Lactobacillus rhamnosus strain A22004 with anti-inflammatory and anti-oxidation functions as well as derivative product and application of lactobacillus rhamnosus strain A22004

Abstract

The invention provides a lactobacillus rhamnosus strain A22004 with anti-inflammatory and anti-oxidation functions as well as a derivative product and application of the lactobacillus rhamnosus strain A22004, and belongs to the technical field of functional microorganisms. A lactobacillus rhamnosus strain A22004 is separated and purified from feces of Guangxi 100-year old people. The lactobacillus rhamnosus strain A22004 has good antioxidant and anti-inflammatory activity, and can be used for preparing probiotics, health care products or related drugs for maintaining intestinal health.

Description

Technical Field

[0001] The invention belongs to the technical field of functional microorganisms, and specifically relates to a Lactobacillus rhamnosus strain A22004 with anti-inflammatory and antioxidant functions and derivative products and applications thereof. Background Art

[0002] Lactobacillus rhamnosus belongs to the genus Lactobacillus and is mostly found in the intestines of humans and animals. It is an anaerobic, acid-resistant, non-spore-forming Gram-positive probiotic with biological characteristics such as acid resistance, bile salt resistance, and resistance to multiple antibiotics. In addition, there are some reports that different strains of Lactobacillus rhamnosus have different characteristics that can be used in the treatment or adjuvant treatment of specific diseases.

[0003] For example, the patent of publication number CN117821337A discloses an acid-resistant Lactobacillus rhamnosus AFY02, which can regulate NF-κB and apoptosis pathways by improving weight loss, increased organ index, shortened colon and increased intestinal index caused by colon cancer, effectively alleviate the process of intestinal inflammation, intervene in the development of intestinal tumors, and prevent / treat colon cancer. For another example, publication number CN115927106A discloses a Lactobacillus rhamnosus DY801, which has the characteristic of producing a large amount of glutathione, thereby improving the oxidative stress of the intestine and can be used to prevent and / or treat radiotherapy and chemotherapy-induced intestinal damage. The patent of publication number CN118064530A discloses a Lactobacillus rhamnosus XY5, which has important potential application value in the fields of whitening and lowering uric acid. However, there is currently no report on a Lactobacillus rhamnosus strain that has both anti-inflammatory and antioxidant functions. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a Lactobacillus rhamnosus strain A22004, which not only has anti-inflammatory function but also has good antioxidant function and has broad application prospects.

[0005] The invention provides a Lactobacillus rhamnosus strain A22004, whose preservation number is GDMCC No: 65645.

[0006] The invention provides a culture solution of the Lactobacillus rhamnosus strain A22004.

[0007] The present invention provides a method for preparing the culture solution, comprising the following steps:

[0008] The activated Lactobacillus rhamnosus strain A22004 is inoculated into an MRS liquid culture medium for anaerobic culture to obtain seed liquid; the seed liquid is inoculated into a fresh MRS liquid culture medium for expanded culture under anaerobic conditions to obtain a culture liquid of the Lactobacillus rhamnosus strain A22004.

[0009] Preferably, the temperature of the anaerobic culture is 36-38°C; the time of the anaerobic culture is 7-9h;

[0010] The temperature of the expanded culture is 36-38°C;

[0011] The time of the expanded culture is 15 to 17 hours.

[0012] The invention provides a probiotic agent, comprising the Lactobacillus rhamnosus strain A22004 or the culture solution.

[0013] Preferably, the viable bacterial concentration of the Lactobacillus rhamnosus strain A22004 is not less than 1×10 7 CFU / mL.

[0014] The invention provides an anti-inflammatory and / or antioxidant medicine, comprising the Lactobacillus rhamnosus strain A22004, the culture solution or the probiotic agent and pharmaceutically acceptable adjuvants.

[0015] The present invention provides the use of the Lactobacillus rhamnosus strain A22004, the culture solution or the probiotic agent in preparing anti-inflammatory and / or antioxidant drugs.

[0016] Preferably, the antioxidant comprises resistance to at least one of the following oxidants: DPPH, hydroxyl radicals, superoxide anions, SOD, GSH-Px and H2O2.

[0017] Preferably, the anti-inflammatory effect includes reducing the expression or secretion of IL-6 in macrophages.

[0018] The invention provides a rhamnosus lactobacillus strain A22004, which has a deposit number of GDMCC No: 65645. In terms of antioxidant activity, the invention detects the scavenging rate of rhamnosus lactobacillus for DPPH, hydroxyl free radicals, and superoxide anions, the activity of SOD, GSH-Px, and the tolerance to H2O2. The results show that the rhamnosus lactobacillus A22004 is stronger than LGG in scavenging DPPH free radicals, OH-free radicals, and O2-free radicals, and is comparable to LGG in terms of SOD activity and GSH-Px activity. In addition, the rhamnosus lactobacillus A22004 exhibits excellent tolerance to high concentrations of H2O2, that is, in an in vivo environment of high oxidative stress, the strain A22004 can maintain a stable growth state and exert its antioxidant function. Therefore, the strain has good antioxidant capacity, and its effect is comparable to that of the control strain LGG. In terms of anti-inflammatory function, the present invention co-cultures rhamnosus Lactobacillus A22004 and RAW264.7 cells under LPS conditions. Compared with the inflammation model group, the rhamnosus Lactobacillus A22004 can reduce the expression ability of the inflammatory factor IL-6 in RAW264.7 cells and has good anti-inflammatory function. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the morphological diagram of the Lactobacillus rhamnosus strain A22004;

[0020] Figure 2 This is the test result of the ability of Lactobacillus rhamnosus strain A22004 to tolerate H2O2;

[0021] Figure 3 The results show that the Lactobacillus rhamnosus strain A22004 inhibited the expression of IL-6 in RAW264.7 cells;

[0022] Figure 4 This is the detection result of Lactobacillus rhamnosus strain A22004 inhibiting the expression of TNF-α in RAW264.7 cells.

[0023] Biomaterial Deposit Information

[0024] Lactobacillus rhamnosus strain A22004 is preserved in the Guangdong Provincial Microbiological Culture Collection Center, referred to as GDMCC, located on the 5th floor of Building 59, No. 100 Xianlie Middle Road, Guangzhou. Its preservation number is GDMCC No: 65645, and the preservation date is December 16, 2024. DETAILED DESCRIPTION

[0025] The invention provides a Lactobacillus rhamnosus strain A22004, whose preservation number is GDMCC No: 65645.

[0026] In the present invention, the strain A22004 is isolated and purified from the feces of healthy centenarians in Guangxi. The morphological characteristics of the strain A22004 on the MRS plate are as follows: the colonies are round, white, convex, smooth, moist, and have neat edges. The strain A22004 is identified by 16S rDNA, and the obtained 16S rDNA (SEQ ID NO: 1) is sequenced to confirm that its species is Lactobacillus rhamnosus.

[0027] The invention provides a culture solution of the Lactobacillus rhamnosus strain A22004.

[0028] The invention provides a method for preparing a culture solution of the rhamnosus lactobacillus strain A22004, comprising the following steps: inoculating activated rhamnosus lactobacillus strain A22004 into an MRS liquid culture medium for anaerobic culture to obtain seed solution; inoculating the seed solution into a fresh MRS liquid culture medium for expanded culture under anaerobic conditions to obtain a culture solution of the rhamnosus lactobacillus strain A22004.

[0029] In the present invention, the activation method of the rhamnosus lactobacillus strain A22004 preferably comprises the following steps: streak culture of the rhamnosus lactobacillus strain A22004 preserved with glycerol on an MRS plate to obtain a single colony. The streak culture temperature is preferably 36 to 38° C., and can be 37° C. The streak culture time is preferably 22 to 26 hours, and can be 24 hours. The streak culture is preferably carried out under anaerobic conditions, preferably in an anaerobic workstation.

[0030] In the present invention, the temperature of the anaerobic culture is preferably 36-38° C., and may be 37° C. The time of the anaerobic culture is preferably 7-9 hours, and may be 8 hours. During the anaerobic culture, the inoculation amount of the activated Lactobacillus rhamnosus strain A22004 is preferably 0.5%-1%, and may be 0.8%.

[0031] In the present invention, the temperature of the enlarged culture is 36-38°C, and can be 37°C. The time of the enlarged culture is preferably 15-17h, and can be 16h. The inoculation amount of the seed liquid during the enlarged culture is preferably 5%-10%, and can be 8%. The enlarged culture is preferably carried out under anaerobic conditions, preferably in an anaerobic workstation.

[0032] The present invention does not impose any special restrictions on the formula of the MRS liquid culture medium and the MRS plate, and the MRS liquid culture medium and the MRS plate formula known in the art can be used, for example, the MRS liquid culture medium formula is 20g / L glucose, 10g / L beef extract, 10g / L peptone, 5g / L yeast powder, 2g / L dipotassium hydrogen phosphate, 2g / L diammonium citrate, 5g / L sodium acetate, 0.58g / L magnesium sulfate, 0.25g / L manganese sulfate, 0.1% Tween-80, and the pH value is 6.6-6.8. The difference between the MRS plate and the MRS liquid culture medium is that 1.5%-2% agar is additionally added.

[0033] The invention provides a probiotic agent, comprising the Lactobacillus rhamnosus strain A22004 or the culture solution.

[0034] In the present invention, the probiotic refers to supplementing the beneficial bacteria in the intestine so that the beneficial bacteria have sufficient quantity and exert unique biological effects, thereby achieving the purpose of health care or disease prevention and treatment. In the present invention, the probiotic includes powder, drops, capsules, tablets and water. The probiotic preferably includes at least one of the following: tablets, drops, capsules and powders. The powder is processed by low-temperature freeze-drying technology to ensure the activity of the probiotics. The auxiliary materials of the powder preferably include freeze-dried agents, such as skim milk powder, etc. The powder is suitable for warm water and is suitable for infants. The tablet is prepared by mixing and tableting the rhamnosus Lactobacillus strain A22004 and auxiliary materials, and is suitable for chewing and taking. The auxiliary materials preferably include sweeteners or cocoa powder, etc. The capsule preferably packs the fermented mucus Lactobacillus strain A22004 alone or mixed with auxiliary materials as the capsule core in the capsule shell. The drops are prepared by mixing the rhamnosus lactobacillus strain A22004 with edible oil to form drops that isolate air and moisture. When taken, the dropper opening is opened to squeeze the encapsulated oil into the mouth. The live bacteria concentration of the rhamnosus lactobacillus strain A22004 is preferably not less than 1×10 7 CFU / mL or 1×10 7 CFU / g, more preferably 5×10 7 CFU / mL, 10×10 7 CFU / mL, 20×10 7 CFU / mL, 50×10 7 CFU / mL, 1×10 9 CFU / mL, 1×10 10 CFU / mL or 5×10 7 CFU / g, 10×10 7 CFU / g, 20×10 7 CFU / g, 50×10 7 CFU / g, 1×10 9CFU / g, 1×10 10 The present invention has no special limitation on the preparation method of the probiotics, and any preparation method of the probiotics known in the art can be used.

[0035] The invention provides an anti-inflammatory and / or antioxidant medicine, comprising the Lactobacillus rhamnosus strain A22004, the culture solution or the probiotic agent and pharmaceutically acceptable adjuvants.

[0036] In the present invention, the dosage form of the anti-inflammatory and / or antioxidant drug preferably includes oral liquid, tablets, chewable tablets, granules, powders, and capsules. The present invention has no special restrictions on the preparation method of the drug, and the preparation method of the drug known in the art can be used. The live bacterial concentration of the Lactobacillus rhamnosus strain A22004 in the drug is not less than 1×10 7 CFU / mL or 1×10 7 CFU / g.

[0037] The present invention provides the use of the Lactobacillus rhamnosus strain A22004, the culture solution or the probiotic agent in preparing anti-inflammatory and / or antioxidant drugs.

[0038] In the present invention, in the organism, the oxidation process can produce free radicals. If these free radicals are not removed in time, they may attack cell structures, including DNA, proteins and lipids, thereby causing a series of health problems, such as aging, cardiovascular disease, cancer, etc. Antioxidants can neutralize free radicals and reduce or prevent their damage to cells. Studies have shown that the human body's antioxidant system is a system with complete and complex functions comparable to the immune system. The stronger the body's antioxidant ability, the healthier it is and the longer its life. DPPH is a 1,1-diphenyl-2-picrylhydrazyl free radical, which is a very stable nitrogen-centered free radical. Hydroxyl free radicals (OH-) are an important type of reactive oxygen, which acts on biological molecules such as proteins, nucleic acids, and lipids in the body, causing damage to cell structure and function, and then leading to metabolic disorders in the body and causing diseases. Superoxide anion free radicals, as a type of free radical released by biological metabolic processes, have the ability to attack biological macromolecules, thereby causing damage to cell structure and function, and are closely related to the aging and disease state of organisms. Superoxide dismutase (SOD), as a key antioxidant enzyme, is widely present in animals, plants, microorganisms and cultured cells, playing an important role in anti-aging, enhancing the resistance of organisms to various diseases and improving their adaptability to the external environment. Glutathione peroxidase (GPx) is a family of enzymes with important peroxidase activity. They play a key role in protecting organisms from oxidative damage by catalyzing the decomposition of peroxides and reducing the accumulation of free radicals.

[0039] In the present embodiment, in order to verify whether the rhamnosus lactobacillus strain A22004 has antioxidant activity, the DPPH free radical scavenging rate, hydroxyl free radical scavenging rate, superoxide anion scavenging rate, SOD, GSH-Px activity determination and high concentration H2O2 tolerance were verified. The results showed that the rhamnosus lactobacillus strain A22004 had the ability to scavenge DPPH free radicals, scavenging OH- free radicals, scavenging O2 - The free radical capacity was better than that of the LGG control strain, and the SOD activity and GSH-Px enzyme activity were similar to those of the control strain LGG.

[0040] In the present invention, hydrogen peroxide (H2O2) is a ubiquitous oxidant widely distributed in organisms. In a healthy human body, the concentration of H2O2 is maintained in a relatively low range, usually between 10 and 100uM. Studies have revealed that hydrogen peroxide of appropriate concentrations can act as a signal molecule, intervening in various physiological mechanisms such as cell proliferation, differentiation, and apoptosis. However, when the concentration of H2O2 is too high, it attacks macromolecular structures such as nucleic acids, proteins, and lipids of cells, disrupts the synthesis process of ATP and hormones and the normal growth of cells, thereby causing cell damage and inducing oxidative damage. The rhamnosus lactobacillus strain A22004 exhibits excellent tolerance to high concentrations of H2O2, that is, in an in vivo environment of high oxidative stress, the strain can maintain a stable growth state and exert its antioxidant function.

[0041] In the present invention, the anti-inflammatory preferably includes reducing the expression or secretion of IL-6 in macrophages. Interleukin 6 (IL-6) is a multifunctional protein that plays a key role in host defense, acute phase response, immunoregulation and hematopoietic function. Its synthesis process is induced and enhanced by a variety of signals, such as LPS, calcium ion groups, IL-1, IL-2, IFN, TNF, PDGF and viruses. RAW264.7 cells, i.e., mouse mononuclear macrophage leukemia cells, produce proinflammatory cytokines such as TNF-α, IL-1β, IL-6, etc. under the stimulation of LPS, and these cytokines play an important role in resisting pathogen invasion. In the experiment, a certain concentration of LPS is usually used to act together with RAW264.7 cells for a period of time to induce their polarization and establish a cell inflammation model.

[0042] In the present embodiment, the LPS-induced RAW264.7 cell inflammation model was used as the object to carry out the anti-inflammatory activity verification experiment of the rhamnosus lactobacillus strain A22004. The results showed that compared with the model group, the rhamnosus lactobacillus strain A22004 can reduce the expression of inflammatory factor IL-6 in RAW264.7 cells and has good anti-inflammatory function.

[0043] The following is a detailed description of the Lactobacillus rhamnosus strain A22004 with anti-inflammatory and antioxidant functions and its derivative products and applications provided by the present invention in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0044] Example 1

[0045] Isolation and identification of Lactobacillus rhamnosus A22004

[0046] The feces of healthy centenarians in Guangxi were diluted, and the fecal dilution was streaked on the MRS plate and cultured in an anaerobic workstation at 37°C. A single colony was picked and continued to be streaked and cultured until a pure culture was obtained. After 16S rDNA identification, its species was confirmed to be Lactobacillus rhamnosus and named strain A22004. The Lactobacillus rhamnosus strain A22004 was streaked and activated from a glycerol storage tube on an MRS plate and cultured in an anaerobic workstation at 37°C for about 24 hours to obtain a single colony. The colony was round, white, convex, with a smooth and moist surface and neat edges. The strain morphology was as follows: Figure 1 Its 16S rRNA sequence is:

[0047]

[0048] Example 2

[0049] Antioxidant function test of Lactobacillus rhamnosus A22004

[0050] The rhamnosus Lactobacillus A22004 strain was streaked and activated from the glycerol storage tube on an MRS plate, cultured in an anaerobic workstation at 37°C for about 24 hours, and a single clone was picked and cultured in an MRS liquid medium at 37°C for 8 hours, and then inoculated in an MRS liquid medium at a 1% inoculum and cultured anaerobically at 37°C for 16 hours. The strain suspension was adjusted to OD 600 At the same time, prepare the bacterial solution of the control strain LGG in the same way.

[0051] The strain was tested according to the instructions of the DPPH free radical scavenging ability kit (Jianglai Biotechnology), and the results are shown in Table 1.

[0052] The strain was tested according to the instructions of the hydroxyl radical (OH-) content determination kit (Jianglai Biological), and the results are shown in Table 1.

[0053] According to the superoxide anion (O2 - ) kit (Greis Biotech) instructions were used to detect the strains, and the results are shown in Table 1.

[0054] The strain was tested according to the instructions of the SOD activity detection kit (Jianglai Biology), and the results are shown in Table 1.

[0055] The strain was tested according to the instructions of the GSH-Px detection kit (Greis Biotech), and the results are shown in Table 1.

[0056] Table 1 Test results of antioxidant activity of strain A22004 and LGG

[0057]

[0058] The results showed that the rhamnosus Lactobacillus strain A22004 had the ability to scavenge DPPH free radicals and OH - Free radical ability, scavenging O2 - The free radical capacity of the strain A22004 is stronger than that of LGG, and the SOD activity and GSH-Px enzyme activity are comparable to those of the control strain LGG. Therefore, the strain A22004 has good antioxidant capacity, and its effect is comparable to that of the control strain LGG.

[0059] Example 3

[0060] Tolerance of Lactobacillus rhamnosus strain A22004 to H2O2

[0061] The strain A22004 was streaked and activated from the glycerol storage tube onto an MRS plate, and a single clone was selected and cultured in MRS liquid medium. H2O2 at concentrations of 0.5mM, 1.0mM, and 2.0mM was added to the medium, and the growth of the strain was monitored using an ELISA instrument at 0h, 2h, 4h, 6h, 8h, 10h, and 12h. After the determination was completed, the data was plotted.

[0062] The results are as follows Figure 2 As shown, the results showed that the rhamnosus Lactobacillus strain A22004 exhibited excellent tolerance to high concentrations of H2O2, that is, in the in vivo environment of high oxidative stress of 1.0 mM H2O2, the strain A22004 was able to maintain a stable growth state and exert its antioxidant function.

[0063] Example 4

[0064] Determination of anti-inflammatory function of Lactobacillus rhamnosus strain A22004

[0065] RAW264.7 cells were revived and passaged in DMEM medium containing 10% fetal bovine serum and 1% double antibody. When the cells were transparent and round without antennae and the cell density reached 80% to 90%, the cells were blown down with PBS and counted with a hemocytometer. The cells were counted at 2×10 5 cells / well / mL were evenly plated in a 24-well plate and cultured overnight in a 37°C three-gas incubator. The next day, the suspension of Lactobacillus rhamnosus A22004 activated three times in MRS liquid medium and the commercial strain LGG were adjusted to OD 600 To 0.5, centrifuged at 12000rpm for 5min, the obtained bacteria were washed twice with PBS, and the washed bacteria were resuspended with 1mL of DMEM medium containing 100ng / mL LPS and added to the well plate as the experimental group. In addition, a negative control group and a model group were set up. The negative control group added 1mL of cell culture medium, and the model group added 1mL of DMEM medium containing 100ng / mL LPS. The well plate was placed in a 37℃ cell culture incubator and cultured overnight in the dark. After 24h, the cell culture supernatant of each well was collected, and the cell precipitate was removed by centrifugation at 4℃ and 1200rpm for 10min. The collected cell supernatant was detected according to the instructions of the IL-6 and TNF-α ELISA kits (both provided by Jianglai Biology).

[0066] The results are as follows Figure 3 As shown, the results showed that the rhamnosus Lactobacillus A22004 could significantly reduce the expression of the pro-inflammatory factor IL-6 in RAW264.7 cells (p < 0.001), had anti-inflammatory function, and the effect was comparable to that of the commercial strain LGG.

[0067] The results are as follows Figure 4 As shown, the results showed that the rhamnosus Lactobacillus A22004 could significantly reduce the expression of the pro-inflammatory factor TNF-α in RAW264.7 cells (p < 0.0001), and had anti-inflammatory function, and the effect was comparable to that of the commercial strain LGG.

[0068] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Lactobacillus rhamnosus strain A22004, characterized in that: The deposit number is GDMCC No: 65645.

2. A culture solution of the Lactobacillus rhamnosus strain A22004 according to claim 1.

3. A method for preparing the culture solution according to claim 2, characterized in that: The following steps are involved: The activated Lactobacillus rhamnosus strain A22004 was inoculated into MRS liquid medium for anaerobically culturing to obtain seed liquid; The seed liquid is inoculated into a fresh MRS liquid culture medium and cultured under anaerobic conditions to obtain a culture liquid of the Lactobacillus rhamnosus strain A22004.

4. The preparation method according to claim 3, characterized in that: The temperature of the anaerobic culture is 36-38°C; the time of the anaerobic culture is 7-9h; The temperature of the expanded culture is 36-38°C; The time of the expanded culture is 15 to 17 hours.

5. A probiotic, characterized in that: The method comprises the Lactobacillus rhamnosus strain A22004 according to claim 1 or the culture solution according to claim 2.

6. The probiotic according to claim 5, characterized in that: The live bacteria concentration of the Lactobacillus rhamnosus strain A22004 is not less than 1×10 7 CFU / mL.

7. An anti-inflammatory and / or antioxidant drug, characterized in that: The invention comprises the Lactobacillus rhamnosus strain A22004 according to claim 1, the culture solution according to claim 2 or the probiotic agent according to claim 5 or 6, and pharmaceutically acceptable excipients.

8. Use of the Lactobacillus rhamnosus strain A22004 according to claim 1, the culture solution according to claim 2 or the probiotic agent according to claim 5 or 6 in the preparation of anti-inflammatory and / or antioxidant drugs.

9. The use according to claim 8, characterized in that: The antioxidant activity includes resistance to at least one of the following oxidants: DPPH, hydroxyl radicals, superoxide anions, SOD, GSH-Px and H2O2.

10. The use according to claim 8, characterized in that: The anti-inflammatory effect includes reducing the expression or secretion of IL-6 in macrophages.

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