcultured bacterial culture, centrifuge at 6000g for 10 minutes to obtain bacterial sludge, and resuspend the sludge in 20 mL of sterile water.

[0039] A 20 mL fermentation system was set up. The amount of yeast protein added was the optimal amount obtained in Example 1, the amount of bacterial strain added was the optimal amount in Example 2, the amount of arabinoxylan added was set to 2%, 5%, 10% and 20% respectively, and the amount of blueberry powder added was 0.2%, 0.5% and 1% respectively. The specific settings of the groups are shown in Table 1. Each group was placed in a shaker at 37℃ for 24 h and then centrifuged at 8000 rpm for 10 min. The supernatant was taken and filtered through a 0.22 μm filter membrane to effectively filter out the bacterial cells, and the fermented probiotic of Lactobacillus rhamnosus was obtained.

[0040] 2. The ACE inhibitory activity of each group with 2% to 20% arabinoxylan and 0.2% to 1% blueberry powder was determined using an angiotensin-converting enzyme (ACE) inhibitor activity assay kit (purchased from Beijing Solarbio Science & Technology Co., Ltd., catalog number: BC5570). The results are shown in Table 1.

[0041] Table 1. Results of ACE inhibitory activity assays for each group in the arabinoxylan and blueberry powder compound system.

[0042] Note: Different lowercase letters in the same column indicate significant differences, as in Table 2.

[0043] Table 1 shows that with a yeast protein addition of 10%, 10 8 At an inoculum size of *Lactobacillus rhamnosus* NKU ML1-2 (CFU / mL), the addition of 2%–10% arabinoxylan and 0.2%–1% blueberry powder consistently maintained an ACE inhibition rate >50%. However, at 2%–5% arabinoxylan levels, the ACE inhibition rate decreased with increasing blueberry powder content, indicating that the carbon-to-nitrogen ratio of yeast protein to arabinoxylan was the most significant factor affecting the ACE inhibition rate. The highest ACE inhibition rates were observed with a ratio of 0.2% blueberry powder, 10% yeast protein, and 2% or 5% arabinoxylan, at 62.47±14.21% and 63.46±8.06%, respectively.

[0044] 3. The antioxidant activity of each group with 2%~20% arabinoxylan and 0.2%~1% blueberry powder was determined by the DPPH free radical scavenging experiment. The results are shown in Table 2.

[0045] Table 2. DPPH free radical scavenging rate determination results of each group in the arabinoxylan and blueberry powder compound system.

[0046] Table 2 shows that: 10% yeast protein addition, 10 8 At an inoculum concentration of *Lactobacillus rhamnosus* (CFU / mL), the addition of 2%–10% arabinoxylan and 0.2%–1% blueberry powder all ensured a DPPH free radical scavenging rate >90%. The highest DPPH free radical scavenging rates were observed with a ratio of 10% yeast protein, 5%–20% arabinoxylan, and 0.2% blueberry powder, at 93.97±0.12%, 94.72±0.474%, and 95.21±0.68%, respectively.

[0047] Based on the comprehensive ACE inhibitory activity, the optimal ratio was set as 10% yeast protein, 5% arabinoxylan, 0.2% blueberry powder, and 10 8 Fermentation with Lactobacillus rhamnosus NKU ML1-2 at CFU / mL.

[0048] Example 4 The fermentation synergy of Lactobacillus rhamnosus NKU ML1-2 was confirmed by the following steps: The following groups were set up, using angiotensin-converting enzyme (ACE) inhibitory activity and product antioxidant activity as standards, to confirm the fermentation synergistic effect of Lactobacillus rhamnosus NKU ML1-2: Take cryopreserved *Lactobacillus rhamnosus* NKU ML1-2 and add 24 mL of MRS medium to a 50 mL centrifuge tube to fully activate the 1 mL of bacterial culture. Incubate at 37°C on a shaker for 24 hours, then subculture. During subculturing, mix 1 mL of bacterial culture with 30 mL of MRS medium and incubate at 37°C on a shaker for 20-24 hours to obtain the subcultured bacterial culture. Take 40 mL of the subcultured bacterial culture, centrifuge at 6000g for 10 minutes to obtain bacterial sludge, and resuspend the sludge in 20 mL of sterile water.

[0049] A 20 mL fermentation system was set up. The amount of yeast protein added was the optimal amount obtained in Example 2, and the amounts of arabinoxylan and blueberry powder added were the optimal amounts obtained in Example 3. Fermentation strains were added or not added; the group with added strains was labeled LFP, and the group without added strains was labeled FP. The mixture was fermented in a shaker at 37°C for 24 h, then centrifuged at 8000 rpm for 10 min. The supernatant was then filtered through a 0.22 μm filter membrane to effectively remove bacterial cells.

[0050] Example 5 Lactobacillus rhamnosus fermentation promotes the release of short-chain fatty acids (SCFAs), and the steps are as follows: 1. Based on the ingredient ratio of 10% yeast protein, 5% arabinoxylan, and 0.2% blueberry powder in Example 4, 10% of each ingredient was added. 6 10 7 10 8 10 9 Lactobacillus rhamnosus NKU ML1-2 at CFU / mL were fermented according to the method in Example 4. The fermented groups were labeled LFP6, LFP7, LFP8, and LFP9, respectively, while the unfermented group was labeled FP.

[0051] 2. The content of short-chain fatty acids in the FP and LFP groups in Example 4 was determined by gas chromatography-mass spectrometry (GC-MS). The experimental method is as follows: The fermentation sample obtained in step 1 was vortexed with 50% sulfuric acid solution for 30 s, shaken for 10 min, and then sonicated for 10 min (placed in ice water). It was centrifuged at 10,000 rpm for 15 min at 4 °C. It was placed at -20 °C for 30 min, and the supernatant was used for GC-MS analysis. An Agilent GC7890B-5977B gas chromatograph-mass spectrometer was used. An HP-FFAP capillary column was used. The sample was injected at a volume of 1 μL in split mode (5:1). Helium was used as the carrier gas, the purge flow rate before the injection port was 3 mL / min, and the column flow rate was 1.2 mL / min. The initial temperature was maintained at 50°C for 1 min, then increased to 150°C for 1 min at a rate of 50°C / min, then to 170°C for 0 min at a rate of 10°C / min, followed by 225°C for 1 min at a rate of 25°C / min, and finally to 240°C for 1 min at a rate of 40°C / min. The temperatures of the injection port, transfer line, quadrupole, and ion source were 220°C, 240°C, 150°C, and 240°C, respectively. The energy in electron bombardment mode was -70 electron volts.

[0052] The results show that Figure 6As shown, fermentation significantly enriches short-chain fatty acids in the system, with acetic acid, propionic acid, isobutyric acid, isovaleric acid, and butyric acid being the most abundant components. SCFAs have significant protective effects against cardiovascular and cerebrovascular diseases. Propionic acid improves vascular endothelial function by inhibiting the NF-κB pathway, reducing the release of inflammatory factors (such as TNF-α and IL-6); acetic acid provides energy substrates for cardiomyocytes, improving cardiac energy supply and reducing oxidative stress damage to cardiomyocytes; butyric acid reduces the release of inflammatory factors and delays lipid deposition and foam cell formation in the arterial wall by inhibiting histone deacetylase (HDAC) activity. Lactobacillus rhamnosus NKU ML1-2 at 10 6 At an inoculum level of CFU / mL (LFP6), the content of short-chain fatty acids is less than 10. 7 (LFP7), 10 8 (LFP8) and 10 9 The inoculum amount was (LFP9) CFU / mL, with the short-chain fatty acid content of the LFP8 group being 319.59 μg / mL, which was slightly higher than that of LPF7 (310.61 μg / mL) and LPF9 (316.37 μg / mL).

[0053] 3. The effects of fermentation on the ACE inhibitory and antioxidant activities of the compound components were determined using an angiotensin-converting enzyme (ACE) inhibitor activity assay kit and a DPPH free radical scavenging rate experiment. The assay method was the same as in Example 2, and the results are as follows: Figure 7 As shown.

[0054] Depend on Figure 7 It can be concluded that with a ratio of 10% yeast protein, 5% arabinoxylan and 0.2% blueberry powder, the compound component has ACE inhibitory and DPPH scavenging activities. However, fermentation with Lactobacillus rhamnosus NKU ML1-2 can significantly enhance these activities, with ACE inhibitory activity enhanced by about 30% and DPPH free radical scavenging activity enhanced by about 40%.

[0055] Example 6 The verification of the biogenic efficacy of Lactobacillus rhamnosus fermentation in enhancing the activity of vascular endothelial cells (HUVECs) was performed using the following steps: DMEM F12 medium was purchased from Gibco, catalog number C11330500BT. The culture of vascular endothelial cells (HUVECs) was as follows: the medium contained 89% DMEM-F12, 1% penicillin-dextrose antibody, and 10% serum. Cells in T25 culture flasks were digested with trypsin, transferred to 15 mL centrifuge tubes, centrifuged at 1500 rpm for 5 min, and resuspended in 6 mL of medium.

[0056] The cell suspension contains 2 × 10 cells 5 / mL, dilute the stock solution to 5.34×104 Cells / mL were seeded at 8000 and 10000 cells per well, respectively, by diluting the stock solution and seeding at 150 μL or 200 μL per well. LFP8 from Example 5 was used as the intervention and diluted 2, 5, 10, 20, 50, and 100 times with complete culture medium. After 24 h of cell adhesion, the culture medium was aspirated, and 150 μL of the diluted intervention was added. After 20 h of intervention culture, 15 μL of CCK-8 reagent solution was added to the original culture medium. After 2 h of culture, cell viability was measured at 450 nm.

[0057] After 20 hours of intervention with different concentrations of LFP8, the effect on vascular endothelial cells (HUVECs) was determined using a CCK-8 cell viability assay kit (Beyotime Biotechnology Co., Ltd., catalog number C0038). The results are as follows: Figure 8 As shown, the cell viability was better when the cell dilution was 150 μL per well than when the cell dilution was 200 μL. Furthermore, when the fermentation broth was diluted to 20, 50, and 100, the cell viability was even better than the blank control group, with cell viability rates of 1.080±0.145, 1.043±0.106, and 1.080±0.169, respectively. At these dilutions, the metabiotic provided nutrients to the cells, which was more conducive to cell proliferation. Therefore, based on this experiment, this embodiment determined the LFP8 dilutions used to be 20-fold, 50-fold, and 100-fold, and set the cell density for 96-well plates to 8000 cells / well for subsequent experiments.

[0058] Example 7 Lactobacillus rhamnosus fermentation postbiotic alleviates oxidized low-density lipoprotein (ox-LDL)-induced inflammatory damage in HUVECs. The culture method for HUVECs was the same as in Example 6. The LFP8 post-biotic prepared in Example 5 was diluted 20-fold, 50-fold, and 100-fold with complete culture medium as high-dose, medium-dose, and low-dose interventions, respectively. Atorvastatin (5 μM) was set up as a positive control group. After HUVECs adhered to the culture vessel for 24 hours, the culture medium was aspirated, and 150 μL of the diluted intervention was added. After 20 hours of intervention culture, ox-LDL reagent (Beijing Solarbio Science & Technology Co., Ltd., catalog number IO1300, specification 2 mg / mL) was added to the original culture medium at a rate of 200 μL / mL. After 24 hours of culture, the cell culture medium was collected for analysis.

[0059] The methods for measuring inflammatory factors are as follows: ELISA kits for TNF-α, IL-1β, and MCP-1 were purchased from Jiangsu Enzyme-Label Biotechnology Co., Ltd., with catalog numbers MB-0122A, MB-0181A, and MB-0081A, respectively. After collecting cell culture medium, the cells were centrifuged at 3000 rpm for 20 min, and the supernatant was collected. The contents of TNF-α, IL-1β, and MCP-1 were measured according to the ELISA kit instructions.

[0060] The results are as follows Figure 9 As shown, the release levels of the three inflammatory factors in the blank control group were as follows: TNF-α: 8.259±0.2589 pg / mL, IL-1β: 6.091±0.4114 pg / mL, and MCP-1: 8.442±0.7159 pg / mL. Compared with the blank control group, the release levels of TNF-α (9.041±0.4331 pg / mL), IL-1β (6.930±0.5645 pg / mL), and MCP-1 (9.377±0.9640 pg / mL) in the ox-LDL model group were significantly increased. P <0.001, P <0.001, P <0.05, while the medium-dose (TNF-α: 8.301±0.4760pg / mL, IL-1β: 6.317±0.3490pg / mL, MCP-1: 7.945±0.7410pg / mL) and high-dose (TNF-α: 7.773±0.6044pg / mL, IL-1β: 5.913±0.3098pg / mL, MCP-1: 7.916±0.7715pg / mL) intervention groups The release of inflammatory factors was significantly reduced in the high-dose intervention group compared to the ox-LDL model group. In fact, the amount of inflammatory factors released by cells in the high-dose intervention group was lower than that in the blank control group, and there was no significant difference compared to the positive control group. In the low-dose intervention group, only the MCP-1 level was significantly reduced (8.395±0.6692 pg / mL), while the levels of TNF-α (8.827±0.3023 pg / mL) and IL-1β (6.836±0.4721 pg / mL) were not significantly different from those in the model group. Therefore, the post-biotic can effectively inhibit the secretion of ox-LDL-induced inflammatory factors TNF-α, IL-1β, and MCP-1, and its intervention effect shows a clear dose-dependent relationship, with the high-dose intervention group showing the best remission effect.

[0061] Example 8 Lactobacillus rhamnosus fermentation postbiotics alleviate ox-LDL-induced oxidative damage in HUVECs The culture and intervention methods for HUVECs were the same as in Example 7. After the intervention, cells were collected by trypsin digestion, and the cell suspension was placed in a 2 mL centrifuge tube and sonicated to disrupt the cells. The disrupted cells were then collected. The SOD and MDA contents in the cells were measured according to the kit requirements. The malondialdehyde (MDA) assay kit was purchased from Nanjing Jiancheng Biotechnology Co., Ltd., catalog number A003-4-1; the superoxide dismutase (SOD) assay kit was purchased from Nanjing Jiancheng Biotechnology Co., Ltd., catalog number A001-3. The measurement results are as follows: Figure 10 As shown.

[0062] Depend on Figure 10 It can be concluded that compared with the blank control group (MDA: 0.2313±0.080 nmol / mg protein, SOD: 33.16±0.6891 U / mg protein), the content of the oxidative damage marker MDA in the ox-LDL model group cells (0.8809±0.3798 nmol / mg protein) was significantly increased. P <0.0001, while the SOD content (6.448±0.3468 U / mg protein) was significantly reduced ( P <0.0001); All three different doses of post-biotic intervention reduced the abnormal increase in MDA and the significant decrease in SOD induced by ox-LDL. Among them, the SOD content in the low-dose intervention group (13.19±0.2852U / mg protein) was significantly increased compared with the model group, while the MDA content (0.7434±0.2566nmol / mg protein) did not change significantly. The medium-dose (MDA: 0.3726±0.079nmol / mg protein, SOD: 12.14±0.3298U / mg protein) and high-dose groups (MDA: 0.2617±0.072nmol / mg protein, SOD: 17.84±0.7915U / mg protein) interventions showed significant effects, exhibiting obvious dose dependence, and the effects were not significantly different from those of the positive control group.

[0063] In summary, it can be concluded that post-biotics can effectively inhibit the abnormal increase in MDA oxidative damage products and ROS levels and the abnormal decrease in SOD in ox-LDL-induced endothelial cells, effectively alleviating ox-LDL-induced oxidative damage in endothelial cells. Moreover, its intervention effect shows a significant dose-dependent effect, with the high-dose intervention group showing the best alleviation effect.

[0064] Example 9 Lactobacillus rhamnosus fermentation post-biotic effect alleviates ox-LDL-induced apoptosis in HUVECs cells The cultivation of HUVECs was the same as in Example 6. LFP8 post-biotics were diluted 20-fold, 50-fold, and 100-fold with complete culture medium as high-, medium-, and low-dose interventions, respectively. After 24 hours of HUVEC culture and cell adhesion, the culture medium was aspirated, and 150 μL of the diluted intervention was added. After 20 hours of intervention culture, ox-LDL reagent (Beijing Solarbio Science & Technology Co., Ltd., catalog number IO1300, specification 2 mg / mL) was added to the original culture medium at a concentration of 200 μL / mL. After 24 hours of culture, cells were collected by trypsin digestion, washed once with PBS, and the supernatant was aspirated. 50 μL of lysis buffer was added, and the cells were lysed on ice for 30 minutes, vortexed 3-4 times, and centrifuged at 4°C and 12000 rpm for 10-15 minutes. The supernatant was carefully transferred to a new tube and placed on ice for later use. Caspase-3 activity was measured according to the kit instructions. The Caspase-3 activity test kit was purchased from Nanjing Jiancheng Biotechnology Co., Ltd., catalog number G015-1. The results are as follows: Figure 11 As shown.

[0065] Figure 11 The results of Caspase-3 assays showed that, compared with the blank control group, the Caspase-3 enzyme activity in the ox-LDL model group was significantly increased. P <0.0001%, approximately 1.44 times that of the blank control group, while medium and high doses of post-biotic intervention significantly inhibited the abnormal activation of Caspase-3 enzyme ( P <0.0001, P The activity of its Caspase-3 enzyme was even lower than that of the blank control group (<0.0001), approximately 0.79 times and 0.65 times that of the blank control group, respectively, indicating a dose-dependent effect of intervention. This suggests that metabiotics can significantly inhibit abnormal apoptosis in vascular endothelial cells.

[0066] Example 10 Lactobacillus rhamnosus fermentation followed by biotic inhibition of ox-LDL-induced ceramide production in HUVECs The culture and intervention methods for HUVECs were the same as in Example 7. After the intervention, cells were collected by trypsin digestion, washed once with PBS, and the supernatant was aspirated. 50 μL of lysis buffer was added, and the cells were lysed on ice for 30 min, vortexed 3-4 times, and centrifuged at 4℃ and 12000 rpm for 10-15 min. The supernatant was carefully aspirated into a new tube and placed on ice for later use. The ceramide level in the cells was measured using an ELISA kit purchased from Jiangsu Enzyme Labeling Reagent Co., Ltd., catalog number MB-4463A. The results are as follows: Figure 12 As shown.

[0067] Previous studies have shown that ceramides are involved in the initiation and acceleration of key pathophysiology in atherosclerosis and have a significant correlation with cardiovascular disease. Figure 12 The results of the cell experiments showed that, compared with the blank control group (6.543±0.4590 pg / mL), ox-LDL stimulation significantly increased ceramide release in cells (7.301±0.5644 pg / mL). P <0.01, the low-dose group (7.064±0.5645 pg / mL) showed no significant difference compared to the model group, while the medium-dose (6.521±0.2940 pg / mL) and high-dose (6.687±0.4639 pg / mL) metabiotics effectively inhibited ox-LDL-induced abnormal release of ceramides. P <0.01, P <0.05).

[0068] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing lactobacillus fermentation precursors, characterized in that, The steps include: preparing Lactobacillus rhamnosus (… Lacticaseibacillus rhamnosus NKU ML1-2 was inoculated into a fermentation system for liquid fermentation to obtain the liquid fermentation product; the biopreservation name of the Lactobacillus rhamnosus NKU ML1-2 is... Lacticaseibacillus rhamnosus NKU ML1-2, taxonomic name is Lacticaseibacillus rhamnosus The deposit date is August 13, 2025. The depositary institution is Guangdong Provincial Center for Microbial Culture Collection (GDMCC). The depositary address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, Institute of Microbiology. The deposit number is GDMCCNo: 66837. The bacterial cells in the liquid fermentation product are removed to obtain the post-fermentation biogener of Lactobacillus; The fermentation system uses water as a solvent and includes yeast protein, arabinoxylan, and blueberry powder. The amount of yeast protein added is 10% to 15% of the mass of the fermentation system, the amount of arabinoxylan added is 2% to 10% of the mass of the fermentation system, and the amount of blueberry powder added is 0.2% to 1% of the mass of the fermentation system. The anthocyanin content in the blueberry powder is 20% to 40%.

2. The preparation method according to claim 1, characterized in that, The inoculation amount of *Lactobacillus rhamnosus* NKU ML1-2 was 10. 7 ~10 9 CFU / mL.

3. The preparation method according to claim 1, characterized in that, The liquid fermentation is a shaking fermentation, the shaking speed is 180~220 rpm, the shaking temperature is 35~40℃, and the shaking fermentation time is 22~26 h.

4. A lactobacillus fermentation precursor, characterized in that, The lactobacillus is prepared by the preparation method according to any one of claims 1 to 3; the fermented lactobacillus contains short-chain fatty acids and / or polypeptides.

5. The lactobacillus fermentation precursor according to claim 4, characterized in that, The short-chain fatty acids include one or more of butyric acid, propionic acid, acetic acid, nonanoic acid, hexanoic acid, heptanoic acid, isobutyric acid, isovaleric acid, valeric acid, decanoic acid, and caprylic acid.

6. The use of one or both of the following biogenic elements derived from Lactobacillus fermentation as described in claim 4 or 5: 1) Application in the preparation of products for the prevention and / or improvement of atherosclerosis; 2) Application in the preparation of products for preventing thrombosis.

7. The application according to claim 6, characterized in that, The products include those that improve vascular endothelial dysfunction.

8. The application according to claim 7, characterized in that, The improvement of vascular endothelial dysfunction includes one or more of the following: improving inflammatory damage to vascular cells, improving oxidative damage to vascular cells, and regulating ceramide metabolism in vascular cells.

9. A product for preventing and / or improving atherosclerosis, characterized in that, The product includes the Lactobacillus fermentation precursor as described in claim 4 or 5.

10. The product according to claim 9, characterized in that, The lactobacillus fermentation product is the only effective component.

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