Phytobacterium plantarum IOB602 and application of metagen thereof in relieving arthritis swelling

Abstract

The invention provides a plant lactobacillus IOB602 and an application of a metagen of the plant lactobacillus IOB602 in relieving arthritis swelling, and belongs to the technical field of microbial fermentation. The phytobacterium plantarum IOB602 and the metagen can be used for inhibiting the foot swelling degree of the mouse; the content of inflammatory factors TNF-alpha and IL-1beta in a mouse body is inhibited, and inflammatory response is relieved; the content of NO in toes of mice is reduced, and the activity of SOD is improved; the content of prostaglandin E2 (PGE2) in toes of mice is inhibited to relieve foot swelling inflammation pain; edema and hyperplasia degree of toes of the mouse are relieved, infiltration of inflammatory cells is reduced, and then arthritis swelling is relieved.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and particularly relates to application of Lactobacillus plantarum IOB602 and postbiotics thereof in relieving arthritis swelling. Background Art

[0002] Arthritis is a common disease that seriously affects human health and quality of life. Its main clinical manifestations are joint swelling, pain, inflammation, and joint dysfunction. With the aging of the global population and changes in lifestyle, the incidence of arthritis has been increasing year by year, bringing a heavy burden to society and families.

[0003] At present, the treatment methods for arthritis mainly include drug therapy, physical therapy and surgical treatment. In terms of drug therapy, non-steroidal anti-inflammatory drugs (NSAIDs) are the most commonly used first-line treatment drugs, which inhibit the activity of cyclooxygenase (COX) and reduce the synthesis of prostaglandins, thereby achieving anti-inflammatory, analgesic and antipyretic effects. However, long-term use of NSAIDs may cause adverse reactions such as gastrointestinal discomfort, liver and kidney damage, and cardiovascular events, which limits its clinical application. Glucocorticoid drugs have strong anti-inflammatory and immunosuppressive effects and can quickly relieve the symptoms of arthritis, but long-term use can bring serious side effects, such as osteoporosis, increased risk of infection, and metabolic disorders. In addition to chemical drug treatment, physical therapy such as hot compress, cold compress, massage, acupuncture, etc. can relieve the symptoms of arthritis to a certain extent, but the effect is often limited and requires long-term treatment. For patients with severe arthritis, surgical treatments such as joint replacement can improve joint function, but the operation is high-risk and expensive, and a long recovery period is required after surgery.

[0004] In recent years, probiotics and their postbiotics have gradually attracted people's attention as a new type of biological treatment. Probiotics are a type of active microorganisms that are beneficial to the host. They exert health benefits by regulating the balance of intestinal flora, enhancing intestinal barrier function, regulating immune response and other ways. Postbiotics refer to biologically active metabolites, cell components or lysates produced by probiotics during the growth and metabolism process, which also have multiple physiological functions such as regulating immunity, anti-inflammatory and anti-oxidation. However, there are relatively few probiotic products on the market for the treatment of arthritis, and the mechanism of action and effect of most products are not clear. Therefore, finding a safe, effective probiotic and its postbiotics with a clear mechanism of action to relieve arthritis swelling and inflammatory response has important clinical significance and market prospects.

[0005] In existing studies, although some probiotics and their postbiotics have been found to have a certain improvement effect on inflammatory bowel disease, the research on probiotics and their postbiotics that can specifically inhibit arthritis-related inflammatory factors, reduce joint swelling and relieve joint pain is still in its infancy. Therefore, there is an urgent need to develop a new probiotic and its postbiotics that can relieve arthritis swelling and inflammatory response. Summary of the invention

[0006] In view of the technical problems existing in the prior art, the present invention aims to provide an application of Lactobacillus plantarum IOB602 and its postbiotics in relieving arthritis swelling.

[0007] Lactobacillus plantarum ( Lactiplantibacillus plantarum )IOB602 was deposited on June 29, 2018 in the General Microbiology Center of China Microorganism Culture Collection, referred to as CGMCC, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.16021.

[0008] One of the purposes of the present invention is to provide an application of Lactobacillus plantarum IOB602 in preparing a medicine for relieving arthritis swelling. The deposit number of Lactobacillus plantarum IOB602 is CGMCC No.16021.

[0009] Preferably, Lactobacillus plantarum IOB602 is used in the form of Lactobacillus plantarum IOB602 powder for preparing a medicine for relieving arthritis swelling. The preparation method of Lactobacillus plantarum IOB602 powder comprises: Inoculate Lactobacillus plantarum IOB602 into a seed culture medium, and culture at 34-38°C for 21-25 hours to prepare an activated bacterial solution; inoculate the prepared activated bacterial solution into the seed culture medium at an inoculation rate of 1%-3% (v / v), and culture in a 34-38°C incubator for 18-22 hours to obtain a primary seed solution; inoculate the primary seed solution into the seed culture medium at an inoculation rate of 1%-3% (v / v), and culture in a 34-38°C incubator for 16-20 hours to obtain a secondary seed solution; inoculate the prepared secondary seed solution at an inoculation rate of 3%-5 The inoculum was inoculated into the fermentation medium at an inoculum rate of % (v / v), and cultured in a 34-38°C incubator for 14-18h to obtain a cultured bacterial solution; the cultured bacterial solution was centrifuged at 2-5°C and 4000-6000rpm / min for 15-25min, the supernatant was discarded, and the bacteria were collected; the precipitate after centrifugation was collected, and placed at -80~-82°C for pre-freezing for 1-3h, and then freeze-dried, the drying temperature was set to -60~-65°C, and the drying time was 28-32h to obtain Lactobacillus plantarum IOB602 probiotics.

[0010] The second object of the present invention is to provide a plant lactobacillus ( Lactiplantibacillus plantarum ) Application of IOB602 postbiotics in the preparation of medicines for relieving arthritis swelling. The preservation number of Lactobacillus plantarum IOB602 is CGMCC No.16021.

[0011] Preferably, the preparation method of Lactobacillus plantarum IOB602 postbiotics comprises: The plant lactobacillus IOB602 is inoculated into the seed culture medium, and the culture is carried out at 34-38°C for 21-25 hours to prepare an activated bacterial solution; the prepared activated bacterial solution is inoculated into the seed culture medium at an inoculation amount of 1%-3% (v / v), and the culture is carried out in a 34-38°C incubator for 18-22 hours to obtain a first-level seed solution; the first-level seed solution is inoculated into the seed culture medium at an inoculation amount of 1%-3% (v / v), and the culture is carried out in a 34-38°C incubator for 16-20 hours to obtain a second-level seed solution; the prepared second-level seed solution is inoculated into the fermentation medium at an inoculation amount of 3%-5% (v / v), and the culture is carried out in a 34-38°C incubator for 14-18 hours to obtain a propagation culture solution; the propagation culture solution is centrifuged at 2-5°C and 4000-6000rpm / min for 15-25 minutes, the supernatant is discarded, and the bacteria are collected; the precipitate after centrifugation is sterilized at 85-95°C for 35-45 min; collect the precipitate after centrifugation, place it at -80~-82℃ for pre-freezing for 1-3h, and then freeze-dry it, the drying temperature is set to -60~-65℃, the drying time is 28-32h, and the postbiotics of Lactobacillus plantarum IOB602 are obtained.

[0012] Preferably, the seed culture medium is composed of: 1-2% peptone, 0.5-1% beef extract, 0.4-0.8% yeast extract, 1.5-2.5% glucose, 0.1-0.5% dipotassium hydrogen phosphate, 0.3-0.8% sodium acetate, 0.003-0.006% manganese sulfate, 0.05-0.15% Tween 80, 0.1-0.3% ammonium citrate, and the rest is water, pH 6.2-6.4.

[0013] Preferably, the fermentation medium consists of 2-3% peptone, 1.5-2.5% yeast extract, 2-3% glucose, 0.5-1% dipotassium hydrogen phosphate, 0.2-0.5% sodium acetate, 0.02-0.04% magnesium sulfate, 0.002-0.005% manganese sulfate, 0.3-0.6% Tween 80, 0.3-0.6% ammonium citrate, and the rest is water, pH 6.2-6.4.

[0014] Beneficial effects of the present invention: The present invention provides an application of Lactobacillus plantarum IOB602 and its postbiotics in the preparation of medicines for relieving arthritis swelling. Lactobacillus plantarum IOB602 and postbiotics can inhibit the degree of swelling in the feet of mice; inhibit the content of inflammatory factors TNF-α and IL-1β in the body of mice, and reduce inflammatory reactions; reduce the content of NO in the toes of mice and increase the activity of SOD; inhibit the content of prostaglandin E2 (PGE2) in the toes of mice to relieve the pain of swelling and inflammation in the feet; reduce the degree of edema and hyperplasia of the toes of mice and reduce the infiltration of inflammatory cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a graph of mouse foot swelling; Figure 2 is the TNF-α content in mouse serum; Figure 3 is the IL-1β content in mouse serum; Figure 4 is the NO content in the toes of mice; Figure 5 is the SOD activity in the toes of mice; Figure 6 is the content of prostaglandin E2 (PGE2) in the toes of mice in serum; Figure 7 This is a pathological section of a mouse toe.

[0016] The present invention provides plant lactobacillus ( Lactiplantibacillus plantarum )IOB602 was deposited on June 29, 2018 in the General Microbiology Center of China Microorganism Culture Collection, referred to as CGMCC, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.16021. DETAILED DESCRIPTION

[0017] The invention provides application of Lactobacillus plantarum IOB602 and postbiotics thereof in preparing medicines for relieving arthritis swelling.

[0018] Example 1 Preparation of Lactobacillus plantarum IOB602 probiotics: (1) Strain activation: inoculate Lactobacillus plantarum IOB602 from a cryopreserved tube into a seed culture medium and culture at 34-38°C for 21-25 hours to prepare an activated bacterial solution; (2) Seed solution preparation: inoculate the prepared activated bacterial solution into the seed culture medium at an inoculation rate of 1-3% (v / v), and culture it in a 34-38°C incubator for 18-22 hours to obtain the first-level seed solution; inoculate the first-level seed solution into the seed culture medium at an inoculation rate of 1-3% (v / v), and culture it in a 34-38°C incubator for 16-20 hours to obtain the second-level seed solution; (3) Cultivation: The prepared secondary seed solution is inoculated into the fermentation medium at an inoculation rate of 3-5% (v / v), and cultured in an incubator at 34-38°C for 14-18 hours to obtain a culture solution; (4) Centrifugation: Centrifuge the cultured bacterial solution at 2-5°C and 4000-6000 rpm / min for 15-25 min, discard the supernatant, and collect the bacterial cells; (5) Drying: Collect the precipitate after centrifugation, place it at -80~-82℃ for pre-freezing for 1-3h, and then freeze-dry it. The drying temperature is set to -60~-65℃ and the drying time is 28-32h to obtain Lactobacillus plantarum IOB602 probiotics.

[0019] Preparation of postbiotics from Lactobacillus plantarum IOB602: (1) Strain activation: inoculate Lactobacillus plantarum IOB602 from a cryopreserved tube into a seed culture medium and culture at 34-38°C for 21-25 hours to prepare an activated bacterial solution; (2) Seed solution preparation: inoculate the prepared activated bacterial solution into the seed culture medium at an inoculation rate of 1-3% (v / v), and culture it in a 34-38°C incubator for 18-22 hours to obtain the first-level seed solution; inoculate the first-level seed solution into the seed culture medium at an inoculation rate of 1-3% (v / v), and culture it in a 34-38°C incubator for 16-20 hours to obtain the second-level seed solution; (3) Cultivation: The prepared secondary seed solution is inoculated into the fermentation medium at an inoculation rate of 3-5% (v / v), and cultured in an incubator at 34-38°C for 14-18 hours to obtain a culture solution; (4) Centrifugation: Centrifuge the cultured bacterial solution at 2-5°C and 4000-6000 rpm / min for 15-25 min, discard the supernatant, and collect the bacterial cells; (5) Inactivation: sterilize the pellet after centrifugation at 85-95°C for 35-45 min; (6) Drying: Collect the precipitate after centrifugation, place it at -80~-82℃ for pre-freezing for 1-3h, and then freeze-dry it. The drying temperature is set to -60~-65℃ and the drying time is 28-32h to obtain Lactobacillus plantarum IOB602 biomass.

[0020] The composition of the seed culture medium is: peptone 1-2%, beef extract 0.5-1%, yeast extract 0.4-0.8%, glucose 1.5-2.5%, dipotassium hydrogen phosphate 0.1-0.5%, sodium acetate 0.3-0.8%, manganese sulfate 0.003-0.006%, Tween 80 0.05-0.15%, ammonium citrate 0.1-0.3%, pH 6.2-6.4.

[0021] The fermentation medium consists of 2-3% peptone, 1.5-2.5% yeast extract, 2-3% glucose, 0.5-1% dipotassium hydrogen phosphate, 0.2-0.5% sodium acetate, 0.02-0.04% magnesium sulfate, 0.002-0.005% manganese sulfate, 0.3-0.6% Tween 80, 0.3-0.6% ammonium citrate, pH 6.2-6.4.

[0022] Example 2 Establishment of foot swelling model and animal grouping Forty-five male KM mice (4-5 weeks, 18-22 g) with normal toes and no damage were randomly divided into normal control group, model control group, positive control group (indomethacin, 10 mg / kg), IOB602 probiotic group (IOB602 probiotic, 10 9 CFU / mL), IOB602 postbiotic group (IOB602 postbiotic, 10 9 CFU / mL), 9 mice in each group, housed in separate cages. One week before modeling, the following drugs were administered by gavage: 0.85% saline in the normal control group and the model control group; the positive control group (10 mg / kg); the IOB602 probiotic group (10 9 CFU / mL), IOB602 epigenetic group (10 9 CFU / mL), and the model was established 0.5h after the last administration. The thickness of the right hind toe of the mice before inflammation was measured with a vernier caliper, and the unit was expressed in mm. Except for the normal control group, 30μL of 8% carrageenan was subcutaneously injected into the middle of the right hind toe of the mice.

[0023] Example 3 Effects of probiotics Lactobacillus plantarum IOB602 and postbiotics on foot swelling in mice The thickness of the right hind paw was measured before and after administration. The degree of swelling of the right hind paw of each group of mice after inflammation was calculated, and the degree of swelling was used to represent the anti-inflammatory effect of probiotics / postbiotics.

[0024] like Figure 1 As shown in the figure, compared with the model group mice, the degree of foot swelling of mice was significantly reduced after being given indomethacin, Lactobacillus plantarum IOB602 and postbiotics for one week before the experiment (P < 0.05, P < 0.01, P < 0.01), relieving the inflammation caused by carrageenan injection in the toes of mice. According to the formula reduction rate = (model group - experimental group) / model group, the IOB602 probiotic group reduced by 46.5%, and the IOB602 postbiotic group reduced by 41.3%.

[0025] Example 4 Effects of probiotics and postbiotics of Lactobacillus plantarum IOB602 on TNF-α and IL-1β levels in mouse serum.

[0026] In this experiment, enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β) in mouse serum.

[0027] like Figure 2 and Figure 3 As shown in the figure, the ELISA detection of TNF-α and 1L-1β in the serum of mice showed that compared with the normal group, the 1L-1β content in the serum of the model group mice increased (P < 0.05), and the TNF-α content increased significantly (P < 0.0001). Compared with the inflammatory mice in the model group, the 1L-1β content in the serum was significantly reduced after administration of indomethacin and Lactobacillus plantarum IOB602 probiotics (P < 0.01). At the same time, after administration of indomethacin and Lactobacillus plantarum IOB602 probiotics and postbiotics, the TNF-α content in the serum was significantly reduced (P < 0.0001, P < 0.001, P < 0.01), and the inflammation level in the mice was alleviated.

[0028] Example 5 Detection of NO content and SOD activity in mouse toes by probiotics Lactobacillus plantarum IOB602 and postbiotics The NO content and SOD activity in the toes of mice were detected using a kit.

[0029] like Figure 4 As shown in the results, the NO content in the toes of mice was detected, and compared with the normal group, the NO content in the toes of mice in the model group was significantly increased (P < 0.05). After the administration of indomethacin, Lactobacillus plantarum IOB602 and postbiotics, the NO content was significantly reduced (P < 0.01, P < 0.01, P < 0.05), which was consistent with the above results and effectively inhibited inflammation. Figure 5 As shown in the figure, the detection of SOD activity in the toes of mice showed that compared with the normal group, the SOD activity in the toes of mice in the model group was reduced to a certain extent. After the administration of indomethacin, although it did not reach a significant level, the SOD activity was also improved to a certain extent.

[0030] Example 6 Detection of prostaglandin E2 (PGE2) content in mouse serum and toes by Lactobacillus plantarum IOB602 probiotics and postbiotics In this experiment, enzyme-linked immunosorbent assay (ELISA) was used to detect the content of prostaglandin E2 (PGE2) in mouse toes.

[0031] like Figure 6As shown in the data, the content of PGE2 in the toes of mice was detected. Compared with the normal group, the content of inflammatory mediator PGE2 in the toes of mice in the model group was significantly increased (P < 0.05). After administration of indomethacin and Lactobacillus plantarum IOB602, the content of PGE2 was significantly decreased (P < 0.05, P < 0.01), which was consistent with the above results and effectively inhibited inflammation and alleviated inflammatory pain.

[0032] Example 7 Effects of probiotics and postbiotics of Lactobacillus plantarum IOB602 on pathological sections of mouse toes like Figure 7 As shown, representative photos of the toe tissues of mice in different groups are shown. Compared with the normal group of mice not injected with carrageenan, the dermis of the toe tissues of mice injected with carrageenan was significantly edematous, with hypervascularization and increased inflammatory cell infiltration. However, when the mice were treated with indomethacin, Lactobacillus plantarum IOB602 and postbiotics before carrageenan injection, the degree of edema and hyperplasia was significantly reduced, and the infiltration of inflammatory cells was also significantly reduced.

[0033] In summary, Lactobacillus plantarum IOB602 probiotics and postbiotics can inhibit the degree of paw swelling in mice, reduce the levels of inflammatory factors TNF-α, IL-1β, and NO in the body to reduce inflammation, reduce the content of prostaglandin E2 to relieve pain, reduce the degree of toe edema, and reduce inflammatory cell infiltration.

Claims

1. A plant lactobacillus ( Lactiplantibacillus plantarum ) Application of IOB602 in the preparation of a drug for relieving arthritis swelling, characterized in that: The deposit number of the Lactobacillus plantarum IOB602 is CGMCC No.16021.

2. The use of Lactobacillus plantarum IOB602 as claimed in claim 1 in the preparation of a medicine for relieving arthritis swelling, characterized in that: The Lactobacillus plantarum IOB602 is used in the form of Lactobacillus plantarum IOB602 bacterial powder for preparing the medicine for relieving arthritis swelling. The preparation method of the Lactobacillus plantarum IOB602 bacterial powder comprises: Inoculate Lactobacillus plantarum IOB602 into a seed culture medium, and culture at 34-38°C for 21-25 hours to prepare an activated bacterial solution; inoculate the prepared activated bacterial solution into the seed culture medium at an inoculation rate of 1%-3% (v / v), and culture in a 34-38°C incubator for 18-22 hours to obtain a primary seed solution; inoculate the primary seed solution into the seed culture medium at an inoculation rate of 1%-3% (v / v), and culture in a 34-38°C incubator for 16-20 hours to obtain a secondary seed solution; inoculate the prepared secondary seed solution at an inoculation rate of 3%-5 The inoculum was inoculated into the fermentation medium at an inoculum rate of % (v / v), and cultured in a 34-38°C incubator for 14-18h to obtain a cultured bacterial solution; the cultured bacterial solution was centrifuged at 2-5°C and 4000-6000rpm / min for 15-25min, the supernatant was discarded, and the bacteria were collected; the precipitate after centrifugation was collected, and placed at -80~-82°C for pre-freezing for 1-3h, and then freeze-dried, the drying temperature was set to -60~-65°C, and the drying time was 28-32h, to obtain Lactobacillus plantarum IOB602 probiotics.

3. A plant lactobacillus ( Lactiplantibacillus plantarum ) Application of IOB602 postbiotics in the preparation of a drug for relieving arthritis swelling, characterized in that: The deposit number of the Lactobacillus plantarum IOB602 is CGMCC No.16021.

4. The plant lactobacillus as claimed in claim 3 ( Lactiplantibacillus plantarum ) Application of IOB602 postbiotics in the preparation of a drug for relieving arthritis swelling, characterized in that: The preparation method of the Lactobacillus plantarum IOB602 postbiotics comprises: The plant lactobacillus IOB602 is inoculated into the seed culture medium, and the culture is carried out at 34-38°C for 21-25 hours to prepare an activated bacterial solution; the prepared activated bacterial solution is inoculated into the seed culture medium at an inoculation amount of 1%-3% (v / v), and the culture is carried out in a 34-38°C incubator for 18-22 hours to obtain a first-level seed solution; the first-level seed solution is inoculated into the seed culture medium at an inoculation amount of 1%-3% (v / v), and the culture is carried out in a 34-38°C incubator for 16-20 hours to obtain a second-level seed solution; the prepared second-level seed solution is inoculated into the fermentation medium at an inoculation amount of 3%-5% (v / v), and the culture is carried out in a 34-38°C incubator for 14-18 hours to obtain a propagation culture solution; the propagation culture solution is centrifuged at 2-5°C and 4000-6000rpm / min for 15-25 minutes, the supernatant is discarded, and the bacteria are collected; the precipitate after centrifugation is sterilized at 85-95°C for 35-45 min; collect the precipitate after centrifugation, place it at -80~-82℃ for pre-freezing for 1-3h, and then freeze-dry it, the drying temperature is set to -60~-65℃, the drying time is 28-32h, and the postbiotics of Lactobacillus plantarum IOB602 are obtained.

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