Probiotic preparation for repairing chemical liver injury and preparation method thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a probiotic preparation for repairing chemical liver injury and a preparation method. The preparation method of the probiotic preparation comprises the following steps: inoculating a fermentation culture medium with activated lactobacillus plantarum liquid, fermenting to obtain a fermentation mixture, centrifuging the fermentation mixture, collecting thallus precipitate, and resuspending by using a freeze-drying protective agent to obtain resuspension liquid; freeze-dried powder obtained by carrying out vacuum freeze-drying on the resuspension is the probiotic preparation; by taking the probiotic preparation disclosed by the invention, the generation of inflammatory factors related to liver injury can be effectively reduced, the liver injury of mice can be relieved by improving oxidative stress, and the probiotic preparation has prevention and treatment effects on chemical liver injury.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a probiotic preparation for repairing chemical liver injury and a preparation method thereof. BACKGROUND

[0002] The liver is the most important metabolic and detoxification organ in the body, playing an important role in the body's metabolism. Chemical liver injury refers to the damage to the structure and function of the liver caused by exogenous chemicals. This injury can be acute or chronic, with severity ranging from mild liver dysfunction to liver failure.

[0003] Probiotics are active microorganisms that are beneficial to the host. In recent years, the role of probiotics in regulating intestinal microecological balance and enhancing the body's immunity has been discovered and valued, and more and more research reports have found an important correlation between intestinal microbiota and liver disease. Studies have shown that probiotics can reduce the level of oxidative stress in the liver through various pathways, including liver detoxification function, restoration of liver microecological balance, and enhancement of liver immune function. These findings provide a new way of thinking for the development of new liver injury treatment methods. It is crucial to screen probiotics that can effectively alleviate chemical liver injury, and the research on probiotics in alleviating intestinal diseases can provide an important reference.

[0004] At present, there are many probiotic products on the market, but there are few probiotic products for repairing chemical liver injury. In addition, the functions and effects of different probiotics also differ. Based on the above purpose, the present application provides a probiotic preparation for repairing chemical liver injury and a preparation method thereof. SUMMARY

[0005] The first object of the present application is to provide a preparation method of a probiotic preparation for repairing chemical liver injury.

[0006] The second object of the present application is to provide a probiotic preparation for repairing chemical liver injury.

[0007] To achieve the above object, the technical solution adopted by the present application is: A preparation method of a probiotic preparation for repairing chemical liver injury, the preparation method of the probiotic preparation comprising the following steps: (1) inoculating Lactobacillus plantarum bacterial solution into fermentation medium for fermentation to obtain a fermentation mixture; (2) centrifuging the fermentation mixture obtained in step (1) to collect bacterial precipitate, and resuspending the bacterial precipitate with a freeze-drying protective agent to obtain a resuspension, the freeze-drying protective agent comprising senecio heptapetalon sugar, glutathione, and water; (3) vacuum freeze-drying the resuspension obtained in step (2) to obtain a freeze-dried powder as the probiotic preparation.

[0008] Further, the concentration of glucose in the fermentation medium in step (1) is 16-21 g / L, the concentration of proteose peptone is 8-11 g / L, the concentration of ammonium nitrate is 2.0-4.0 g / L, the concentration of tyrosine is 3.0-5.6 g / L, the concentration of 5-glutelin-3-ol is 0.5-1.2 g / L, the concentration of sodium acetate is 3.8-6.0 g / L, the concentration of yeast extract is 3.5-5.0 g / L, the concentration of magnesium sulfate is 0.3-0.6 g / L, the concentration of zinc sulfate is 0.10-0.23 g / L, the concentration of ferric sulfate is 0.1-0.3 g / L, the concentration of manganese sulfate is 0.20-0.25 g / L, the concentration of potassium dihydrogen phosphate is 3.0-4.2 g / L, and the concentration of Tween-80 is 0.5-2.0 g / L, all in terms of final concentration.

[0009] Further, the concentration of heptonic sugar in the freeze-drying protective agent in step (2) is 12.0-16.0 g / L, and the concentration of glutathione is 5.2-7.8 g / L.

[0010] Further, the mass ratio of the freeze-drying protective agent to the resuspended bacterial pellet in step (2) is (1-3):1.

[0011] Further, the preparation process of the Lactobacillus plantarum bacterial solution in step (1) is as follows: inoculate Lactobacillus plantarum into MRS liquid medium at an inoculation amount of 2-4% (v / v), and culture at 35-39°C for 22-26 h to obtain the Lactobacillus plantarum bacterial solution.

[0012] Further, the viable bacterial count of the Lactobacillus plantarum bacterial solution is ≥1×10 9 CFU / mL.

[0013] Further, the inoculation amount of the Lactobacillus plantarum bacterial solution in step (1) is 3-5% (v / v), the fermentation temperature is 35-39°C, and the fermentation time is 20-30 h.

[0014] A probiotic preparation for repairing chemical liver injury, which is prepared according to the preparation method of the probiotic preparation for repairing chemical liver injury.

[0015] Compared with the prior art, the probiotic preparation for repairing chemical liver injury has the following beneficial effects: This invention provides a method for preparing a probiotic formulation for repairing chemically induced liver injury. By adding 5-glutenin-3-ol to the fermentation medium and sedoheptulose to the freeze-drying protectant, the number of viable *Lactobacillus plantarum* P-8 bacteria in the probiotic formulation is increased, and bacterial death during the freeze-drying process is reduced. Experimental results show that the probiotic formulation prepared by this invention can effectively reduce the production of inflammatory factors associated with liver injury, alleviate liver injury in mice by improving oxidative stress, and has preventive and therapeutic effects on chemically induced liver injury. Attached Figure Description

[0016] Figure 1 The image shows the results of malondialdehyde (MDA) content detection in mouse liver. Figure 2 The image shows the results of catalase (CAT) content detection in mouse liver; Figure 3 The image shows the results of detecting tumor necrosis factor-α (TNF-α) levels in mouse livers. Detailed Implementation

[0017] The technical solution of the present invention will be further described below with reference to specific embodiments. However, those skilled in the art should understand that the following embodiments are only for illustrating the present invention and should not be regarded as limiting the present invention. Specific conditions not specified in the embodiments are performed according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the reagents or instruments used are all conventional products obtained through commercial channels.

[0018] The *Lactobacillus plantarum* P-8 used in this invention, with accession number CGMCC No. 6312, was purchased from the China General Microbiological Culture Collection Center (CGMCC). Example 1

[0019] A method for preparing a probiotic preparation for repairing chemically induced liver damage, specifically including the following steps: (1) Inoculate Lactobacillus plantarum P-8 into MRS liquid medium at an inoculation rate of 3% (v / v) and incubate at 37°C for 24 h to obtain Lactobacillus plantarum bacterial suspension. The viable count of the Lactobacillus plantarum bacterial suspension shall not be less than 1×10⁻⁶. 9 CFU / mL.

[0020] (2) Take glucose, peptone, ammonium nitrate, tyrosine, 5-gluten-3-alcohol, sodium acetate, yeast extract, magnesium sulfate, zinc sulfate, ferric sulfate, manganese sulfate, potassium dihydrogen phosphate, Tween-80, according to the respective concentration usage, fully dissolved in deionized water, adjust pH to 6.0, sterilize at 115℃ for 20min to obtain the fermentation medium. Among them, the final concentration of glucose in the fermentation medium is 18.0g / L, peptone is 9.0g / L, ammonium nitrate is 3.0g / L, tyrosine is 4.3g / L, 5-gluten-3-alcohol is 0.8g / L, sodium acetate is 4.9g / L, yeast extract is 4.2g / L, magnesium sulfate is 0.5g / L, zinc sulfate is 0.16g / L, ferric sulfate is 0.2g / L, manganese sulfate is 0.22g / L, potassium dihydrogen phosphate is 3.6g / L, and Tween-80 is 1.2g / L.

[0021] (3) The Lactobacillus plantarum bacterial solution obtained in step (1) is inoculated into the fermentation medium obtained in step (2) at an inoculation amount of 4% (v / v) and fermented at 37℃ for 25h to obtain a fermentation mixture. The obtained fermentation mixture is centrifuged at 4℃, 6000xg for 20min, the supernatant is discarded, and the bacterial cell precipitate is gently resuspended with a freeze-drying protective agent. The mass ratio of the freeze-drying protective agent to the bacterial cell precipitate resuspension is 2:1, the concentration of the sedoheptulose in the freeze-drying protective agent is 14.0g / L, and the concentration of glutathione is 6.5g / L, and the solvent is water.

[0022] (4) The resuspension solution obtained in step (3) is vacuum freeze-dried to obtain a freeze-dried powder as a probiotic preparation. Example 2

[0023] A method for preparing a probiotic preparation for repairing chemical liver injury, specifically comprising the following steps: (1) Lactobacillus plantarum P-8 is inoculated into MRS liquid medium at an inoculation amount of 3% (v / v), and cultured at 37℃ for 22h to obtain a Lactobacillus plantarum bacterial solution, wherein the viable bacterial number of the Lactobacillus plantarum bacterial solution is not less than 1×10 9 CFU / mL.

[0024] (2) Take glucose, peptone, ammonium nitrate, tyrosine, 5-gluten-3-alcohol, sodium acetate, yeast extract, magnesium sulfate, zinc sulfate, ferric sulfate, manganese sulfate, potassium dihydrogen phosphate, Tween-80, according to the respective concentration usage, fully dissolved in deionized water, adjust pH to 6.0, sterilize at 115℃ for 20min to obtain the fermentation medium. Among them, the final concentration of glucose in the fermentation medium is 16.0g / L, peptone is 8.0g / L, ammonium nitrate is 2.0g / L, tyrosine is 3.0g / L, 5-gluten-3-alcohol is 0.5g / L, sodium acetate is 3.8g / L, yeast extract is 3.5g / L, magnesium sulfate is 0.3g / L, zinc sulfate is 0.10g / L, ferric sulfate is 0.1g / L, manganese sulfate is 0.20g / L, potassium dihydrogen phosphate is 3.0g / L, and Tween-80 is 0.5g / L.

[0025] (3) The Lactobacillus plantarum bacterial solution obtained in step (1) is inoculated into the fermentation medium obtained in step (2) at an inoculation amount of 3% (v / v) and fermented at 35℃ for 20h to obtain a fermentation mixture. The obtained fermentation mixture is centrifuged at 4℃, 6000xg for 20min, the supernatant is discarded, and the bacterial cell precipitate is gently resuspended with a freeze-drying protective agent. The mass ratio of the freeze-drying protective agent to the bacterial cell precipitate resuspension is 2:1, the concentration of the heptonic sugar in the freeze-drying protective agent is 12.0g / L, and the concentration of glutathione is 5.2g / L, and the solvent is water.

[0026] (4) The resuspension solution obtained in step (3) is vacuum freeze-dried to obtain a freeze-dried powder as a probiotic preparation. Example 3

[0027] A method for preparing a probiotic preparation for repairing chemical liver injury, specifically comprising the following steps: (1) Lactobacillus plantarum P-8 is inoculated into MRS liquid medium at an inoculation amount of 3% (v / v), and cultured at 37℃ for 26h to obtain a Lactobacillus plantarum bacterial solution, wherein the viable bacterial number of the Lactobacillus plantarum bacterial solution is not less than 1×10 9 CFU / mL.

[0028] (2) Take glucose, peptone, ammonium nitrate, tyrosine, 5-glutenin-3-ol, sodium acetate, yeast extract, magnesium sulfate, zinc sulfate, ferric sulfate, manganese sulfate, potassium dihydrogen phosphate, and Tween-80, and dissolve them thoroughly in deionized water according to their respective concentrations and usage amounts. Mix well, adjust the pH to 6.0, and sterilize at 115℃ for 20 min to obtain the fermentation medium. The final concentrations of the fermentation medium are as follows: glucose 21 g / L, peptone 11 g / L, ammonium nitrate 4.0 g / L, tyrosine 5.6 g / L, 5-glutenin-3-ol 1.2 g / L, sodium acetate 6.0 g / L, yeast extract 5.0 g / L, magnesium sulfate 0.6 g / L, zinc sulfate 0.23 g / L, ferric sulfate 0.3 g / L, manganese sulfate 0.25 g / L, potassium dihydrogen phosphate 4.2 g / L, and Tween-80 2.0 g / L.

[0029] (3) The *Lactobacillus plantarum* bacterial culture obtained in step (1) was inoculated into the fermentation medium obtained in step (2) at an inoculation rate of 5% (v / v) and fermented at 39°C for 30 h to obtain a fermentation mixture. The fermentation mixture was centrifuged at 4°C and 6000×g for 20 min, the supernatant was discarded, the bacterial precipitate was collected, and it was gently resuspended with a lyophilization protectant to obtain a resuspension. The mass ratio of the lyophilization protectant to the bacterial precipitate resuspension was 2:1. The concentration of sedoheptulose in the lyophilization protectant was 16.0 g / L, the concentration of glutathione was 7.8 g / L, and the solvent was water.

[0030] (4) The resuspended liquid obtained in step (3) is subjected to vacuum freeze-drying to obtain freeze-dried powder as a probiotic preparation.

[0031] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that 5-glutenin-3-ol is omitted from the fermentation medium prepared in step (2), while the rest are the same as in Example 1.

[0032] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that the sedoheptulose was omitted from the freeze-drying protectant components, while all other components were the same as in Example 1.

[0033] Comparative Example 3 The difference between Comparative Example 3 and Example 1 is that the sedoheptulose in the freeze-drying protectant is replaced with trehalose, while all other components are the same as in Example 1.

[0034] Experimental Example 1 The effect of different culture medium components on the viable cell count of Lactobacillus plantarum in culture: Weigh 1 mL of the fermentation mixtures prepared in step (3) of Examples 1-3 and Comparative Example 1, place them in 9 mL of sterile physiological saline, and dilute 10-fold. Then, dilute sequentially to the desired gradient. The total number of viable bacteria in the fermentation mixtures of different groups was determined by the plate count method, and the results are shown in Table 1.

[0035]

[0036] As shown in Table 1, compared with Comparative Example 1, the probiotic preparations prepared in Examples 1-3 of this invention have a higher total number of effective live bacteria. This indicates that the 5-glutenin-3-ol nutrient in the fermentation medium plays a very important role in fermentation and can significantly increase the total number of effective live bacteria in the probiotic preparations.

[0037] Experimental Example 2 The effect of freeze-drying agent on strain survival rate: Take 1 mL of the resuspended liquid prepared in step (3) of Examples 1-3 and Comparative Examples 1-3, and determine the number of viable bacteria using the plate colony counting method as the number of viable bacteria in the material before freeze-drying; dissolve the freeze-dried powder obtained after freeze-drying in sterile distilled water, and determine the number of viable bacteria using the plate colony counting method as the number of viable bacteria in the freeze-dried powder; the survival rate of the strain during the freeze-drying stage is calculated by the following formula: survival rate (%) = (number of viable bacteria in the freeze-dried powder / number of viable bacteria in the freeze-dried powder) × 100%, and the results are shown in Table 2.

[0038]

[0039] The results are shown in Table 2. Compared with Comparative Examples 2 and 3, the freeze-drying survival rate of probiotic powder was improved after freeze-drying with the freeze-drying protectants prepared in Examples 1-3 of this invention. This indicates that sedoheptulose can improve the survival rate of the strains during the freeze-drying process.

[0040] Experimental Example 3 Eighty 8-week-old male SD rats were selected for the experiment and housed in a controlled environment with an indoor temperature maintained at 22°C and humidity at 60%, following a 12-day light / dark cycle. They were allowed free access to food and water. After one week of acclimatization, the rats were randomly divided into four groups: control group, model group, Example 1 group, Example 2 group, Example 3 group, Comparative Example 1 group, Comparative Example 2 group, and Comparative Example 3 group, with 10 rats in each group. The control group was given physiological saline (20 mL / kg body weight) by gavage daily for 8 weeks; the model group was given physiological saline (20 mL / kg body weight) by gavage daily and subcutaneously injected once daily with 5% (w / v) D-galactose (500 mg / kg body weight) dissolved in physiological saline for 8 weeks; 1 g of the probiotic preparations prepared in Examples 1-3 and Comparative Examples 1-3 were dissolved in 100 mL of physiological saline and administered by gavage; the mice in the other experimental groups were given the corresponding probiotic preparations prepared in this invention by gavage daily (20 mL / kg body weight) and subcutaneously injected once daily with 5% (w / v) D-galactose (500 mg / kg body weight) dissolved in physiological saline for 8 weeks.

[0041] (1) Analysis of oxidative stress markers in rat liver: After the 8-week experiment, male SD rats were sacrificed, and their livers were removed, rinsed with enzyme-free nucleic acid water, and then mixed with sterile saline solution to prepare a 10% (w / v) homogenate. The supernatant was collected, and malondialdehyde (MDA), catalase (CAT), and other liver antioxidant indicators were detected using a kit to evaluate the antioxidant capacity and degree of liver damage in SD rats. The results are as follows: Figure 1 , Figure 2 As shown, elevated malondialdehyde (MDA) levels indicate increased lipid peroxidation in the liver and are positively correlated with liver damage; decreased catalase (CAT) activity indicates a reduced ability of the liver to scavenge free radicals and impaired antioxidant defense system.

[0042] Depend on Figure 1 , Figure 2 The graph shows the statistical results of malondialdehyde (MDA) and catalase (CAT) levels in rat liver, primarily used to assess liver function. Figure 1 and Figure 2 It was found that, compared with the control group, the malondialdehyde (MDA) level in the liver of rats in the model group was significantly increased, and the catalase (CAT) activity was significantly decreased. After oral administration of the probiotic preparation prepared in this invention, compared with comparative groups 1-3, the probiotic preparation prepared in Examples 1-3 of this invention could significantly reduce the MDA content in the liver of rats and significantly increase the catalase (CAT) activity in the liver of rats. This indicates that the probiotic preparation of this invention can alleviate liver damage by improving oxidative stress.

[0043] (2) Analysis of inflammatory factors in rat liver: After the 8-week experiment, male SD rats were sacrificed, and their livers were removed. After rinsing with sterile nucleic acid water, a 10% (w / v) homogenate was prepared by adding sterile saline solution. The supernatant was collected, and the content of the rat liver inflammatory factor tumor necrosis factor-α (TNF-α) was detected using a kit. The results are as follows: Figure 3 As shown, tumor necrosis factor-α (TNF-α) is a core inflammatory factor for assessing liver injury, and its level changes are closely related to the severity, mechanism, and repair process of liver injury.

[0044] Depend on Figure 3 The graph shows the statistical results of tumor necrosis factor-α (TNF-α) levels in rat liver. Compared with the control group, the tumor necrosis factor-α (TNF-α) level in the model group was significantly increased. After oral administration of the probiotic preparation prepared in this invention, compared with comparative groups 1-3, the probiotic preparations prepared in Examples 1-3 of this invention significantly reduced the content of tumor necrosis factor-α (TNF-α) in rat liver. This indicates that the probiotic preparation of this invention can effectively reduce the production of inflammatory factors related to liver injury.

[0045] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. The basic principles and main features of the present invention have been described above with specific implementation schemes. Based on the present invention, some modifications or substitutions can be made, but these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of protection claimed by the present invention.

Claims

1. A method for preparing a probiotic preparation for repairing chemically induced liver damage, characterized in that, The preparation method of the probiotic preparation includes the following steps: (1) The Lactobacillus plantarum culture was inoculated into a fermentation medium to obtain a fermentation mixture; the fermentation medium contained glucose at a concentration of 16-21 g / L, peptone at a concentration of 8-11 g / L, ammonium nitrate at a concentration of 2.0-4.0 g / L, tyrosine at a concentration of 3.0-5.6 g / L, 5-glutenin-3-ol at a concentration of 0.5-1.2 g / L, sodium acetate at a concentration of 3.8-6.0 g / L, yeast extract at a concentration of 3.5-5.0 g / L, magnesium sulfate at a concentration of 0.3-0.6 g / L, zinc sulfate at a concentration of 0.10-0.23 g / L, ferric sulfate at a concentration of 0.1-0.3 g / L, manganese sulfate at a concentration of 0.20-0.25 g / L, potassium dihydrogen phosphate at a concentration of 3.0-4.2 g / L, and Tween-80 at a concentration of 0.5-2.0 g / L. (2) Centrifuge the fermentation mixture obtained in step (1) to collect the cell precipitate, and resuspend it with a freeze-drying protectant to obtain a resuspension. The freeze-drying protectant includes sedoheptulose, glutathione, and water. The concentration of sedoheptulose in the freeze-drying protectant is 12.0-16.0 g / L, and the concentration of glutathione is 5.2-7.8 g / L. (3) The freeze-dried powder obtained by vacuum freeze-drying the resuspended liquid obtained in step (2) is a probiotic preparation.

2. The method for preparing a probiotic preparation for repairing chemically induced liver damage according to claim 1, characterized in that, In step (2), the mass ratio of the freeze-drying protectant to the bacterial cell precipitate resuspension is (1-3):

1.

3. The method for preparing a probiotic preparation for repairing chemically induced liver damage according to claim 1, characterized in that, The preparation process of Lactobacillus plantarum bacterial solution in step (1) is as follows: Lactobacillus plantarum is inoculated into MRS liquid medium at an inoculation rate of 2-4% (v / v) and cultured at 35-39℃ for 22-26 h to obtain Lactobacillus plantarum bacterial solution.

4. The method for preparing a probiotic preparation for repairing chemically induced liver damage according to claim 3, characterized in that, The viable count of the *Lactobacillus plantarum* bacterial solution is ≥1×10⁻⁶. 9 CFU / mL.

5. The method for preparing a probiotic preparation for repairing chemically induced liver damage according to claim 1, characterized in that, In step (1), the inoculation amount of Lactobacillus plantarum culture is 3-5% (v / v), the fermentation temperature is 35-39℃, and the fermentation time is 20-30h.

6. A probiotic preparation for repairing chemically induced liver damage, characterized in that, The probiotic preparation is prepared according to the method of any one of claims 1-5 for preparing a probiotic preparation for repairing chemically induced liver damage.

Images