A composite probiotic for alleviating liver damage caused by oxidative stress and its application

Abstract

The present invention relates to a composite probiotic for alleviating liver damage caused by oxidative stress and its application. The composite probiotic for alleviating liver damage caused by oxidative stress is composed of animal Bifidobacterium lactis subsp. Bifidobacterium animalis subsp. lactis BLa36 strain and Lactobacillus paracasei Lactobacillus cheese LC86 strain composition. The present invention found that the above two strains can cooperate with each other, promote each other, and synergize in the effect of preventing and treating liver damage caused by oxidative stress, which is specifically manifested in: effectively reducing the content of malondialdehyde in the liver in the D-galactose-induced acute liver injury rat model, increasing the total superoxide dismutase, catalase and glutathione peroxidase activities in the liver, and enhancing its antioxidant capacity; effectively reducing the content of TNF-α in the liver in the D-galactose-induced acute liver injury rat model and improving the body's immune level.

Description

Technical Field

[0001] The invention belongs to the technical field of probiotics and relates to a composite probiotic for alleviating liver damage caused by oxidative stress and an application thereof. Background Art

[0002] Oxidative stress is a physiological reaction of the body under various internal and external environmental stimuli. It will lead to the generation of a large number of oxygen free radicals. These free radicals are highly oxidative and can damage liver cells, thereby causing liver dysfunction. Liver damage caused by oxidative stress is one of the important factors in the onset of liver disease.

[0003] Traditional treatments for liver damage caused by oxidative stress mainly include drug therapy and lifestyle intervention. However, drug therapy is often accompanied by certain side effects, and the long-term efficacy is not stable. Although lifestyle intervention is important, it is difficult to adhere to in practice, and the effect varies from person to person.

[0004] In recent years, the role of probiotics in regulating the balance of intestinal microecology and enhancing the body's immunity has gradually been discovered and paid attention to. In particular, probiotics have shown significant effects in the adjuvant treatment of liver disease. Specific types of probiotics can reduce the level of oxidative stress in the liver through various pathways, including improving liver detoxification function, restoring liver microecological balance, and enhancing liver immune function. These findings provide ideas for the development of new treatments for liver injury.

[0005] However, there are many types of probiotic products on the market, but there are few professional products targeting liver damage caused by oxidative stress. In addition, the functions and effects of different probiotic strains are also different. How to select and combine these strains to achieve the best therapeutic effect is also an urgent problem to be solved. Summary of the invention

[0006] In view of the deficiencies of the prior art, the object of the present invention is to provide a composite probiotic for alleviating liver damage caused by oxidative stress and its application.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a composite probiotic for alleviating liver damage caused by oxidative stress, wherein the composite probiotic for alleviating liver damage caused by oxidative stress is prepared from Bifidobacterium animalis subsp. lactis with a preservation number of CGMCC No. 24029. Bifidobacterium animalis subsp. lactis BLa36 strain and Lactobacillus paracasei with a deposit number of CGMCC No.1.12731 Lactobacillus paracasei LC86 strain composition.

[0009] The present invention creatively develops a new probiotic compounding method and a new strategy for preventing and treating liver damage caused by oxidative stress, namely, Bifidobacterium animalis subsp. Bifidobacterium animalis subsp. lactis BLa36 strain and Lactobacillus paracasei Lactobacillus paracasei The LC86 strain was used in combination, and it was found that the two strains can cooperate with each other, promote each other, and synergize in the prevention and treatment of liver damage caused by oxidative stress, which is specifically manifested in: (1) effectively reducing the content of malondialdehyde (MDA) in the liver of rats with acute liver damage induced by D-galactose, increasing the activities of total superoxide dismutase, catalase and glutathione peroxidase in the liver, and enhancing its antioxidant capacity; (2) effectively reducing the content of TNF-α in the liver of rats with acute liver damage induced by D-galactose, and improving the body's immune level. When the amount of bacteria used is the same, compared with the intervention method without any one of the bacteria, the combination of the two bacteria has a significantly improved effect in improving liver damage caused by oxidative stress. At the same time, both bacteria are probiotics, the product is highly safe, and it is not easy to develop resistance.

[0010] Preferably, the ratio of the number of viable cells of the BLa36 strain to that of the LC86 strain is 1:5-5:1, for example, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, etc. Other specific point values ​​within the above numerical range can be selected and will not be described here one by one.

[0011] In a second aspect, the present invention provides a probiotic for alleviating liver damage caused by oxidative stress, wherein the strain in the probiotic is the composite probiotic described in the first aspect.

[0012] Preferably, the live bacteria content of the composite probiotics in the probiotic agent is not less than 1×10 9 CFU / g or 1×10 9 CFU / mL, for example 1×10 9 CFU / g (CFU / mL), 1×10 10 CFU / g (CFU / mL), 5×10 10 CFU / g (CFU / mL), 1×10 11 CFU / g (CFU / mL), 3×10 11 CFU / g (CFU / mL), 5×10 11 CFU / g (CFU / mL), 1×10 12 CFU / g (CFU / mL), 1×10 13 CFU / g (CFU / mL), etc. Other specific point values ​​within this numerical range can be selected and will not be described here one by one.

[0013] Preferably, the dosage form of the probiotics includes solutions, lyophilized powders, capsules, tablets or granules. The dosage form of the probiotics involved in the present invention is not limited, including the most commonly used solutions, lyophilized powders, or further prepared capsules, tablets or granules.

[0014] Preferably, the probiotic is in the form of a solution, which is prepared by the following method:

[0015] The BLa36 strain and the LC86 strain are inoculated into a culture medium respectively for activation and fermentation culture in sequence to obtain a fermentation broth; the fermentation broth is centrifuged and resuspended with a solvent to obtain a BLa36 bacterial suspension and a LC86 bacterial suspension; the BLa36 bacterial suspension and the LC86 bacterial suspension are mixed according to the ratio of the number of live bacteria to obtain the probiotic agent.

[0016] Preferably, the probiotic is in the form of a freeze-dried powder, which is prepared by the following method:

[0017] The BLa36 strain and the LC86 strain are inoculated into a culture medium respectively for activation and fermentation culture in sequence to obtain a fermentation broth; the fermentation broth is centrifuged respectively, mixed with a protective agent and freeze-dried to obtain BLa36 bacterial powder and LC86 bacterial powder; the BLa36 bacterial powder and the LC86 bacterial powder are mixed according to the ratio of the number of live bacteria to obtain the probiotic agent.

[0018] In a third aspect, the present invention provides use of the composite probiotic described in the first aspect or the probiotic agent described in the second aspect in the preparation of a medicament for preventing, improving or treating liver damage caused by oxidative stress.

[0019] Preferably, the liver damage caused by oxidative stress includes acute liver damage, non-alcoholic fatty liver disease, alcoholic liver disease or viral hepatitis.

[0020] Preferably, the drug further contains excipients;

[0021] The auxiliary materials include any one of fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, colorants, pH regulators, antioxidants, antibacterial agents or buffers, or a combination of at least two of them.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention creatively develops a new probiotic compounding method and a new strategy for preventing and treating liver damage caused by oxidative stress, namely, Bifidobacterium animalis subsp. Bifidobacterium animalis subsp. lactis BLa36 strain and Lactobacillus paracasei Lactobacillus paracaseiThe LC86 strain was used in combination, and it was found that the two strains can cooperate with each other, promote each other, and synergize in the prevention and treatment of liver damage caused by oxidative stress, which is specifically manifested in: (1) effectively reducing the content of malondialdehyde (MDA) in the liver of rats with acute liver damage induced by D-galactose, increasing the activities of total superoxide dismutase, catalase and glutathione peroxidase in the liver, and enhancing its antioxidant capacity; (2) effectively reducing the content of TNF-α in the liver of rats with acute liver damage induced by D-galactose, and improving the body's immune level. When the amount of bacteria used is the same, compared with the intervention method without any one of the bacteria, the combination of the two bacteria has a significantly improved effect in improving liver damage caused by oxidative stress. At the same time, both bacteria are probiotics, the product is highly safe, and it is not easy to develop resistance.

[0024] The BLa36 strain involved in the present invention is classified as Bifidobacterium animalis subsp. lactis Bifidobacterium animalis subsp. lactis The depository is the General Microbiology Center of China Microorganism Culture Collection Administration. The deposit date is December 2, 2021. The deposit number is CGMCC No.24029. The address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0025] The LC86 strain involved in the present invention is classified and named as Lactobacillus paracasei Lactobacillus paracasei The depository is the General Microbiology Center of China Microbiological Culture Collection Administration. The deposit date is July 20, 2020. The deposit number is CGMCC No.1.12731. The address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is the statistical result diagram of malondialdehyde (MDA) level in the liver of rats in each group;

[0027] Figure 2 It is the statistical result diagram of total superoxide dismutase (T-SOD) activity in the liver of rats in each group;

[0028] Figure 3 is the statistical result diagram of catalase (CAT) activity in the liver of rats in each group;

[0029] Figure 4 is the statistical result diagram of glutathione peroxidase (GSH-Px) activity in the liver of rats in each group;

[0030] Figure 5 It is a statistical result graph of tumor necrosis factor-α (TNF-α) level in the liver of rats in each group. DETAILED DESCRIPTION

[0031] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0032] The culture medium formula involved in the following examples is as follows:

[0033] MRS medium: peptone 10g / L, beef extract 10g / L, glucose 20g / L, sodium acetate 2g / L, yeast powder 5g / L, diammonium hydrogen citrate 2g / L, K2PO4·3H2O 2.6g / L, MgSO4·7H2O 0.1g / L, MnSO4 0.05g / L, Tween 801mL / L, cysteine ​​hydrochloride 0.5g / L.

[0034] The BLa36 strain involved in the following examples is named as Bifidobacterium animalis subsp. lactis Bifidobacterium animalis subsp. lactis The deposit date is December 2, 2021, and the deposit number is CGMCC No.24029.

[0035] The LC86 strain involved in the following examples is classified and named as Lactobacillus paracasei Lactobacillus paracasei The deposit date is July 20, 2020, and the deposit number is CGMCC No.1.12731.

[0036] The following method for preparing a bacterial suspension involves: taking out the desired strain from a -80°C refrigerator, inoculating it into an MRS liquid culture medium at an inoculum size of 2% (v / v), culturing it at 37°C for 24 hours for activation, and continuously activating it twice to obtain an activated solution; inoculating the activated solution into an MRS liquid culture medium at an inoculum size of 2% (v / v), culturing it at 37°C for 24 hours to obtain a bacterial solution; centrifuging the bacterial solution at 5000 rpm at 4°C for 10 min, filtering it to obtain bacterial bodies, and resuspending the bacterial bodies in a PBS solution to obtain the product.

[0037] The experimental results were statistically analyzed using ggplot2 in R language. Compared with the control group, ### represents p<0.001, ## represents p<0.01, and # represents p<0.05; compared with the model group, *** represents p<0.001, ** represents p<0.01, * represents p<0.05, and NS represents no significant difference. Example 1

[0038] Free Radical Scavenging Activity Evaluation:

[0039] (1) The BLa36 strain and the LC86 strain were cultured in MRS liquid medium at 37°C for 24 hours, and then the cells were collected by centrifugation (12,000 g, 5 minutes, 4°C). The cell pellets were washed twice with PBS and resuspended to adjust the cell concentration to 10 9 CFU / mL. Take 1 mL of bacterial suspension and add it to 2 mL of 0.2 mmol / L DPPH anhydrous ethanol solution. Mix well and react at room temperature in the dark for 30 min. Centrifuge at 10,000 r / min for 2 min, take the supernatant and measure its absorbance at 517 nm. The scavenging rate of the probiotic strain on DPPH free radicals is calculated by the following formula.

[0040] DPPH removal rate (%) = [1-(A sample group-A blank group) / A control group] × 100

[0041] The difference between the blank group and the sample group was that an equal volume of anhydrous ethanol was used instead of the DPPH solution, and the difference between the control group and the sample group was that an equal volume of distilled water was used instead of the DPPH solution.

[0042] The results are shown in Table 1. Both the BLa36 strain and the LC86 strain involved in the present invention have excellent DPPH free radical scavenging effects.

[0043] Table 1

[0044]

[0045] (2) The BLa36 strain and the LC86 strain were cultured in MRS liquid medium at 37°C for 18 h, respectively. The precipitates were collected by centrifugation (12,000 g, 5 min, 4°C) and resuspended in phosphate buffered saline (PBS). The viable cell count was adjusted to 10 8 CFU / mL was used to obtain intact viable cells (IC). IC was disrupted by sonication, and the precipitate was removed by centrifugation (8000 g, 10 min, 4 °C), and the supernatant was collected as the cell-free extract (CFE).

[0046] (2.1) Determination of hydroxyl radical scavenging activity: Take 1 mL of o-phenanthroline (0.1%, w / v), 1 mL of PBS (pH 7.4), 1 mL of 2.5 mM FeSO4, 1 mL of 20 mM H2O2, and 0.5 mL of cell-free extract (CFE) or 0.5 mL of live bacterial suspension (IC) (1×10 8 CFU / mL) in a test tube and then incubated in a water bath at 37°C for 1.5 hours. The absorbance was measured at 536 nm, and the hydroxyl radical scavenging rate was calculated as follows.

[0047] Clearance rate (%) = [(A sample - A0) / (A1 - A0)] × 100

[0048] Wherein, A sample is the absorbance value after CFE or IC treatment, A0 is the absorbance value when 1 mL of H2O2 is replaced by 1 mL of distilled water, and A1 is the absorbance value when 0.5 mL of sample is replaced by 0.5 mL of distilled water.

[0049] (2.2) Determination of superoxide anion scavenging activity: Tris-HCl buffer (0.05 M, pH 8.2, 2.8 mL), pyrogallol (0.05 M, 0.1 mL) and sample (CFE or IC) (0.1 mL) were mixed and incubated in the dark at 25°C for 4 min. The reaction was stopped by adding HCl (8 M, 1 mL). The absorbance at 320 nm was measured and the superoxide anion scavenging rate was calculated according to the following formula.

[0050] Clearance rate (%) = [1-(A sample / A blank)] × 100

[0051] Wherein, A sample is the absorbance value after CFE or IC treatment, and A blank is the absorbance value after 0.1 mL of sample is replaced by 0.1 mL of distilled water.

[0052] The results are shown in Table 2. Both the BLa36 strain and the LC86 strain involved in the present invention have excellent scavenging effects on hydroxyl free radicals and superoxide anions.

[0053] Table 2

[0054]

[0055] Example 2

[0056] Improvement effect on acute liver injury rat model:

[0057] (1) Experimental animals: Healthy 8-week-old male SD rats (n=64) were housed in a controlled environment with room temperature maintained at 21-23°C, humidity at 50-60%, and a 12-h light / dark cycle. They were allowed to eat and drink as they pleased.

[0058] (2) Animal grouping: After one week of adaptive feeding, the rats were randomly divided into 8 groups, with 8 rats in each group: control group (Con), model group (D-gal), BLa36 group (intervention with BLa36 bacterial solution), LC86 group (intervention with LC86 bacterial solution), BLa36+LC86 group 1 (combined intervention with BLa36 bacterial solution and LC86 bacterial solution, with a live bacteria count ratio of 1:1), BLa36+LC86 group 2 (combined intervention with BLa36 bacterial solution and LC86 bacterial solution, with a live bacteria count ratio of 5:1), BLa36+LC86 group 3 (combined intervention with BLa36 bacterial solution and LC86 bacterial solution, with a live bacteria count ratio of 1:5), ATCC700541+LC86 group (combined intervention with commercially available animal Bifidobacterium lactis subsp. ATCC700541 bacterial solution and LC86 bacterial solution, with a live bacteria count ratio of 1:1).

[0059] (3) Animal modeling and intervention methods:

[0060] Control group (Con): normal saline (20 ml / kg body weight) was given by gavage every day;

[0061] Model group (D-gal): normal saline (20 ml / kg body weight) was administered by gavage daily, and 5% (w / v) D-galactose (500 mg / kg body weight) dissolved in normal saline was injected subcutaneously once a day for 8 weeks;

[0062] Probiotic intervention group: Each group was gavaged with probiotic solution (total bacterial count was 10 9 CFU / day / kg body weight) and injected subcutaneously once a day with 5% (w / v) D-galactose (500 mg / kg body weight) dissolved in saline for 8 weeks.

[0063] (4) Indicator analysis:

[0064] (4.1) Analysis of oxidative stress markers in rat liver:

[0065] After the test results, the rats were killed, and the rat livers were removed and rinsed with enzyme-free nucleic acid water. A 10% (w / v) homogenate was prepared by adding sterile 0.9% saline solution. The supernatant was taken and the total superoxide dismutase (T-SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) antioxidant indicators of the liver were detected using a kit to evaluate the antioxidant capacity of rat liver and the degree of liver damage.

[0066] The results are as follows Figures 1 - 4As shown in the figure, compared with the control group, the level of malondialdehyde (MDA) in the model group was significantly increased, and the activities of total superoxide dismutase (T-SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) were significantly reduced. After the intervention of various probiotics, the level of malondialdehyde (MDA) in the liver of rats decreased to varying degrees, and the activities of total superoxide dismutase (T-SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) increased to varying degrees, especially the combined intervention of BLa36 and LC86 had a more prominent effect.

[0067] (4.2) Analysis of inflammatory factors in rat liver:

[0068] After the test results, the rats were killed, and the rat livers were removed and rinsed with enzyme-free nucleic acid water. A 10% (w / v) homogenate was prepared by adding sterile 0.9% saline solution to the liver. The supernatant was taken and the content of rat liver inflammatory factor tumor necrosis factor-α (TNF-α) was detected using a kit.

[0069] The results are as follows Figure 5 As shown in the figure, compared with the control group, the level of tumor necrosis factor-α (TNF-α) in the model group was significantly increased. After the intervention of various probiotics, the level of tumor necrosis factor-α (TNF-α) in the liver of rats decreased to varying degrees, especially the combined intervention of BLa36 and LC86, which had a more prominent effect and was closer to the control group.

[0070] The applicant declares that the present invention illustrates the technical solution of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0071] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0072] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A probiotic for alleviating liver damage caused by oxidative stress, characterized in that: The strain in the probiotic is Bifidobacterium animalis subsp. lactis with a deposit number of CGMCC No. 24029. Bifidobacterium animalis subsp. lactis BLa36 strain and Lactobacillus paracasei with a deposit number of CGMCC No.1.12731 Lactobacillus paracasei LC86 strain composition.

2. The probiotic for alleviating liver damage caused by oxidative stress according to claim 1, characterized in that: The ratio of the number of viable bacteria of the BLa36 strain to that of the LC86 strain is 1:5-5:

1.

3. The probiotic according to claim 1, characterized in that The live bacteria content of the strain in the probiotic is not less than 1×10 9 CFU / g or 1×10 9 CFU / mL.

4. The probiotic according to claim 1, characterized in that The dosage form of the probiotics includes solution, lyophilized powder, capsule, tablet or granule.

5. The probiotic according to claim 4, characterized in that The dosage form of the probiotic is a solution, which is prepared by the following method: The BLa36 strain and the LC86 strain are inoculated into a culture medium respectively for activation and fermentation culture in sequence to obtain a fermentation broth; the fermentation broth is centrifuged and resuspended with a solvent to obtain a BLa36 bacterial suspension and a LC86 bacterial suspension; the BLa36 bacterial suspension and the LC86 bacterial suspension are mixed according to the ratio of the number of live bacteria to obtain the probiotic agent.

6. The probiotic according to claim 4, characterized in that The dosage form of the probiotic is a freeze-dried powder, which is prepared by the following method: The BLa36 strain and the LC86 strain were inoculated into a culture medium respectively for activation and fermentation culture in sequence to obtain fermentation broth; the fermentation broth was centrifuged respectively, mixed with a protective agent and freeze-dried to obtain BLa36 bacterial powder and LC86 bacterial powder; The BLa36 bacterial powder and the LC86 bacterial powder are mixed according to the ratio of the number of live bacteria to obtain the probiotic agent.

7. Use of the probiotic according to any one of claims 1 to 6 in the preparation of a medicament for preventing, improving or treating acute liver injury.

8. The use according to claim 7, characterized in that: The drug also contains excipients; The auxiliary materials include any one of fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, colorants, pH regulators, antioxidants, antibacterial agents or buffers, or a combination of at least two of them.

Images