Application of fermented lactobacillus grx08 in the preparation of food, functional food and medicine for alleviating alcoholic liver damage and related symptoms

Abstract

The present invention relates to the use of fermented lactobacillus grx08 in the preparation of food, functional food and medicine with the effect of alleviating alcoholic liver damage and related symptoms thereof, and the preservation number of the fermented lactobacillus grx08 is CGMCC No: 7695. The fermented lactobacillus grx08 has the effect of reducing alcohol dehydrogenase and alleviating and restoring the balance of ethanol metabolism. The fermented lactobacillus grx08 has the effect of reducing alanine aminotransferase, reducing liver lipid accumulation, reducing liver lipid peroxidation products, and alleviating liver damage. Animal experiments have shown that fermented lactobacillus grx08 (CGMCC No: 7695) can restore the metabolic balance of rat alcohol dehydrogenase, reduce the level of alanine aminotransferase, reduce lipid accumulation and lipid peroxides in the liver, and alleviate liver damage. In addition, fermented lactobacillus grx08 can also alleviate kidney damage, enhance the tight connectivity of rat intestines, reduce inflammatory factors in the body, and alleviate the increase in blood sugar caused by acute alcohol intervention.

Description

Technical Field

[0001] The present invention relates to application of Lactobacillus fermentum grx08 in preparing food, functional food and medicine capable of alleviating alcoholic liver damage and related symptoms thereof, and belongs to the field of biotechnology. Background Art

[0002] Alcohol is metabolized through three primary pathways in the body, one of which is the ADH pathway within liver cells. ADH activity can affect the liver's normal alcohol metabolism, leading to the accumulation of ethanol and its metabolites in the liver and causing liver damage. The liver is the primary organ for ethanol metabolism, metabolizing over 90% of ethanol. Alcohol damages the liver, causing deformation and necrosis of liver cells, and impairing the liver's ability to metabolize sugars, proteins, fats, and other substances, as well as its detoxification function.

[0003] In the pathogenesis of alcoholic liver disease, alcohol not only causes the accumulation of adipose tissue and the generation of oxidative stress through various pathways, but also causes endotoxin translocation, which in turn stimulates inflammatory cells to release various cytokines and inflammatory mediators, leading to liver cell damage and hepatic inflammatory reactions. Furthermore, acute alcohol damage can also cause some kidney damage and blood sugar fluctuations.

[0004] Probiotics have antioxidant, gut microbiome-modifying, and immune-enhancing properties. As research deepens, the use of probiotics to prevent and treat alcoholic liver injury is increasingly attracting attention. Researchers have found that some probiotics can alleviate alcoholic liver injury by alleviating oxidative stress, regulating gut microbiota, and suppressing endotoxemia. This study examined the in vivo efficacy of the screened Lactobacillus fermentum grx08 strain in alleviating alcoholic liver injury, laying the foundation for the development of foods, functional foods, and pharmaceuticals that alleviate alcoholic liver injury and its associated symptoms. Summary of the Invention

[0005] The purpose of the present invention is to address the problems existing in the prior art and provide a method for preparing foods, functional foods and medicines that can alleviate alcoholic liver damage and related symptoms by using Lactobacillus fermentum grx08.

[0006] The objective of the present invention is achieved through the following technical solution: Lactobacillus fermentum grx08 is used in the preparation of foods, functional foods and medicines that can alleviate alcoholic liver damage and related symptoms. The preservation number of the Lactobacillus fermentum grx08 is CGMCC No: 7695.

[0007] The fermented lactobacillus grx08 has the effects of reducing alcohol dehydrogenase and alleviating and restoring the balance of ethanol metabolism.

[0008] The fermented lactobacillus grx08 has the effects of lowering alanine aminotransferase, reducing liver lipid accumulation, reducing liver lipid peroxidation products, and alleviating liver damage.

[0009] The fermented lactobacillus grx08 has the effects of lowering the levels of urea nitrogen and blood creatinine and alleviating kidney damage.

[0010] The fermented lactobacillus grx08 has the functions of reducing endotoxin and D-lactic acid levels and enhancing intestinal tight connectivity.

[0011] The fermented lactobacillus grx08 has the effects of reducing the level of inflammatory factors and alleviating the body's inflammatory response.

[0012] The fermented lactobacillus grx08 has the effect of lowering blood sugar levels and alleviating the increase in blood sugar caused by acute alcohol damage.

[0013] The present invention is scientifically sound and discloses the use of Lactobacillus fermentum grx08 (CGMCC No. 7695) in the preparation of foods, functional foods, and pharmaceuticals that alleviate alcoholic liver damage and its associated symptoms. Animal experiments have demonstrated that Lactobacillus fermentum grx08 (CGMCC No. 7695) can restore the metabolic balance of alcohol dehydrogenase in rats, while simultaneously lowering alanine aminotransferase levels, reducing lipid accumulation and lipid peroxides in the liver, and alleviating liver damage. Furthermore, Lactobacillus fermentum grx08 can alleviate kidney damage, enhance intestinal tight junctions in rats, reduce inflammatory factors, and alleviate the rise in blood sugar associated with acute alcohol exposure. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a diagram of the determination of alcohol metabolizing enzymes in rats;

[0015] *Indicates significant difference compared with the model group (P<0.05).

[0016] Figure 2 This is a graph showing the determination of liver function in rats;

[0017] *Indicates significant difference compared with the model group (P<0.05).

[0018] Figure 3 This is a graph showing the determination of lipids and lipid peroxidation products in rat liver;

[0019] *Indicates significant difference compared with the model group (P<0.05).

[0020] Figure 4 This is a graph showing the rat renal function test;

[0021] *Indicates significant difference compared with the model group (P<0.05).

[0022] Figure 5 This is a graph showing the determination of intestinal permeability in rats;

[0023] *Indicates significant difference compared with the model group (P<0.05).

[0024] Figure 6 This is a diagram showing the determination of inflammation in rats;

[0025] *Indicates significant difference compared with the model group (P<0.05).

[0026] Figure 7 This is a graph showing the measurement of blood glucose in rats;

[0027] *Indicates significant difference compared with the model group (P<0.05). DETAILED DESCRIPTION

[0028] Chinese Patent ZL201310309222.7 has discovered that Lactobacillus fermentum grx08 (CGMCC No.: 7695) has the function of assisting in lowering blood lipids. Through further research, the present invention found that this strain also has the effect of alleviating alcoholic liver damage and its related symptoms, and used animal experiments to study the effect of Lactobacillus fermentum grx08 in alleviating alcoholic liver damage and its related symptoms in vivo. The rats were gavaged with Lactobacillus fermentum grx08 suspension and an acute alcoholic liver injury model was used. After alcohol injury, the levels of acetaldehyde dehydrogenase (ADH), alcohol dehydrogenase (ALDH), liver injury indicators alanine aminotransferase (ALT) and aspartate aminotransferase (AST), kidney injury indicators urea nitrogen (Urea), uric acid (UA) and creatinine (Crea), inflammatory factors tumor necrosis factor-ɑ (TNF-ɑ), interleukin-6 (IL-6) and nuclear factor κB (NF-κB), as well as endotoxin (LPS), D-lactic acid (D-LA) and blood glucose (GLU) in the serum were measured. The levels of total cholesterol (TC), triglyceride (TG) and malondialdehyde (MDA) in the liver were also measured to evaluate the effect of Lactobacillus fermentum grx08 in alleviating alcoholic liver injury and its related symptoms in vivo.

[0029] Specific steps

[0030] 1. Preparation of oral gavage samples

[0031] The preserved Lactobacillus fermentum grx08 was activated and inoculated into MRS liquid culture medium at a rate of 3%, cultured at 37° C. for 18 h, and then centrifuged at 4000×g for 10 min to collect the bacteria.

[0032] Lactobacillus fermentum grx08 suspension: collect the bacteria with physiological saline and count the colonies. Dilute with physiological saline to 1×10 9CFU / mL.

[0033] Lactobacillus fermentum grx08 heat-killed bacterial suspension: The prepared Lactobacillus fermentum grx08 bacterial suspension was heat-killed at 80°C for 20 minutes.

[0034] For the preparation of the gavage solution for the drug control group, take 5 tablets of Dongbao Gantai and dissolve them in 50 mL of normal saline for immediate use.

[0035] 2 Animal grouping and modeling

[0036] Twenty-five SPF male Wistar rats were housed in a well-ventilated, light-permeable, and clean animal room at a room temperature of 23.0±1.0°C and a humidity of 50±5%. They were acclimated to a basal diet (20% flour, 10% rice flour, 20% corn, 26% drum skin, 20% soybeans, 2% fish meal, and 2% bone meal) for one week and acclimated to a regular 12-hour day / 12-hour night cycle. The rats were then regrouped based on the lack of significant differences in mean body weight between groups. The intervention group received daily oral gavage of a Lactobacillus fermentum grx08 suspension and heat-killed bacteria, while the blank and model control groups received normal saline. Animals were weighed twice weekly to adjust the dose of the test samples. On day 28 (week 4) of the acute injury experiment, all groups were fasted for 16 hours overnight. The alcoholic liver injury model and intervention groups received a single oral gavage of 12 mL / kg body weight of 50% ethanol, while the blank control group received distilled water. The intervention group continued to receive the Lactobacillus fermentum grx08 suspension until the end of the experiment (at least 4 hours after ethanol gavage). The animals were sacrificed at 24 hours, blood was collected for serum separation, and liver samples were harvested for marker analysis. Grouping and treatment are shown in Table 1.

[0037] Table 1 Animal groups and treatments

[0038]

[0039] Note: The oral volume is 1 mL per 100 g of rat body weight.

[0040] 3 rat alcohol metabolizing enzymes

[0041] ADH and ALDH are important enzymes in the alcohol metabolism process. ADH is responsible for oxidizing ethanol to acetaldehyde. The generated acetaldehyde is further converted into harmless acetic acid under the catalysis of ALDH as a substrate. Rat serum was collected and the activities of alcohol metabolism enzymes ADH and ALDH in rat serum were measured using an alcohol dehydrogenase assay kit and an acetaldehyde dehydrogenase assay kit. Figure 1It can be seen that compared with the blank group, the serum ALDH content of rats in the model group was significantly decreased (P < 0.05), and the ADH activity was significantly increased (P < 0.05). Compared with the model group, Lactobacillus fermentum grx08 significantly reduced the ADH activity (P < 0.05), recovering from 0.32 U / g to 0.25 U / g, the same as the blank group.

[0042] 4 Liver function of rats

[0043] The liver is the main organ for ethanol metabolism, and more than 90% of ethanol is metabolized in the liver. Long-term and excessive alcohol intake can seriously damage the liver. ALT and AST are important indicators of liver damage. When the liver is damaged, it will cause an increase in ALT and AST in the blood. The serum of rats was taken and the content of ALT and AST in the serum of each group of rats was measured using an automatic biochemical analyzer. Figure 2 It can be seen that there was no significant change in serum AST levels in all test groups after acute injury (P>0.05). In rats with acute injury, Lactobacillus fermentum grx08 significantly reduced the ALT content of rats compared with the model group (P<0.05), from 55.67U / L to 38.00U / L.

[0044] 5. Lipids and lipid peroxides in rat liver

[0045] Alcohol can cause lipid accumulation and lipid oxidative damage in the liver through various pathways. MDA is a product of lipid peroxidation caused by free radicals, and its content can reflect the degree of oxidative damage in the body. Rat liver tissue was taken and tissue homogenate was prepared. The contents of TC and TG were measured using an automatic biochemical analyzer, and the content of MDA was measured using a malondialdehyde assay kit. Figure 3 It can be seen that compared with the blank group, the contents of TC, TG and MDA in the liver of rats in the model group were significantly increased (P < 0.05). Compared with the model group, the contents of TC, TG and MDA were significantly decreased after intervention with Lactobacillus fermentum grx08 (P < 0.05), from 0.63mmol / L, 1.09mmol / L and 399.87nmol / g to 0.46mmol / L, 0.93mmol / L and 212.90nmol / g, respectively.

[0046] 6. Rat renal function

[0047] Alcohol intake can also cause kidney damage. Crea, Urea and UA are important indicators of kidney function. Kidney damage will cause their levels to increase in the blood. Rat serum was collected and the levels of Crea, Urea and UA in the serum of each group of rats were measured using an automatic biochemical analyzer. Figure 4It can be seen that in the case of acute alcohol injury, the levels of Crea and Urea in the model group rats were significantly increased compared with the blank group (P < 0.05), and the UA content was significantly decreased (P < 0.05). After intervention with Lactobacillus fermentum grx08, the levels of Crea and Urea in the rats were significantly decreased compared with the model group (P < 0.05), from 36.50 μmol / L and 8.60 mmol / L to 28.67 μmol / L and 7.27 mmol / L, respectively.

[0048] 7. Rat intestinal permeability test

[0049] Alcohol intake can lead to damage to the intestinal barrier, increased intestinal mucosal permeability, increased LPS and D-LA produced by intestinal bacterial metabolism entering the portal vein, and thus the production of inflammatory cytokines. Rat serum was collected and the levels of LPS and D-LA in the serum were determined using an ELISA kit. Figure 5 Compared with the blank group, the serum LPS and D-LA levels in the model group were significantly increased (P < 0.05), indicating endotoxin translocation and intestinal barrier damage. After Lactobacillus fermentum grx08 intervention, the serum LPS and D-LA levels in the rats were significantly decreased (P < 0.05), from 45.53 pg / mL and 45.53 μmol / L to 36.71 pg / mL and 36.71 μmol / L, respectively.

[0050] 8 Detection of inflammatory factors in rats

[0051] Cytokines are an important part of the body's defense system. They are both products of immune responses and can enhance immune responses. TNF-α and IL-6 have a wide range of biological activities and are considered to be important indicators of the severity of inflammatory responses. NF-κB, as a multifunctional cellular transcription factor, plays an important role in pathophysiology such as inflammatory responses and cell apoptosis. The levels of inflammatory factors TNF-α, IL-6, and NF-κB in rat serum were determined. Figure 6 Compared with the blank group, the levels of TNF-ɑ, IL-6, and NF-κB in the serum of rats in the model group were significantly increased (P < 0.05). Compared with the model group, Lactobacillus fermentum grx08 intervention significantly reduced the levels of TNF-ɑ and NF-κB in the serum of rats (P < 0.05), from 249.26 and 1209.60 pg / mL to 219.15 and 825.21 pg / mL, respectively.

[0052] 9 Effects of blood glucose on rats

[0053] Acute alcohol damage can sometimes cause fluctuations in blood sugar levels. Figure 7It can be seen that during acute alcohol injury, the blood glucose level of rats in the model group was significantly higher than that in the blank group (P < 0.05). After intervention with Lactobacillus fermentum grx08, the blood glucose level of rats was significantly lower than that in the model group (P < 0.05), from 7.19 mmol / L to 5.29 mmol / L.

[0054] Application Examples

[0055] 1 Preparation of Lactobacillus fermentum grx08 probiotic solid beverage

[0056] Lactobacillus fermentum grx08 and one or more of xylooligosaccharides, fructooligosaccharides, turmeric extract, xylitol and natural fruit powder are used as raw materials, and the specific proportions (mass percentage) are as follows: Lactobacillus fermentum grx08 powder (viable bacteria count 10 11 CFU / g), 5%-10%, xylo-oligosaccharides 15%-25%, fructo-oligosaccharides 10%-15%, turmeric extract 5%-10%, natural fruit powder 15%-20%, and xylitol 20%-25%. The raw materials are mixed, crushed, sieved, and thoroughly mixed before packaging.

[0057] 2 Preparation of fermented milk by Lactobacillus fermentum grx08

[0058] Heat the whole milk to about 50°C, add 6% sucrose, and preheat the whole milk to about 60°C after it is fully dissolved. Homogenize it under a pressure of 20-25 MPa, heat treat it at 95°C for 5 minutes, cool it to about 40°C, inoculate it with Lactobacillus fermentum grx08 starter at a 3% inoculum rate, ferment it at 37°C to a pH of 4.2-4.5, and store it at 4°C after cooling.

[0059] 3 Preparation of fermented milk beverage with Lactobacillus fermentum grx08

[0060] 350 kg of reconstituted skim milk (containing 12% skim milk powder) is fully dissolved, heat-treated at 95° C. for 5 minutes, cooled to 37-40° C., inoculated with a 3.5% inoculum of a Lactobacillus fermentum grx08 starter, and fermented at 37° C. for about 24-26 hours, with an end-point acidity controlled at about 160-170° F. 650 kg of a mixed solution of sugar and stabilizer heat-treated at 110° C. for 5 seconds (the mixed solution consists of 12-14% sucrose, 0.1-0.15% monoglyceride, 0.1-0.15% sucrose ester, and 0.4-0.8% pectin) is added, and the mixture is homogenized under a pressure of 20-25 MPa, cooled to about 15-20° C., and aseptically filled to obtain 1000 kg of a Lactobacillus fermentum grx08 fermented milk beverage, which is stored at 4° C.

Claims

1. Application of Lactobacillus fermentum grx08 in the preparation of food and medicine for alleviating alcoholic liver damage and related symptoms thereof, wherein the accession number of the Lactobacillus fermentum grx08 is CGMCC No: 7695; The fermented lactobacillus grx08 has the effect of reducing alcohol dehydrogenase and relieving and restoring the balance of ethanol metabolism; The fermented lactobacillus grx08 has the effects of reducing alanine aminotransferase, reducing liver lipid accumulation, reducing liver lipid peroxidation products, and alleviating liver damage; The fermented lactobacillus grx08 has the effect of reducing the levels of urea nitrogen and blood creatinine and alleviating kidney damage; The fermented lactobacillus grx08 has the effects of reducing endotoxin and D-lactic acid levels and enhancing intestinal tight connectivity; The fermented lactobacillus grx08 has the effect of reducing the level of inflammatory factors and alleviating the body's inflammatory response; The fermented lactobacillus grx08 has the effect of lowering blood sugar levels and alleviating the increase in blood sugar caused by acute alcoholic injury.

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