Application of lactobacillus paracasei N1115 in aspect of adjusting inflammation balance

Abstract

The invention discloses application of lactobacillus paracasei N1115 in the aspect of adjusting inflammation balance, and belongs to the technical field of microbial engineering. The lactobacillus paracasei N1115 is used for preparing a composition for regulating the expression of TLR-4, Myd88, IL-6 and iNOS. According to the lactobacillus paracasei N1115 product provided by the invention, when cells are in an inflammatory state, the expression of TLR-4, Myd88, IL-6 and iNOS can be effectively inhibited, so that an anti-inflammatory effect is achieved; when the cells are in a normal state, the expression of TLR-4, IL-6 and iNOS can be promoted, and the effect of regulating immunity is achieved. The method is suitable for preparing probiotic products for regulating inflammation balance.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial engineering, and relates to a new application of Lactobacillus paracasei N1115, in particular to the application of Lactobacillus paracasei N1115 in regulating inflammation balance. Background Art

[0002] Inflammation is a complex defense response of the body to various endogenous and exogenous damaging factors. Under normal physiological conditions, the inflammatory response is precisely regulated by the body and is in a balanced state to maintain the body's health. However, when this balance is broken, it may cause a variety of diseases and pose a serious threat to human health.

[0003] Traditional anti-inflammatory drugs, such as nonsteroidal anti-inflammatory drugs and glucocorticoids, are effective in relieving inflammatory symptoms, but long-term use is often accompanied by serious side effects: nonsteroidal anti-inflammatory drugs may cause adverse reactions such as gastrointestinal discomfort and bleeding; glucocorticoids may cause immunosuppression, osteoporosis, hypertension and other problems. As a new generation of anti-inflammatory treatment, biological agents have better targeting, but they are expensive and have potential immunogenicity, which limits their widespread application.

[0004] Lactobacillus paracasei is a type of facultative anaerobic probiotic with multiple functions such as relieving constipation, regulating intestinal health, and improving intestinal immunity. Probiotics are used to regulate inflammatory balance, often without side effects. Although there are studies on the regulation of inflammatory factors by Lactobacillus paracasei in the prior art, for example, the Chinese invention patent with application number "202410069815.9" and name "A kind of Lactobacillus paracasei LP-116 and its application in the preparation of products regulating damaged intestinal barriers", the prior art often studies the application of Lactobacillus paracasei in inhibiting pro-inflammatory gene expression and reducing body inflammation, without its exploration in promoting the expression of certain pro-inflammatory genes. And usually for the application of probiotics, live bacteria or heat-killed bacteria are often used, without exploring the properties of bacterial lysates. As a result, the scope of application of probiotics is limited. Summary of the invention

[0005] The purpose of the present invention is to provide an application of Lactobacillus paracasei N1115 in regulating inflammatory balance, so as to solve the problem that the prior art lacks research on the application of Lactobacillus paracasei in promoting the expression of certain pro-inflammatory genes and bacterial lysates, and to expand the application scenarios of Lactobacillus paracasei.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] The invention discloses an application of Lactobacillus paracasei N1115 in regulating inflammatory balance. The Lactobacillus paracasei N1115 is used for preparing a composition for regulating the expression of TLR-4, Myd88, IL-6 and iNOS.

[0008] Among them, Lactobacillus paracasei N1115 was deposited in the China General Microbiological Culture Collection Center on March 17, 2011, with the deposit number: CGMCC No.4691. The strain was first disclosed in the Chinese invention patent with application number "201110357058.8" and the name "Lactobacillus casei N1115, its immunoregulatory effect and application".

[0009] As a limitation, the regulation of TLR-4, Myd88, IL-6 and i NOS expression specifically includes: when the body is in an inflammatory state, Lactobacillus paracasei N1115 inhibits the expression of TLR-4, Myd88, IL-6 and i NOS; when the body is in a normal state, Lactobacillus paracasei N1115 promotes the expression of TLR-4, IL-6 and i NOS.

[0010] As another limitation, the Lactobacillus paracasei N1115 includes bacterial lysate.

[0011] The present invention also provides a dairy product for regulating inflammation balance. The raw materials for preparing the effective ingredients thereof include, by weight, 1 part of bacterial lysate of Lactobacillus paracasei N1115 and 1-2 parts of nutrient solution;

[0012] The nutrient solution contains 100-120 g / L of skim milk, 60-70 g / L of mushroom puree and 60-70 g / L of okra puree.

[0013] Among them, shiitake mushrooms are rich in protein, vitamins, minerals and polysaccharides. Trehalose can reduce the denaturation of protein during freezing and thawing, and vitamin C can reduce protein oxidation, thereby protecting the protein structure. At the same time, the shiitake polysaccharide contained in shiitake mushrooms has a positive effect on enhancing human immune function, activating immune cells, anti-tumor and anti-virus, and has an immunomodulatory effect.

[0014] Okra is rich in nutrients such as protein, vitamins, minerals, amino acids and polysaccharides. The rich vitamin E and vitamin C in okra can reduce protein oxidation. Amino acid ions have acidic and alkaline properties and can inhibit the pH change of the solution during the freeze-drying process, thereby protecting the active components. The mucus protein and polysaccharide components also protect the active ingredients in the lysate. At the same time, the flavonoids and polysaccharides in okra can keep the immune response in a balanced state by regulating the production of cytokines.

[0015] By using a reasonable ratio of shiitake mushrooms and okra, the damage of active substances in the bacterial lysate during the freeze-drying process is reduced, thereby improving the stability and effectiveness of the composition; and the three components also play a role in regulating inflammation.

[0016] As a limitation, it is prepared by the following method: activating Lactobacillus paracasei N1115, proliferating and culturing until the number of viable bacteria reaches 10 6 ~10 8 CFU / mL, inactivate, centrifuge to obtain bacterial sludge, lyse the bacterial sludge, add nutrient solution, vacuum freeze-dry, grind to obtain the dairy product.

[0017] As a further limitation, the inactivation process is performed at 98°C for 10 min; the centrifugation process is performed at 4°C, with a rotation speed of 12,000 rpm for 5 min; and the lysis process is performed with an ultrasonic power of 200 to 500 W, a frequency of 20 to 50 kHz, 3 to 5 s of ultrasound, and an interval of 3 to 8 s, in a cycle for 5 to 10 min.

[0018] As a further limitation, the vacuum freeze-drying specifically includes: pre-freezing at -40 to -80°C, heating to -26 to -35°C at a rate of 5°C / 30min until the vacuum degree is ≤0.02MPa, and heating to 10°C until the water content is 3.5% to 5%.

[0019] Due to the adoption of the above technical solution, the present invention has the following technical advances compared with the prior art:

[0020] ① The present invention discloses the application of Lactobacillus paracasei N1115 in regulating inflammatory balance. When cells are in an inflammatory state, Lactobacillus paracasei N1115 can effectively inhibit the expression of TLR-4, Myd88, IL-6 and iNOS, thereby playing an anti-inflammatory role; when cells are in a normal state, it can promote the expression of TLR-4, IL-6 and iNOS, and play an immune-regulating role;

[0021] ② The bacterial lysate of Lactobacillus paracasei N1115 provided by the present invention has the most prominent effect of inhibiting the expression of TLR-4, Myd88 and i NOS compared with live bacteria or heat-killed bacteria when the cells are in an inflammatory state, and there is a significant difference. Therefore, the bacterial lysate has a better effect on regulating the balance of inflammation, and the properties of the bacterial lysate are more stable, and there is no need to consider the maintenance of the amount of live bacteria, which is of great significance to the preservation and stability of the product;

[0022] ③ The present invention provides a dairy product for regulating inflammation balance, which utilizes a reasonable ratio of shiitake mushrooms and okra to reduce the damage of active substances in bacterial lysates during the freeze-drying process, thereby improving the stability and effectiveness of the dairy product and playing a coordinated and synergistic role in regulating inflammation; and the preparation process is simple, the formula is clean, and it is suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The results of gene expression of TLR-4 and Myd88 in Example 1 are shown in FIG. Figure 1 A is the gene expression result of TLR-4, Figure 1 B is the gene expression result of Myd88;

[0024] Figure 2 This is the IL-6 expression result diagram in Example 1;

[0025] Figure 3 This is a graph showing the iNOS expression results in Example 1. DETAILED DESCRIPTION

[0026] The present invention is further described in detail below by specific examples. It should be understood that the described examples are only used to explain the present invention, and are not intended to limit the present invention.

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods in the art.

[0028] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0029] Example 1

[0030] This example discloses the application of Lactobacillus paracasei N1115 in regulating inflammatory balance, and experimentally verifies the effects of live bacteria, heat-killed bacteria, and bacterial lysates of Lactobacillus paracasei N1115 on the expression of TLR-4, Myd88, IL-6, and iNOS, specifically comprising the following steps:

[0031] S1. Sample preparation

[0032] 1) Live Lactobacillus paracasei N1115: Lactobacillus paracasei N1115 was inoculated into MRS liquid medium for activation, and then inoculated into medium 1 for expansion culture until the number of live bacteria reached 10 6 CFU / mL, to obtain live bacterial solution;

[0033] Among them, medium 1 contains 20 g / L glucose, 20 g / L peptone, 10 g / L sodium acetate, 1 g / L potassium dihydrogen phosphate, 0.1 g / L citric acid and MnSO 4 ·5H 2O 50 mg / L;

[0034] 2) Heat-killed Lactobacillus paracasei N1115: The live bacteria liquid was treated at 98° C. for 10 min to obtain a heat-killed bacteria liquid;

[0035] 3) Lactobacillus paracasei N1115 bacterial lysate: The heat-killed bacterial solution was centrifuged at low temperature and high speed (4° C., 12000 rpm) for 5 min, the supernatant was discarded to obtain bacterial mud, and the bacterial mud was lysed into bacterial lysate using an ultrasonic cell crusher (ultrasonic power of 200 W, frequency of 20 kHz, ultrasound for 3 s, interval of 3 s, cycle, time for 5 min).

[0036] S2. Experimental Procedure

[0037] J774A.1 cells were spread in culture medium and plated on a 6-well cell culture plate. When the confluence reached about 80%, the culture medium was removed and the cells were divided into two parts, one of which was added with only DMEM and the other was added with DMEM containing 500 ng / mL LPS. Live bacteria, heat-killed bacteria and bacterial lysate of Lactobacillus paracasei N1115 were added to the two parts of cells, mixed, and incubated with the cells for 4 hours; a control group without the addition of Lactobacillus paracasei N1115 was set up;

[0038] After incubation, the cells were washed twice with PBS, and the cells were recovered with a cell scraper. The total RNA of each group of cells was extracted with a kit (the kit was purchased from Beijing Tiangen Biochemical Technology Co., Ltd.), and then reverse transcribed into cDNA. Fluorescence quantitative PCR was used to determine the expression of TLR-4, Myd88, inflammatory cytokine IL-6 and inducible nitric oxide synthase iNOS. GAPDH was used as an internal reference. The primer sequences are shown in Table 1. The reverse transcription and qRT-PCR parameter settings were set according to the kit instructions:

[0039] Table 1 Gene and primer sequences

[0040]

[0041]

[0042] S3. Experimental Results

[0043] TLR-4 (Toll-like receptor 4) is a receptor that recognizes pathogen-associated molecular patterns. Myd88 (myeloid differentiation factor 88) is an adaptor protein molecule downstream of TLR-4, which participates in signal transmission. When TLR-4 is activated, it can recruit Myd88 to trigger the signal cascade, thereby activating the key transcription factor NF-κB (nuclear factor κB). The activation of NF-κB can lead to the expression of inflammation-related cytokines, including IL-6 (interleukin 6) and iNOS (inducible nitric oxide synthase). IL-6 is a pro-inflammatory cytokine, while iNOS catalyzes the synthesis of nitric oxide (NO). Both play an important role in inflammation and immune response.

[0044] This example, through a large number of experimental studies, proves that Lactobacillus paracasei N1115 regulates the TLR-4 and Myd88 signaling pathways, further affecting the expression of IL-6 and iNOS, thereby regulating the body's inflammatory balance.

[0045] The results of TLR-4 and Myd88 gene expression levels were as follows Figure 1 As shown;

[0046] Depend on Figure 1 It can be seen that LPS significantly increased the gene expression levels of TLR-4 and Myd88, putting the cells in an inflammatory state, while the live bacteria, heat-killed bacteria and bacterial lysates of Lactobacillus paracasei N1115 could significantly inhibit the expression levels of TLR-4 and MyD88 genes stimulated by LPS, among which the inhibitory effect of bacterial lysates was more significant to maintain the homeostasis of cells.

[0047] In the absence of LPS stimulation, that is, when the cells are in a normal state, live bacteria, heat-killed bacteria, and bacterial lysates of Lactobacillus paracasei N1115 can also promote the expression of TLR-4 genes, thereby regulating immune function and eliminating pathogens.

[0048] The expression level of IL-6 was shown in Figure 2 As shown;

[0049] Depend on Figure 2 It can be seen that when the cells are in an inflammatory state, live bacteria, heat-killed bacteria and bacterial lysates of Lactobacillus paracasei N1115 can significantly inhibit the expression of pro-inflammatory cytokine IL-6 induced by LPS stimulation, among which the bacterial lysate has a better inhibitory effect.

[0050] When the cells are in a normal state, live bacteria, heat-killed bacteria and bacterial lysates of Lactobacillus paracasei N1115 can significantly stimulate the expression of IL-6 and play an immune-regulating role.

[0051] The expression level of iNOS was as follows Figure 3 As shown;

[0052] Depend on Figure 3 It can be seen that when cells are in an inflammatory state, live bacteria, heat-killed bacteria and bacterial lysates of Lactobacillus paracasei N1115 can significantly inhibit the expression of i NOS induced by LPS stimulation, among which the inhibitory effect of bacterial lysates is more significant;

[0053] When cells are in a normal state, live bacteria, heat-killed bacteria and bacterial lysates of Lactobacillus paracasei N1115 can significantly stimulate the expression of i NOS and play an immune-regulating role.

[0054] In summary, live bacteria, heat-killed bacteria, and bacterial lysates of Lactobacillus paracasei N1115 can effectively inhibit the activation of TLR-4 and Myd88 genes and the production of downstream proinflammatory cytokines in J774A.1 cells induced by LPS, and play an anti-inflammatory role; while in the normal cell state, it can mildly activate the expression of TLR-4 gene and the production of downstream proinflammatory cytokines, playing a role in regulating immunity.

[0055] Example 2

[0056] This embodiment prepares a dairy product for regulating inflammation balance, which specifically includes the following steps:

[0057] S1. Bacterial activation and proliferation culture

[0058] Lactobacillus paracasei N1115 was inoculated into MRS liquid medium for activation, and then inoculated into medium 1 for expansion culture until the number of viable bacteria reached 10 6 CFU / mL, bacterial solution A was obtained;

[0059] Among them, medium 1 contains 20 g / L glucose, 20 g / L peptone, 10 g / L sodium acetate, 1 g / L potassium dihydrogen phosphate, 0.1 g / L citric acid and MnSO 4 ·5H 2 O 50 mg / L;

[0060] S2. Inactivation of bacteria

[0061] Bacterial solution A was treated at 98°C for 10 min to obtain bacterial solution B;

[0062] S3. Collecting bacteria

[0063] The bacterial solution B was centrifuged at low temperature and high speed (4°C, 12000 rpm) for 5 min, and the supernatant was discarded to obtain bacterial sludge C;

[0064] S4. Bacterial lysis

[0065] The bacterial sludge C was lysed into bacterial lysate D using an ultrasonic cell pulverizer;

[0066] The ultrasonic power was 200 W, the frequency was 20 kHz, the ultrasonic was applied for 3 s, the interval was 8 s, the cycle was repeated, and the duration was 5 min.

[0067] S5. Freeze drying

[0068] 1) Pre-freeze

[0069] Take 1 part of bacterial lysate D, add it to 1 part of nutrient solution E (containing 100 g / L skim milk, 60 g / L mushroom puree and 70 g / L okra puree), mix well to obtain material F, pre-freeze material F at a plate temperature of -40°C, and completely freeze material F when the temperature of the material reaches -35°C or below;

[0070] 2) Main drying stage

[0071] At a heating rate of 5°C / 30min, slowly raise the plate temperature to -26°C, and maintain this temperature until the heat exchange between the plate and the material F is basically completed, the vacuum degree has dropped to ≤0.02MPa, and the fluctuation range is ≤0.002MPa, the temperature of the cold well drops to -70°C, and the main drying stage is completed;

[0072] 3) Analysis and drying stage

[0073] The temperature was directly raised to 10°C to evaporate the remaining water until the water content was 3.5%, and the vacuum freeze drying was completed to obtain the freeze-dried product G;

[0074] S6. Grinding

[0075] The freeze-dried product G is ground and sieved (40 meshes) to obtain the dairy product.

[0076] Example 3

[0077] This embodiment prepares a dairy product for regulating inflammation balance, which specifically includes the following steps:

[0078] S1. Bacterial activation and proliferation culture

[0079] Lactobacillus paracasei N1115 was inoculated into MRS liquid medium for activation, and then inoculated into medium 1 for expansion culture until the number of viable bacteria reached 10 8 CFU / mL, bacterial solution A was obtained;

[0080] Among them, medium 1 contains 20 g / L glucose, 20 g / L peptone, 10 g / L sodium acetate, 1 g / L potassium dihydrogen phosphate, 0.1 g / L citric acid and MnSO 4 ·5H 2 O 50 mg / L;

[0081] S2. Inactivation of bacteria

[0082] Bacterial solution A was treated at 98°C for 10 min to obtain bacterial solution B;

[0083] S3. Collecting bacteria

[0084] The bacterial solution B was centrifuged at low temperature and high speed (4°C, 12000 rpm) for 5 min, and the supernatant was discarded to obtain bacterial sludge C;

[0085] S4. Bacterial lysis

[0086] The bacterial sludge C was lysed into bacterial lysate D using an ultrasonic cell pulverizer;

[0087] The ultrasonic power was 500 W, the frequency was 30 kHz, the ultrasonic was applied for 5 s, the interval was 3 s, and the cycle lasted for 10 min.

[0088] S5. Freeze drying

[0089] 1) Pre-freeze

[0090] Take 1 part of bacterial lysate D, add it to 1.5 parts of nutrient solution E (containing 120 g / L skim milk, 65 g / L mushroom puree and 60 g / L okra puree), mix well to obtain material F, pre-freeze material F at a plate temperature of -80°C, and when the temperature of the material reaches -35°C or below, material F is completely frozen;

[0091] 2) Main drying stage

[0092] At a heating rate of 5°C / 30min, slowly raise the plate temperature to -35°C, and maintain this temperature until the heat exchange between the plate and the material F is basically completed, the vacuum degree has dropped to ≤0.02MPa, and the fluctuation range is ≤0.002MPa, the temperature of the cold well drops to -70°C, and the main drying stage is completed;

[0093] 3) Analysis and drying stage

[0094] The temperature was directly raised to 10°C to evaporate the remaining water until the water content was 5%, and the vacuum freeze drying was completed to obtain the freeze-dried product G;

[0095] S6. Grinding

[0096] The freeze-dried product G is ground and sieved (40 meshes) to obtain the dairy product.

[0097] Example 4

[0098] This embodiment prepares a dairy product for regulating inflammation balance, which specifically includes the following steps:

[0099] S1. Bacterial activation and proliferation culture

[0100] Lactobacillus paracasei N1115 was inoculated into MRS liquid medium for activation, and then inoculated into medium 1 for expansion culture until the number of viable bacteria reached 10 7 CFU / mL, bacterial solution A was obtained;

[0101] Among them, medium 1 contains 20 g / L glucose, 20 g / L peptone, 10 g / L sodium acetate, 1 g / L potassium dihydrogen phosphate, 0.1 g / L citric acid and MnSO 4 ·5H 2 O 50 mg / L;

[0102] S2. Inactivation of bacteria

[0103] Bacterial solution A was treated at 98°C for 10 min to obtain bacterial solution B;

[0104] S3. Collecting bacteria

[0105] The bacterial solution B was centrifuged at low temperature and high speed (4°C, 12000 rpm) for 5 min, and the supernatant was discarded to obtain bacterial sludge C;

[0106] S4. Bacterial lysis

[0107] The bacterial sludge C was lysed into bacterial lysate D using an ultrasonic cell pulverizer;

[0108] The ultrasonic power was 300 W, the frequency was 50 kHz, the ultrasonic was applied for 4 seconds, the interval was 5 seconds, and the cycle lasted for 8 minutes.

[0109] S5. Freeze drying

[0110] 1) Pre-freeze

[0111] Take 1 part of bacterial lysate D, add it to 2 parts of nutrient solution E (containing 110 g / L skim milk, 70 g / L mushroom puree and 63 g / L okra puree), mix well to obtain material F, pre-freeze material F at a plate temperature of -60°C, and when the temperature of the material reaches -35°C or below, material F is completely frozen;

[0112] 2) Main drying stage

[0113] At a heating rate of 5°C / 30min, slowly raise the plate temperature to -30°C, and maintain this temperature until the heat exchange between the plate and the material F is basically completed, the vacuum degree has dropped to ≤0.02MPa, and the fluctuation range is ≤0.002MPa, the temperature of the cold well drops to -70°C, and the main drying stage is completed;

[0114] 3) Analysis and drying stage

[0115] The temperature was directly raised to 10°C to evaporate the remaining water until the water content was 4%. The vacuum freeze drying was completed to obtain the freeze-dried product G;

[0116] S6. Grinding

[0117] The freeze-dried product G is ground and sieved (40 meshes) to obtain the dairy product.

Claims

1. An application of Lactobacillus paracasei N1115 in regulating inflammatory balance, characterized in that: The Lactobacillus paracasei N1115 is used for preparing a composition for regulating the expression of TLR-4, Myd88, IL-6 and iNOS.

2. The use according to claim 1, characterized in that: The regulating the expression of TLR-4, Myd88, IL-6 and iNOS specifically includes: when the body is in an inflammatory state, Lactobacillus paracasei N1115 inhibits the expression of TLR-4, Myd88, IL-6 and iNOS; when the body is in a normal state, Lactobacillus paracasei N1115 promotes the expression of TLR-4, IL-6 and iNOS.

3. The use according to claim 1 or 2, characterized in that: The Lactobacillus paracasei N1115 comprises bacterial lysate.

4. A dairy product for regulating inflammation balance, characterized in that: The raw materials for making the effective component thereof include, by weight, 1 part of bacterial lysate of Lactobacillus paracasei N1115 and 1-2 parts of nutrient solution; The nutrient solution comprises 100-120 g / L of skim milk, 60-70 g / L of mushroom puree and 60-70 g / L of okra puree.

5. The dairy product according to claim 4, characterized in that The invention is prepared by the following method: activating Lactobacillus paracasei N1115, and multiplying and culturing the viable bacteria to 10 6 ~10 8 CFU / mL, inactivate, centrifuge to obtain bacterial sludge, lyse the bacterial sludge, add nutrient solution, vacuum freeze-dry, grind to obtain the dairy product.

6. The dairy product according to claim 5, characterized in that The inactivation process is performed at 98°C for 10 minutes; the centrifugation process is performed at 4°C, with a rotation speed of 12,000 rpm for 5 minutes; and the lysis process is performed at an ultrasonic power of 200-500 W, a frequency of 20-50 kHz, 3-5 seconds of ultrasonication, and an interval of 3-8 seconds, in a cycle for 5-10 minutes.

7. The dairy product according to claim 5 or 6, characterized in that The vacuum freeze drying specifically includes: pre-freezing at -40~-80°C, heating to -26~-35°C at a rate of 5°C / 30min until the vacuum degree is ≤0.02MPa, and heating to 10°C until the water content is 3.5%~5%.

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