Application of Lactobacillus plantarum JF4 in the preparation of uric acid-lowering and anti-aging food and medicine

Abstract

The present invention belongs to the field of probiotics and their application technology, and is particularly related to the application of Lactobacillus plantarum JF4 in the preparation of uric acid-lowering and anti-aging food and medicine. In order to further explore the probiotic function of Lactobacillus plantarum, the present invention has found through research that Lactobacillus plantarum JF4 strain has multiple probiotic effects, manifested in inhibiting the activity of xanthine oxidase, producing and secreting glutathione, and inhibiting renin activity. Therefore, Lactobacillus plantarum JF4 strain has multiple functions such as preventing and alleviating hyperuricemia and gout; anti-aging, whitening and anti-oxidation; lowering blood pressure and protecting the kidneys. It can be seen that Lactobacillus plantarum JF4 strain has multiple probiotic effects and can be used in the fields of uric acid-lowering and anti-aging. For example, it can be made into uric acid-lowering and anti-aging medicines, and has important application value and economic value.

Description

Technical Field

[0001] The invention belongs to the technical field of probiotics and their applications, and particularly relates to the application of Lactobacillus plantarum JF4 in the preparation of uric acid-lowering and anti-aging food and medicine. Background Art

[0002] Lactobacillus plantarum, also known as Lactobacillus plantarum, belongs to the genus Lactobacillus of the family Lactobacillaceae. It is a Gram-positive bacterium that is either anaerobic or facultatively anaerobic, with an optimal culture pH of 6.5. Its cells are straight or curved rods, growing singly, sometimes in pairs, or in chains. Lactobacillus plantarum is a probiotic found in the human gastrointestinal tract. It produces a variety of natural antibacterial substances, including organic acids, bacteriocins, hydrogen peroxide, and bisacetylphthalide. It has multiple functions, including maintaining intestinal flora balance, lowering cholesterol levels, enhancing immunity, and promoting nutrient absorption.

[0003] Lactobacillus plantarum is an edible lactobacillus that is closely related to human life and is commonly found in fermented products such as dairy products, meat, and many vegetables. It can colonize the intestines through the stomach and exert beneficial effects, having a significant impact on intestinal microorganisms. It has a wide range of applications in food fermentation, industrial lactic acid fermentation, and healthcare. As is well known, lactobacillus is part of the normal human flora. As an important component of the normal intestinal microbial system and a lifelong companion to the host, it is of great significance for maintaining the balance of the intestinal microecology. As an edible lactic acid bacterium, Lactobacillus plantarum is also an important intestinal probiotic that can help intestinal motility, enhance the body's digestive ability, and improve immunity. In recent years, Lactobacillus plantarum has become a research hotspot as a probiotic lactobacillus with great potential, and is being continuously produced into probiotic preparations suitable for humans and animals.

[0004] Studies have found that different strains of Lactobacillus plantarum have different probiotic functions, such as: (1) Anti-pathogenic bacteria: Studies have found that Lactobacillus plantarum has a strong inhibitory effect on Salmonella and also has an inhibitory effect on Helicobacter pylori. (2) Immunity regulation: Studies have shown that Lactobacillus plantarum HK-LP has a strong induction effect on IL-12 in mice. Feeding HK-LP can inhibit the production of immunoglobulins, resist food allergies caused by natural diet in mice, and inhibit tumor growth and tumor cell transplantation in mice. (3) Cadmium excretion effect: Studies have shown that Lactobacillus plantarum CCFM8610 is acid-resistant and has good tolerance and adsorption capacity for cadmium ions in vitro. It can reduce the cadmium content in the liver and kidneys of cadmium-exposed mice and promote the excretion of cadmium through feces in cadmium-exposed mice. (4) Inhibition of Escherichia coli: Studies have shown that Lactobacillus plantarum N13 has acid and bile salt resistance, has high adhesion to intestinal epithelial cell line HT-29 cells, can inhibit the growth of enteroinvasive Escherichia coli, and prevent the invasion of HT-29. (5) Proteolytic activity: Studies have shown that Lactobacillus plantarum CW006 has proteolytic activity and can ferment raw milk to produce angiotensin-converting enzyme inhibitory peptides, thereby achieving an anti-hypertensive effect.

[0005] Because plant lactobacillus has source diversity, it has genetic diversity and functional diversity. At present, although a small number of studies have begun to pay attention to the development and utilization of plant lactobacillus, the research on the separation and identification, prebiotic properties and metabolic mechanism of plant lactobacillus is still relatively small. Moreover, although the functions and applications of a few plant lactobacillus have been developed, their potential efficacy is still far from being fully and deeply excavated. Therefore, it is necessary to further develop its prebiotic function according to the different sources of plant lactobacillus so that it can play a greater role in human health. In summary, further in-depth research on the efficacy and application of prebiotic plant lactobacillus has a relatively broad development space. Summary of the Invention

[0006] In order to overcome the deficiencies of the above-mentioned prior art, the present invention has discovered through research that the Lactobacillus plantarum JF4 strain can inhibit the activity of xanthine oxidase, produce and secrete glutathione, and inhibit renin activity, and has important potential application value in the fields of lowering uric acid and anti-aging.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is:

[0008] In a first aspect, the present invention provides the use of the Lactiplantibacillus plantarum JF4 strain in the preparation of a xanthine oxidase inhibitor. The Lactiplantibacillus plantarum JF4 strain was isolated from preserved plums in Guangdong, China, and has a deposition number of CCTCC NO: M 20221496. For details, see Chinese invention patent "CN116656526B."

[0009] Studies have found that the probiotic Lactobacillus plantarum JF4 strain can inhibit the activity of xanthine oxidase (XOD), suggesting that Lactobacillus plantarum JF4 strain can be used to inhibit the activity of xanthine oxidase through XOD activity, reduce purine in the body, reduce uric acid production, thereby controlling uric acid levels and preventing gout attacks.

[0010] The second aspect of the present invention provides the use of Lactiplantibacillus plantarum JF4 strain in producing reduced glutathione.

[0011] Research has found that the probiotic Lactobacillus plantarum JF4 strain can produce reduced glutathione (GSH), suggesting that the Lactobacillus plantarum JF4 strain can be used to produce GSH, and through this property of producing GSH, it can be used in the fields of antioxidant whitening, anti-aging, immunity enhancement, anti-tumor and anti-allergy.

[0012] A third aspect of the present invention provides use of Lactiplantibacillus plantarum JF4 strain in the preparation of a renin inhibitor.

[0013] Studies have found that the probiotic Lactobacillus plantarum JF4 strain can effectively inhibit renin activity, suggesting that Lactobacillus plantarum JF4 strain can be used as a renin inhibitor to inhibit renin activity, thereby preventing and treating hypertension and other cardiovascular diseases and kidney diseases.

[0014] The fourth aspect of the present invention provides a method for preparing reduced glutathione, which comprises the following steps: inoculating Lactobacillus plantarum JF4 strain into MRS liquid culture medium, culturing to a stable phase, collecting the bacterial suspension, and finally separating and purifying the reduced glutathione in the bacterial suspension.

[0015] Preferably, the culture conditions are: constant temperature 37°C and anaerobic.

[0016] Preferably, the Lactiplantibacillus plantarum JF4 strain is first cultured in MRS liquid culture medium to a stable phase, and then expanded into a new MRS liquid culture medium at a dilution multiple of 1:20-1000.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention has been found through research that the Lactobacillus plantarum JF4 strain has multiple beneficial effects, including inhibiting the activity of xanthine oxidase, producing and secreting glutathione (GSH), and inhibiting renin activity. Therefore, the Lactobacillus plantarum JF4 strain has multiple functions, including preventing and alleviating hyperuricemia and gout; anti-aging, whitening, and antioxidant effects; lowering blood pressure and protecting the kidneys. Thus, the Lactobacillus plantarum JF4 strain has multiple beneficial effects and can be used in fields such as uric acid reduction and anti-aging. For example, it can be made into uric acid reduction and anti-aging drugs, and has important application value and economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The JF4 strain of Lactobacillus plantarum can inhibit XOD activity;

[0020] Figure 2 Because Lactobacillus plantarum JF4 strain can produce and secrete GSH;

[0021] Figure 3 The fermentation broth of Lactobacillus plantarum JF4 can secrete substances that significantly inhibit renin activity. DETAILED DESCRIPTION

[0022] The following is a further description of specific embodiments of the present invention. It should be noted that the description of these embodiments is intended to facilitate understanding of the present invention and does not constitute a limitation of the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

[0023] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0024] The experimental materials involved in the following examples are as follows:

[0025] (1) Strain: Lactobacillus plantarum JF4 strain was isolated from pickled mustard greens in Guangdong, China. For details, see Chinese invention patent “CN116656526B”. Lactobacillus plantarum JF4 strain was deposited in China Center for Type Culture Collection with the accession number: CCTCC NO: M 20221496.

[0026] (2) Kits: Xanthine oxidase activity assay kit (Box Biotech, Cat: AKAO006M), trace reduced glutathione (GSH) assay kit (Nanjing Jiancheng, Cat: A006-2-1), and renin inhibitor screening kit (abnova, Cat: KA1361).

[0027] (3) MRS plate: beef extract 10 g, peptone 10 g, yeast extract 5 g, ammonium citrate tribasic 2 g, sodium acetate 5 g, glucose 20 g, potassium dihydrogen phosphate 2 g, Tween 80 1 mL, magnesium sulfate 0.58 g, manganese sulfate 0.25 g, agar 15 g, ddH2O to 1 L, adjust pH to 6.2-6.6, autoclave at 121°C for 20 min, and prepare MRS plate.

[0028] (4) MRS liquid culture medium: beef extract 10 g, peptone 10 g, yeast extract 5 g, ammonium citrate tribasic 2 g, sodium acetate 5 g, glucose 20 g, potassium dihydrogen phosphate 2 g, Tween 80 1 mL, magnesium sulfate 0.58 g, manganese sulfate 0.25 g, ddH2O to 1 L, adjust pH to 6.2-6.6, autoclave at 121 °C for 20 min to prepare MRS liquid culture medium.

[0029] Example: Function and application of Lactiplantibacillus plantarum JF4 strain

[0030] (1) Lactobacillus plantarum JF4 strain can inhibit xanthine oxidase (XOD) activity

[0031] The plant lactobacillus JF4 strain, which was cultured to the stable phase in MRS liquid medium, was expanded into a new MRS liquid medium at a dilution ratio of 1:30. The bacterial suspension was collected 24 hours after culture to the stable phase, and the fermentation supernatant was collected after centrifugation at 10,000×g and 4°C for 10 minutes. The activity of xanthine oxidase in the fermentation supernatant was then determined using a xanthine oxidase activity assay kit (Box Biotechnology, Cat: AKAO006M). The results showed that compared with the blank medium MRS, which had no inhibitory effect on xanthine oxidase activity, the fermentation supernatant of strain JF4 had a significant inhibitory effect on xanthine oxidase activity, with an inhibition rate of 82.6% (*P<0.05), indicating that plant lactobacillus JF4 can produce and secrete metabolites during the stable phase to inhibit the activity of xanthine oxidase (XOD) ( Figure 1 ).

[0032] Xanthine oxidase is a key enzyme in the purine catabolism process, catalyzing the direct production of uric acid from hypoxanthine and xanthine. Therefore, when xanthine oxidase activity in the body is abnormally high, it can lead to the production of large amounts of uric acid, which can cause hyperuricemia or gout.

[0033] Xanthine oxidase inhibitors, such as allopurinol, inhibit xanthine oxidase activity, preventing the metabolism of hypoxanthine and xanthine into uric acid, thereby reducing uric acid production and improving gout and hyperuricemia. Xanthine oxidase inhibitors can also reduce stress and tissue damage caused by free radicals, and are expected to be clinically useful in treating gout and diseases caused by peroxide free radicals. Currently, allopurinol is one of the main treatments for hyperuricemia and gout, and is the only chemical drug clinically available that inhibits uric acid production. However, this drug has numerous side effects, including fever, allergic rash, abdominal pain, diarrhea, leukopenia and thrombocytopenia, and multiple organ damage, with reports of even fatalities. Its safety has been questioned, but its potent inhibitory effect on xanthine oxidase has led to its continued use. Therefore, the development of new, low-toxic and highly effective xanthine oxidase inhibitors is of paramount importance.

[0034] Therefore, the probiotic Lactobacillus plantarum JF4 strain is expected to reduce purine in the body and reduce uric acid production by inhibiting the activity of xanthine oxidase, thereby controlling uric acid levels and preventing gout attacks.

[0035] (2) Lactobacillus plantarum JF4 strain can produce and secrete reduced glutathione (GSH)

[0036] The plant lactobacillus JF4 strain, which had been cultured to the stable phase in MRS liquid medium, was expanded into new MRS liquid medium at a dilution ratio of 1:30. The bacterial suspension was collected 24 hours after culture to the stable phase, and the fermentation supernatant was collected after centrifugation at 10,000×g and 4°C for 10 minutes. The GSH concentration in the fermentation supernatant was then determined using a reduced glutathione (GSH) assay kit (A006-2-1). The results showed that compared with the low concentration of GSH in the blank medium MRS, the GSH concentration in the fermentation supernatant of strain JF4 was 290.1 ​​μmol / L, indicating that the concentration of GSH increased significantly after JF4 fermentation (****P<0.0001), indicating that plant lactobacillus JF4 can produce and secrete glutathione (GSH) during the stable phase ( Figure 2 ).

[0037] Glutathione (GSH) is a tripeptide composed of glutamic acid, cysteine, and glycine, and contains a γ-amide bond and a sulfhydryl group. It has antioxidant and integrated detoxification effects. The sulfhydryl group on cysteine ​​is the active group of glutathione (hence glutathione is often abbreviated as GSH). Glutathione helps maintain normal immune system function and has antioxidant and integrated detoxification effects. It plays an important role in various cellular biochemical processes, such as free radical neutralization, detoxification, cysteine ​​transportation and storage, maintenance of cellular redox function, and regeneration of ascorbic acid and vitamin E. It mainly includes the following aspects:

[0038] ① Detoxification: combining with poisons or drugs to eliminate their toxic effects;

[0039] ② Participate in redox reactions: As an important reducing agent, it participates in various redox reactions in the body;

[0040] ③ Protect the activity of sulfhydryl enzymes: keep the active group (-SH) of sulfhydryl enzymes in a reduced state;

[0041] ④ Maintain the stability of the red blood cell membrane structure: eliminate the destructive effects of oxidants on the red blood cell membrane structure.

[0042] Therefore, the multiple biological functions of GSH give it a variety of effects and uses, mainly manifested in:

[0043] 1) Antioxidants: They remove free radicals from the human body and protect the sulfhydryl groups in many proteins and enzymes from being oxidized by harmful substances, thereby ensuring the normal physiological functions of proteins and enzymes. Human red blood cells contain a lot of glutathione, which is important for protecting the sulfhydryl groups of proteins on the red blood cell membrane in a reduced state and preventing hemolysis. They can also prevent skin aging and pigmentation, reduce the formation of melanin, improve the skin's antioxidant capacity, and make the skin shiny.

[0044] 2) Clinical drugs: Its thiol group is used to chelate heavy metals, fluoride, mustard gas and other toxins to prevent poisoning; it can also be used as a treatment or auxiliary treatment for hepatitis, hemolytic diseases, keratitis, cataracts and retinal diseases; it can also correct the imbalance of acetylcholine and cholinesterase and play an anti-allergic role.

[0045] 3) Food additives: strengthen food nutrition, stabilize vitamin C, and enhance flavor.

[0046] In summary, glutathione can not only be used in medicine, but also as a base material for functional foods. It has wide application value in functional food fields such as antioxidant whitening, anti-aging, immunity enhancement, anti-tumor and anti-allergy.

[0047] Therefore, the probiotic Lactobacillus plantarum JF4 strain can produce GSH and thus exert the above multiple functions.

[0048] (3) The fermentation broth of Lactobacillus plantarum JF4 strain can effectively inhibit renin activity

[0049] The plant lactobacillus JF4 cultured to the stable phase in MRS liquid medium was expanded into a new MRS liquid medium at a dilution ratio of 1:30. The bacterial suspension was collected 24 hours after the culture reached the stable phase. The fermentation supernatant was collected after centrifugation at 10,000×g and 4°C for 10 minutes. The inhibitory ability of the fermentation supernatant on renin was then determined using a renin inhibitor screening kit (KA1361). The results showed that compared with the blank medium MRS, which had no inhibitory effect, the fermentation supernatant of strain JF4 had the ability to inhibit renin, with an inhibition rate of approximately 24.48% (**P<0.01), indicating that the fermentation broth of plant lactobacillus JF4 can effectively inhibit renin ( Figure 3 ).

[0050] Renin is an approximately 40 kDa aspartic protease that converts angiotensinogen to angiotensin I. Angiotensin-converting enzyme (ACE), a monomeric zinc metalloenzyme found in the vascular endothelium, converts angiotensin I to angiotensin II, the final active messenger of the renin-angiotensin system (RAS) pathway. Angiotensin II can inhibit renin secretion by directly acting on juxtaglomerular cells. Angiotensin II has many physiological effects, the most important of which is its role as a potent vasoconstrictor, increasing blood pressure by altering peripheral vascular resistance. Since angiotensinogen is the only known renin substrate, and its cleavage by renin is crucial for influencing the final activity of the RAS pathway, renin inhibition is an attractive strategy for controlling hypertension. Furthermore, renin inhibitors can block the formation of angiotensin I and angiotensin II. Therefore, the inhibition of renin activity by renin inhibitors could be used to prevent and treat hypertension, as well as other cardiovascular and renal diseases.

[0051] It can be seen that the probiotic Lactobacillus plantarum JF4 strain has the ability to inhibit renin, which makes it a potential probiotic for lowering blood pressure and protecting the kidneys.

[0052] In summary, Lactobacillus plantarum JF4 strain has multiple beneficial effects: (1) it can inhibit the activity of xanthine oxidase; (2) it can produce and secrete glutathione (GSH); and (3) it can inhibit the activity of renin. Therefore, Lactobacillus plantarum JF4 strain has important application value and economic value in the fields of uric acid reduction and anti-aging.

[0053] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. It is apparent to those skilled in the art that various changes, modifications, substitutions, and variations of these embodiments may be made without departing from the principles and spirit of the present invention, and the changes still fall within the scope of protection of the present invention.

Claims

1. Lactobacillus plantarum ( Lactiplantibacillus plantarum ) The use of the JF4 strain in the preparation of a renin inhibitor, characterized in that: The Lactobacillus plantarum JF4 strain has a preservation number of CCTCC NO: M 20221496. Lactobacillus plantarum JF4 cultured in MRS liquid medium to a stable phase is diluted and then expanded into a new MRS liquid medium. When the culture reaches the stable phase, the bacterial suspension is collected, and the fermentation supernatant is collected by centrifugation and used to prepare a renin inhibitor.

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