TWI715177B - A lactobacillus rhamnosus gklc1, a composition and its use for improving alcoholic injury in liver, stomach and/or intestine - Google Patents

Abstract

The present invention provides a Lactobacillus rhamnosusGKLC1, a composition and its use for improving alcoholic injury in liver, stomach and/or intestine. The composition comprises Lactobacillus paracaseiGKS6, Lactobacillus fermentumGKF3, Bifidobacterium lactisGKK2, Lactobacillus rhamnosusGKLC1, Lactobacillus plantarumGKM3 or the combination thereof.

Description

Lactobacillus rhamnosus GKLC1, composition and its use for improving alcoholic liver injury, stomach injury and/or intestinal injury

This disclosure relates to a lactic acid bacteria for improving alcoholic liver damage, stomach damage and/or intestinal damage, a composition containing the same, and uses thereof; more specifically, it relates to a lactic acid bacteria containing Lactobacillus embryonicum GKM3 and Bifidobacterium lactis GKK2, Lactobacillus rhamnosus GKLC1, Lactobacillus paracasei GKS6, Lactobacillus fermentum GKF3, or a combination thereof, and administer the composition to a recipient for improving alcoholic liver injury, stomach injury, and / Or symptoms of bowel damage.

Alcohol metabolism

The liver is the main organ for alcohol metabolism. There are three enzyme systems that metabolize alcohol: (1) alcohol dehydrogenase-acetaldehyde dehydrogenase, (2) microsomal alcohol oxidation system (MEOS) , microsomal ethanol oxidizing system) and (3) Catalase system. Ethanol is metabolized in the body to produce acetaldehyde, which is the most toxic product to the liver during alcohol metabolism. Acetaldehyde forms a covalent bond with macromolecular products and proteins in the liver, which affects normal metabolism, such as albumin, lipoproteins, mitochondrial proteins and amino acids (Lin et al., 1988).

Alcoholic Liver damage, stomach damage and / Or intestine damage

Alcoholic liver disease (ALD) is a disease caused by long-term drinking. Its symptoms include fatty liver, lipid degeneration, liver fibrosis, liver cirrhosis, or elevated liver index AST and ALT. In the pathogenic mechanism, ethanol consumes the microsomal ethanol oxidation system (MEOS) and induces the expression of hepatic cytochrome P4502E1 (CYP2E1), leading to the production of free radicals, which in turn causes liver damage (Nagata et al., 2007). Some literatures also pointed out that ethanol can cause lipid accumulation, tissue damage and liver dysfunction in the liver (Lieber, 1997). Other documents indicate that ethanol can also damage the intestinal barrier and increase the concentration of endotoxins in the circulatory system, leading to the activation of Kupffer cells in the liver and the secretion of pro-inflammatory cytokines (Wheeler, 2003).

In addition to causing damage to the liver, long-term alcohol consumption can also cause damage to other organs. Under normal circumstances, alcohol is not easy to burn the esophagus, but alcohol still has a certain irritation and damage to the esophageal mucosa, which can cause the mucosa to deteriorate. Therefore, under the long-term stimulation of high concentration of alcohol, the esophageal mucosal cells may also have lesions. Furthermore, because alcohol relaxes the muscles, excessive alcohol intake may reduce the contractility of the cardia sphincter muscle located below the esophagus, causing gastric acid to easily flow back into the esophagus and trigger gastroesophageal reflux. Alcohol may also cause gastric mucosal damage and cause gastric ulcers, and even Marvel's syndrome with gastric bleeding due to a laceration of the esophageal mucosa located above the cardia. Alcohol may also cause the pancreatic duct to produce protein plugs and block it, causing the digestive enzymes secreted by the pancreas to return to the pancreas, causing self-digestion and causing acute pancreatic inflammation. Alcohol can also cause intestinal leakage, which in turn can cause harm to the human body, such as malabsorption of nutrients, food intolerance, autoimmune reaction, systemic inflammation or endotoxemia.

This disclosure provides a Lactobacillus rhamnosus GKLC1, which is deposited with the Food Industry Development Institute under the deposit number BCRC910828.

The present disclosure provides a composition for effectively improving alcoholic liver injury, stomach injury and/or intestinal injury, which comprises Lactobacillus embryonicum GKM3, Bifidobacterium lactis GKK2, Lactobacillus rhamnosus GKLC1, Lactobacillus paracasei GKS6, and fermented lactic acid. Bacillus GKF3 or a combination thereof.

The present disclosure provides a composition for effectively improving alcoholic liver injury, stomach injury and/or intestinal injury, which comprises: Lactobacillus rhamnosus GKLC1 and; Any one of the group consisting of Lactobacillus germ GKM3, Bifidobacterium lactis GKK2, Lactobacillus paracasei GKS6, and Lactobacillus fermentum GKF3.

The present disclosure provides a composition for effectively improving alcoholic liver injury, stomach injury and/or intestinal injury, which comprises Lactobacillus embryonicum GKM3, Bifidobacterium lactis GKK2, Lactobacillus rhamnosus GKLC1, Lactobacillus paracasei GKS6, and fermented lactic acid. The active substance of Bacillus GKF3 or a combination thereof, wherein the active substance is prepared by the following method: (A) Take a colony of a strain and inoculate it on a solid medium for solid culture; and (B) Inoculating the bacteria cultured in step (a) into a liquid medium for liquid culture.

Preferably, the Lactobacillus rhamnosus GKLC1 is deposited with the Food Industry Development Institute under the deposit number BCRC910828.

Preferably, the method further includes the following steps: (C) Centrifuge the liquid medium containing bacteria in step (b) to obtain bacteria sludge; and (D) freeze-drying the bacterial paste obtained in step (c) as a freeze-dried powder.

Preferably, the freeze-drying temperature is -196 to -40°C.

Preferably, the method further includes the following steps: (E) Centrifuge the liquid medium containing bacteria in step (b) to obtain the fermentation supernatant.

Preferably, the above composition includes an additive selected from the following group: excipients, preservatives, diluents, fillers, absorption enhancers, sweeteners, or combinations thereof.

Preferably, the aforementioned composition is a medicine, feed, beverage, nutritional supplement, dairy product, food or health food.

Preferably, the above composition is in the form of powder, lozenge, granulation, suppository, microcapsule, ampoule, liquid spray or suppository.

The present disclosure has been experimentally confirmed that the composition comprising Lactobacillus germ GKM3, Bifidobacterium lactis GKK2, Lactobacillus rhamnosus GKLC1, Lactobacillus paracasei GKS6, Lactobacillus fermentum GKF3 or a combination thereof can be used to improve alcoholic liver injury, stomach Use for injury and/or intestinal injury.

Preferably, the liver injury is fatty liver, lipid degeneration, liver fibrosis, liver cirrhosis, increased liver index or Kühler cell activation.

Preferably, the liver injury is the accumulation of lipids in liver cells.

Preferably, the gastric injury is gastroesophageal reflux, gastric ulcer or Marvel's syndrome.

Preferably, the gastric injury is necrosis or disease of gastric mucosal epithelial cells.

Preferably, the intestinal injury is intestinal leakage or endotoxemia.

Preferably, the intestinal injury is necrosis or disease of intestinal mucosal epithelial cells.

Source and characteristics of strains

The samples of Lactobacillus rhamnosus GKLC1 were collected from breast milk samples donated by people in Taoyuan City, Taiwan. After the isolation of a single strain, multiple strains of strains were successfully isolated, among which strains with larger colonies and milky white cream-like appearance can be observed. The strain has high growth characteristics, high activity, rapid reproduction and strong growth ability. In the test of co-cultivation with gastrointestinal cells, the strain has the characteristics of significantly improving cell viability and cell survival rate. It also has the characteristics of strengthening the gastrointestinal tract and can even effectively reduce the damage of alcohol or hydrogen peroxide to enhance cell viability. After microscopic appearance inspection, the strain showed a thin rod-shaped, facultative anaerobic, non-motility, negative catalyst test, no spores, no motility, it is a characteristic of typical Lactobacillus rhamnosus, so it was named GKLC1. Observed from the characteristics of the strain, the strain can grow well in MRS medium at 37°C, and has excellent acid and bile salt resistance. It can effectively improve the intestinal flora through the gastrointestinal tract. The antibiotic resistance is in line with EU EFSA It is recognized as a characteristic strain with high potential and high maintenance of gastrointestinal function.

Genotype analysis

Further identification by molecular biology and comparison of 16sRNA sequencing results with the database, the strain was identified as Lactobacillus rhamnosus with a similarity rate of 99%, and its scientific name was Lactobacillus rhamnosus .

In order to confirm the uniqueness of the GKLC1 strain, the GKLC1 recN sequence was compared with the L. rhamnosus BCRC 18879 strain (purchased from the Bioresource Conservation and Research Center of the Food Industry Development Institute) and the L. rhamnosus ATCC 53103 strain.

After the strain was activated and amplified to extract gDNA, the primer pairs in the following table 1 were used to react at 94°C for 3 minutes, followed by 35 cycles of 94°C for 30 seconds, 52°C for 30 seconds, and 72°C for 1 minute and 10 seconds. , And finally the polymerase chain reaction was performed at 72°C for 5 minutes.

Table 1: The primer pairs of recN in polymerase chain reaction name sequence Lrh-recN-F 5'- ACGCATCTAGGTTTATTGGA-3' Lrh-recN-R 5'- GCGCTTTATGGGTTAGTTTA-3'

The samples after the reaction were sent for sequencing, and the rec N sequences of 3 strains were obtained. On the other hand, the known Lactobacillus rhamnosus strain DS4 (NZ QAZI01000013), Lrh29 (JTIA01000077), ATCC were downloaded from the NCBI database 8530 (CP003094), BPL15 (CBZU010000001) and the rec N sequence of Lactobacillus casei strain Lbs2 (JPKN02000062) outside the group. Based on the above data, the evolution tree is drawn using the Neighbor-Joining mode of MEGA X software. The alignment result of recN gene sequence is shown in Figure 1. In the evolution tree of rec N gene, the position of GKLC1 in the evolution tree can be separated from other strains and form a line on its own, indicating the difference between GKLC1 and the existing Lactobacillus rhamnosus strain. It is a novel Lactobacillus rhamnosus strain .

After confirming that GKLC1 is a novel strain, the strain was deposited with the Food Industry Development Institute under the name " Lactobacillus rhamnosus GKLC1" on February 12, 2018, and obtained the deposit number BCRC 910828, and it was completed in 2018 Confirmation of the survival of the strain was completed on February 26th.

Phenotypic analysis - Acid resistance test

Compare the acid tolerance of GKLC1 relative to other strains. GKLC1, BCRC-18879, BCRC-18880 and ATCC-53103 purchased from the Biological Resources Conservation and Research Center of the Food Industry Development Institute were activated. The pH of the original MRS liquid medium is pH 6.5. By adding hydrochloric acid to the MRS liquid medium, the pH of the medium is adjusted to three different pH levels: pH 3.2, pH 2.4, and pH 2.0. The strains were inoculated on media with different pH values and incubated at 37°C for 3 hours, and the number of colonies formed was counted.

The result is shown in Figure 2. Under the original pH culture (approximately pH 6.5), the bacterial count of GKLC1 and the other three strains can reach the power of 10. At pH 3.2, the bacterial count of all strains decreased slightly, and there was no significant difference between GKLC1 and the other three strains. When the pH dropped to pH 2.4 and pH 2.0, the bacterial count of BCRC-18879, BCRC-18880 and ATCC-53103 dropped sharply to the 4th power, which was significantly lower than GKLC1 (P> 0.05). Based on this, it is known that the number of viable bacteria of GKLC1 is significantly higher than that of other strains in an acidic environment, indicating that GKLC1 has better acid resistance and better resistance to gastric acid when passing through the stomach.

Phenotypic analysis - Bile salt tolerance test

Compare the bile salt tolerance of GKLC1 relative to other strains. GKLC1, BCRC-18879, BCRC-18880 and ATCC-53103 purchased from the Biological Resources Conservation and Research Center of the Food Industry Development Institute were activated. These strains were inoculated into MRS liquid medium containing 0.3% bile salts, and after soaking for half an hour at 37°C, the number of colonies formed was observed and counted.

The results are shown in Figure 3. The cell counts of GKLC1, BCRC-18879, BCRC-18880 and ATCC-53103 are all close to 9 x 10 ⁹ in the original MRS liquid medium. In the MRS with 0.3% bile salt, the bacterial counts of BCRC-18879, BCRC-18880 and ATCC-53103 were significantly lower than those of GKLC1 (P>0.05). Based on this, it is known that in the bile salt environment, the number of viable bacteria of GKLC1 is significantly higher than that of other strains, indicating that GKLC1 has better acid tolerance and better ability to resist bile salts when passing through the digestive tract in the body.

Phenotypic analysis - Heat resistance test

Compare the heat resistance of GKLC1 relative to other strains. GKLC1, BCRC-18879, BCRC-18880 and ATCC-53103 purchased from the Biological Resources Conservation and Research Center of the Food Industry Development Institute were activated. After heating these bacteria in a water bath at 70°C for 5, 10, and 15 minutes, observe and count the number of colonies formed.

The result is shown in Figure 4. The cell counts of GKLC1, BCRC-18879, BCRC-18880 and ATCC-53103 are all close to 9x10 ⁹ under the original MRS liquid culture medium. After heating at 70°C for 5 minutes, the bacterial counts of all strains dropped to the 7th power. When heated at 70°C for 15 minutes, the bacterial counts of BCRC-18879, BCRC-18880 and ATCC-53103 dropped to about 6th power. Significantly lower than GKLC1 (P>0.05), which maintains the number of bacteria to the 7th power. Based on this, it is known that the number of viable bacteria of GKLC1 is significantly greater than that of other strains in a high temperature environment, indicating that GKLC1 has better heat resistance and better strain stability in the environment.

Test substance

The strains used in the examples of the present disclosure as test substances were purchased from the Biological Resources Conservation and Research Center of the Food Industry Development Institute. The names, deposit numbers and deposit dates of these strains are shown in Table 2 below. However, the strains mentioned in this disclosure are not limited to those obtained through this channel.

Table 2: Source of strains name Deposit number Deposit date Lactobacillus plantarum GKM3 BCRC 910787 July 14, 106 Bifidobacterium lactis GKK2 BCRC 910826 February 12, 107 Lactobacillus rhamnosus GKLC1 BCRC 910828 February 12, 107 Lactobacillus paracasei GKS6 BCRC 910788 July 14, 106 Lactobacillus fermentum GKF3 BCRC 910824 February 12, 107

Strain culture

The colonies of GKM3, GKK2, GKLC1, GKS6 and GKF3 mentioned above were picked up and inoculated on a solid medium to activate the strain. In a preferred embodiment, the solid medium is MRS agar. After the growth of the bacteria is completed, the fresh bacteria and the solid culture medium are inserted into the conical flask containing the liquid culture medium for liquid culture. In a preferred embodiment, the liquid culture is carried out at a temperature of 35 to 50° C., an aeration rate of 0 to 1 vvm nitrogen or carbon dioxide, and a rate of 10 to 100 rpm. In a preferred embodiment, the time for liquid culture is 16 to 24 hours, more preferably 18 hours. In a preferred embodiment, the liquid medium is MRS liquid medium. In a preferred embodiment, the formula of the liquid culture medium is shown in Table 3 below.

table 3 ingredient Proportion (weight percentage) glucose 1~10% Yeast extract 0.1~5% Egg White 0.1~5% Trace elements 0.01~2% Cysteine 0.01~0.1% Tween-80 0.05~1%

Fermentation supernatant preparation

After the strain to be activated completes the cultivation of liquid fermentation, the strain is removed by centrifugation to obtain the fermentation supernatant and save it. In a preferred embodiment, the liquid fermentation culture of the bacterial species selects MRS liquid medium. In a preferred embodiment, the culture time of liquid fermentation is about 24 hours. In a preferred embodiment, the liquid culture medium containing the bacteria is centrifuged at a speed of 1000-15000 rpm. In a preferred embodiment, the storage temperature of the fermentation supernatant is about -20°C. The preserved fermentation supernatant was used as the test substance for the following cell experiments as the active substance.

Freeze-dried powder preparation

After the bacterial species have grown in the liquid culture, the liquid medium containing the bacterial cells is collected and centrifuged to obtain bacterial sludge. In a preferred embodiment, the liquid medium containing the bacteria is centrifuged at a rate of 1000 to 15000 rpm. Mix the obtained bacterial paste with protective agent (protective agent is 6-30% skimmed milk powder), freeze-dry, freeze-dry and store at low temperature. In a preferred embodiment, the freeze-drying temperature is set at -196 to -40°C. In a preferred embodiment, the freeze-drying time is 16 to 72 hours. In a preferred embodiment, the storage temperature is -30°C to 0°C. The preserved freeze-dried powder is used as the active substance in the following animal experiments.

The active substance used in the test substance is not limited to the form of the aforementioned fermentation supernatant and freeze-dried powder without affecting the activity, but also includes the aforementioned state of the culture solution containing the bacterial cells obtained after the bacterial cells are cultured in a liquid state.

Cell test

The human hepatocyte cell line HepG2 was cultured in DMEM (purchased from Sigma) medium containing 10% fetal bovine serum (purchased from HyClone™). The medium is supplemented with 1 mM sodium pyruvate and 1% antibiotics (including penicillin 100 units/mL and streptomycin 100 μg/mL). The culture medium is placed in a constant temperature incubator with a temperature of 37°C and a gas atmosphere of 5% carbon dioxide, and subcultures once every 2 to 3 days.

The human hepatocytes HepG2 were seeded into 96-well flat-bottomed culture plates at a cell number of 1×10 ⁴ /well, and after 24 hours of culture, the cells were attached to the culture plate. In the experimental group, the cells were placed in an incubator with a temperature of 37 ℃ and a gas atmosphere of 5% carbon dioxide with concentrations of 0.31%, 0.63%, 1.25%, 2.5%, 5%, and 10% alcohol. After 24 or 48 hours, Remove the medium and add 100 µl of MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution at a concentration of 0.5 mg/mL to each well. Place the cells in an incubator with a temperature of 37°C and a gas atmosphere of 5% carbon dioxide for 1 hour. Next, remove the MTT solution and add 50 µl of DMSO to each well to dissolve the formazan. Measure the absorbance at 570 nm with a spectrophotometer (ELISA reader) (purchased from Multiskan™ FC, ThermoFisher, USA). In the step of adding alcohol to the experimental group, the control group only added the solvent DMSO (Dimethyl sulfoxide) (purchased from Sigma) without adding alcohol, and the rest of the experimental steps of the control group were the same as the experimental group. Calculate the cell survival rate percentage (%) of the experimental group based on the control group by the following formula:

Percentage of survival rate = [absorbance value of the experimental group (570 nm) / absorbance value of the control group (570 nm)] × 100%.

The human hepatocytes HepG2 were seeded into 96-well flat-bottomed culture plates at a cell number of 1×10 ⁴ /well, and after 24 hours of culture, the cells were attached to the culture plate. In the experimental group, 5% of the fermentation supernatant was added (percentage concentration relative to the volume of the culture medium), and the cells were placed in an incubator with a temperature of 37 ℃ and a gas atmosphere of 5% carbon dioxide for 2 hours. Then, one group was treated with 5% alcohol and the other group with solvent DMSO (Dimethyl sulfoxide) (purchased from Sigma) in an incubator with a temperature of 37 ℃ and a gas atmosphere of 5% carbon dioxide for 24 hours, and then removed Medium and add 100 µl of MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution at a concentration of 0.5 mg/mL to each well. Place the cells in an incubator with a temperature of 37°C and a gas atmosphere of 5% carbon dioxide for 1 hour. Next, remove the MTT solution and add 50 µl of DMSO to each well to dissolve the formazan. Measure the absorbance at 570 nm with a spectrophotometer (ELISA reader) (purchased from Multiskan™ FC, ThermoFisher, USA). In the step of adding lyophilized powder back to solution or alcohol in the experimental group, the positive control group only added the solvent DMSO (Dimethyl sulfoxide) (purchased from Sigma), while the negative control group did not add the lyophilized powder back to the solution but added alcohol. The rest of the test steps of the negative control group are the same as those of the experimental group. Calculate the cell survival rate percentage (%) of the experimental group based on the control group by the following formula:

Percentage of survival rate = [absorbance value of the experimental group (570 nm) / absorbance value of the control group (570 nm)] × 100%.

Statistical Analysis

All result data are mean ± standard deviation (mean ± SD). The fold change is to compare the change of gene activity between the control group and the experimental group. When analyzing the statistical difference between the experimental group and the control group, if p>0.05, it is regarded as a statistically significant difference.

Results of cell viability

The survival rate test results of cells treated with different concentrations of alcohol for different times are shown in Figure 11. The survival rate of human hepatocytes HepG2 treated with alcohol at concentrations of 0.31%, 0.63% and 1.25 for 24 or 48 hours did not change significantly compared with the cell survival rate of the control group. The cell survival rate of human hepatocytes HepG2 treated with 2.5%, 5%, and 10% alcohol for 24 or 48 hours compared with the cell survival rate of the control group decreased significantly with the increase of alcohol concentration and treatment time. This result shows that as the alcohol concentration becomes higher, the higher the alcohol concentration and the longer the treatment time, the more obvious the damage to the cells. Among them, the survival rate of human liver cells HepG2 treated with 5% alcohol for 24 hours was significantly lower than that of the control group to about 60%. Therefore, follow-up experiments use this concentration and time to evaluate the improvement effect of the invention on the liver cells damaged by alcohol.

The survival rate test results of the cells treated with the freeze-dried powder of each single species of bacteria are shown in Figure 12. Compared with the positive control group treated with alcohol, the experimental group administered with a single strain (GKM3, GKS6, GKK2, GKF3 and GKLC1) lyophilized powder was re-dissolved and then treated with alcohol. The results showed that, except for GKLC1, the rest The cell survival rate did not increase significantly after single strain treatment. This result shows that the administration of the other single strains except GKLC1 did not show the effect of repairing and improving the cells damaged by alcohol (P>0.05).

The survival rate test results of the cells treated with freeze-dried powders of different combinations of strains are shown in Figure 13. Compared with the positive control group treated with alcohol, in the experimental group administered with a combination of different strains of freeze-dried powder and then treated with alcohol, the experimental group of GKM3, GKS6, GKK2, and GKF3 combined with GKLC1, the cells The survival rate has improved significantly. This result shows that the combination of specific strains can show repair and improvement effects on alcohol-injured cells (P>0.05).

Test animal

The test animals purchased 42 C57BL/6N (B6) male rats from BioLASCO, each weighing about 20-25 grams. The mouse is kept in a conventional cage, the room temperature is maintained at 22 ± 3°C, the humidity is 55 ± 1 5%, and the time is 12 hours of light and darkness. The feed and sterile reverse osmosis water are allowed to eat freely by the mice. The test was performed in accordance with Taiwan-US standard operating procedures SOPA-303. New animals need to be quarantined for 7 days to ensure that there are no abnormalities before testing. Each breeding cage is affixed with an experimental animal identification card, and the animal number, species/line, number/sex, source, IACUC number and the person in charge of the experiment are indicated.

Test design

Grouping: Divide 42 mice into 7 groups with 6 animals in each group, including positive control group, negative control group, GKS6 lyophilized powder, GKF3 lyophilized powder, GKK2 lyophilized powder, GKLC1 lyophilized powder, GKM3 lyophilized powder Dry powder. Dosage conversion: The oral dose for mice is calculated based on the daily intake of adults, and then is converted according to the metabolic ratio coefficient of the mouse to the human body 12.3 to calculate the daily oral dose for mice. Taking an adult taking 4 grams per person per day as an example, the daily oral dose for mice is 0.82 g/kg B.W. (4 g/60 kg × 12.3 = 0.82 g/kg). The detailed grouping and dosage are shown in Table 4:

Table 4 Test group Feed Substance Dose ( g/kg ) Relative human dose ( g/60kg ) Positive control group Normal liquid feed LD101 Reverse osmosis water - - Negative control group Alcohol liquid feed LD101A Reverse osmosis water - - Experimental group GKS6 GKS6 freeze-dried powder 0.82 4.0 Experimental group GKF3 GKF3 freeze-dried powder Experimental group GKK2 GKK2 freeze-dried powder Experimental group GKLC1 GKLC1 freeze-dried powder Experimental group GKM3 GKM3 freeze-dried powder

The feed used in this experiment refers to Lieber-DeCarli's formula LD101A for inducing alcoholic fatty liver, replacing carbohydrates with alcohol to provide 36% of the total energy required by the animal (Lieber et al., 1982). After the mice were fed for 6 weeks, their fatty liver pathological examination results showed that in addition to the normal control group, the livers of the negative control group and the 5 groups of test substance groups showed extensive accumulation of hepatocyte fatty oil droplets, and the pathological fraction of the lesion The degree ranges from mild to extremely severe. During the experiment, a proper amount of the test substance was taken every day and used only on the same day. The plastic syringe was covered with a feeding needle for tube feeding once a day for a total of 8 weeks during the experiment. The total volume of each dose group and the control group was 10 mL/kg of the test substance or control substance by tube every day. When the test substance was administered for 8 weeks, the mice were sacrificed and sectioned for analysis.

Tissue biopsy

Take the liver tissue, stomach tissue, and intestine tissue that have been immersed in 10% formalin solution and have been roughly repaired. After dehydration, clarification, paraffin infiltration, and embedding, they are processed into paraffin tissue blocks, which are then treated with a microtome ( Leica RM 2145, Nussloch, Germany) Cut tissue sections with a thickness of 5 μm, stain them with Hematoxylin & Eosin (H&E), and observe the pathological changes of mice in each group under an optical microscope (Opticphot-2, Nikon, Tokyo, Japan). The veterinarian in the Pathological Toxicology Laboratory of the Biomedical Department of Lesco Biotechnology Co., Ltd. was commissioned to score histopathological lesions for fatty liver, alcohol-induced gastric injury and intestinal leakage.

Statistical Analysis

All data are expressed as mean ± standard deviation (Mean ± S.D.). The test results were analyzed using the One-way ANOVA of the statistical software SPSS 16.0 and Duncan's multiple range test. When p>0.05, it indicates that there is a significant difference between the groups. Part of the test group data was analyzed using the Independent-Samples T Test of the statistical software SPSS 16.0 and the negative control group, and displayed as a P value.

Liver tissue section results

For semi-quantitative evaluation of fatty liver tissue pathology, according to the Health Food Announcement No. 1031304063 of the Department of Health, the liver cell fatty oil droplet accumulation lesions are divided into 5 grades, as shown in Table 5 below.

Table 5: Scoring table of liver tissue score Extent of disease 0 Normal (0%) 1 Slight (> 10%) 2 Moderate (10~33%) 3 Serious (33~66%) 4 Extremely serious (66~100%)

As shown in Figure 5, (A) the hepatocytes of the positive control group showed a slight accumulation of lipid oil droplets, which is a spontaneous animal disease. (B) The hepatocytes of the negative control group showed extensive accumulation of mild to very severe lipid oil droplets, and the pathological fraction of the lesion was significantly different from that of the positive control group (p> 0.05). (C) Experimental group GKS6 showed extensive normal to slight accumulation of lipid oil droplets, (D) Experimental group GKF3 exhibited extensive normal to moderate accumulation of lipid oil droplets, (E) Experimental group GKK2 liver showed extensive normal to moderate lipid accumulation Oil droplet accumulation, (F) experimental group GKLC1 showed extensive normal to moderate accumulation of lipid oil droplets and (G) experimental group GKM3 showed extensive normal to moderate accumulation of lipid oil droplets. Compared with the negative control group, the degree of fatty liver tissue lesions in the five experimental groups were lower (p> 0.05). The statistical results are shown in Figure 6, showing that the administration of these five strains can all improve fatty liver caused by alcohol symptom.

Stomach biopsy results

The pathology of alcohol-induced gastric injury is based on Shackelford et al.'s publication in Toxicologic Pathology (2002), which is divided into 4 grades, as shown in Table 6 below.

Table 6: Stomach tissue score table score Extent of disease 1 Very slight (> 10%) 2 Mild (10~39%) 3 Moderate (40~79%) 4 Serious (80~100%)

As shown in Figure 7, (A) the gastric mucosal epithelial cells in the positive control group showed very slight lesions and necrotic lesions, which are spontaneous animal lesions. (B) The minimal to moderate degeneration and necrosis of gastric mucosal epithelial cells in the negative control group were significantly different from those in the positive control group (p> 0.05). (C) GKS6 gastric mucosal epithelial cells in the experimental group had minimal to mild degeneration and necrosis, (D) GKF3 gastric mucosal epithelial cells in the experimental group were normal to mild degeneration and necrosis, (E) GKK2 hepatic and gastric mucosal epithelial cells in the experimental group were normal To mild to mild degeneration and necrosis, (F) the experimental group GKLC1 gastric mucosal epithelial cells normal to mild degeneration and necrosis and (G) the experimental group GKM3 gastric mucosal epithelial cells normal to moderate degeneration and necrosis. Compared with the negative control group, the gastric mucosal lesions and necrosis in the experimental group GKF3, GKK2 and GKLC1 animals were milder (p> 0.05). The statistical results are shown in Figure 8, showing that the experimental group strains GKF3, GKK2 and GKLC1 can improve alcohol The resulting gastric mucosal damage.

Intestinal tissue section results

The pathology of alcohol-induced intestinal leakage is based on Shackelford et al.'s publication in Toxicologic Pathology (2002). The score is divided into 4 grades, as shown in Table 7 below.

Table 7: Intestinal tissue score table score Extent of disease 1 Very slight (> 10%) 2 Mild (10~39%) 3 Moderate (40~79%) 4 Serious (80~100%)

As shown in Figure 9, (A) the intestinal mucosal epithelial cells in the positive control group showed very slight degeneration and necrosis lesions, which are spontaneous animal diseases. (B) The intestinal mucosal epithelial cells in the negative control group had minimal to moderate degeneration and necrosis, and the pathological fraction of the lesion was significantly different from that of the positive control group (p> 0.05). (C) GKS6 intestinal mucosal epithelial cells in the experimental group with normal to minimal degeneration and necrosis, (D) GKF3 intestinal mucosal epithelial cells in the experimental group with normal to mild degeneration and necrosis, (E) GKK2 hepatic intestinal mucosal epithelial cells in the experimental group are normal to mild Degree of degeneration and necrosis, (F) normal to minimal degeneration and necrosis of GKLC1 intestinal mucosal epithelial cells in the experimental group, and (G) normal to minimal degeneration and necrosis of GKM3 intestinal mucosal epithelial cells in the experimental group. Compared with the negative control group, the degree of intestinal mucosal tissue lesions in the 5 experimental groups were lower (p> 0.05). The statistical results are shown in Figure 10, showing that the administration of these 5 strains can all improve the intestinal leakage caused by alcohol symptom.

Based on the above experimental results, the administration of strains GKF3, GKK2, GKLC1 can simultaneously reduce the severity of liver fatty liver, gastric mucosal damage and intestinal leakage caused by alcohol intake; the beneficial strains GKS6 and GKM3 can simultaneously reduce alcohol intake, causing liver fatty liver and intestine The severity of the leakage. It shows that the probiotics mentioned in this case have the ability to improve alcoholic liver damage, intestinal damage and/or stomach damage. The results are summarized in Table 7 below.

Table 7: Integration of scoring results GKS6 GKF3 GKK2 GKLC1 GKM3 Fatty liver ↓ ↓ ↓ ↓ ↓ Stomach injury ↓ ↓ ↓ Intestinal leakage ↓ ↓ ↓ ↓ ↓

↓: After one-way ANOVA and Duncan's multiple range test, the negative control group has a significant decrease (p>0.05).

The present disclosure provides a composition comprising at least one selected from the group consisting of Lactobacillus germ GKM3, Bifidobacterium lactis GKK2, Lactobacillus rhamnosus GKLC1, Lactobacillus paracasei GKS6, and Lactobacillus fermentum GKF3. The composition has improved alcoholic properties The efficacy of liver damage, intestinal damage and/or stomach damage.

The composition further contains additives. In a preferred embodiment, the additive may be an excipient, preservative, diluent, filler, absorption enhancer, sweetener, or a combination thereof. The excipient can be selected from sodium citrate, calcium carbonate, calcium phosphate, sucrose or a combination thereof. The preservative can prolong the shelf life of the pharmaceutical composition, such as benzyl alcohol and parabens. The diluent may be selected from water, ethanol, propylene glycol, glycerin or a combination thereof. The filler may be selected from lactose, nougat, high molecular weight ethylene glycol, or a combination thereof. The absorption enhancer may be selected from dimethyl sulfide (DMSO), azolinone, propylene glycol, glycerin, polyethylene glycol, or a combination thereof. The sweetener may be selected from Acesulfame K, aspartame, saccharin, sucralose, neotame, or a combination thereof. In addition to the additives listed above, other suitable additives can be selected according to requirements without affecting the medical effect of the composition.

The composition can be developed into different commodities in the medical field. In a preferred embodiment, the composition is a medicine, feed, beverage, nutritional supplement, dairy product, food or health food.

The composition can adopt different forms according to the needs of the recipient. In a preferred embodiment, the composition is in the form of powder, lozenge, granulation, suppository, microcapsule, ampoule/ampule, liquid spray or suppository.

The composition of the present disclosure can be used in animals or humans. Under the premise of not affecting the effect of the lactic acid bacteria, the composition containing the lactic acid bacteria can be prepared into any pharmaceutical form, and administered to the animal or human by an applicable route according to the pharmaceutical form.

Composition preparation

If the strain of the present disclosure is applied to food use, the following compositions 1 to 4 are taken as illustrative examples.

Composition 1: Take the combination of GKM3 and GKLC1 freeze-dried powder as the test substance (20 wt%), mix it with benzyl alcohol (8 wt%) as preservative and glycerin (7 wt%) as diluent, and dissolve in pure Store in water (65 wt%) at 4°C for later use. The aforementioned wt% refers to the proportion of each component to the total weight of the composition.

Compositions 2, 3, 4: The GKM3 of composition 1 was replaced with GKS6, GKK2, GKF3, and the lyophilized powder of GKLC1 was combined as the test substance (20 wt%), and the rest of the composition was the same as that of composition 1.

If the strains of the present disclosure are applied in liquid form for medical purposes, the following compositions 5 to 8 are taken as illustrative examples.

Composition 5: Take a combination of GKM3 and GKLC1 lyophilized powder as the test substance (20 wt%), and benzyl alcohol (8 wt%) as a preservative, glycerin (7 wt%) as a diluent, and sucrose as a diluent ( 10 wt%) mixed thoroughly, dissolved in pure water (55 wt%), and stored at 4°C for later use. The aforementioned wt% refers to the proportion of each component to the total weight of the composition.

Compositions 6, 7, and 8: The GKM3 of composition 5 was replaced with GKS6, GKK2, GKF3, and GKLC1 freeze-dried powder combination as the test substance (20 wt%), and the rest of the composition was the same as composition 5.

no

Figure I shows Lactobacillus rhamnosus (Lactobacillus rhamnosus) GKLC1 with other strains rec N gene evolution tree.

Figure 2 shows the acid tolerance of Lactobacillus rhamnosus GKLC1 and other strains.

Figure 3 shows the bile tolerance of Lactobacillus rhamnosus GKLC1 and other strains.

Figure 4 shows the heat resistance of Lactobacillus rhamnosus GKLC1 and other strains.

Figure 5 shows alcohol-induced fatty liver mice (A) positive control group, (B) negative control group, (C) experimental group GKS6, (D) experimental group GKF3, (E) experimental group GKK2, (F) experimental group GKLC1, (G) experimental group GKM3 liver tissue section staining results.

Figure 6 shows the statistical results of liver tissue slice lesions in the positive control group, negative control group, experimental group GKS6, experimental group GKF3, experimental group GKK2, experimental group GKLC1, and experimental group GKM3 in alcohol-induced fatty liver mice.

Figure 7 shows alcohol-induced gastric injury mice (A) positive control group, (B) negative control group, (C) experimental group GKS6, (D) experimental group GKF3, (E) experimental group GKK2, (F) experimental group GKLC1 and (G) experimental group GKM3 gastric tissue section staining results.

Figure 8 shows the statistical results of gastric tissue slice lesions in the positive control group, negative control group, experimental group GKS6, experimental group GKF3, experimental group GKK2, experimental group GKLC1, and experimental group GKM3 in alcohol-induced gastric injury mice.

Figure 9 shows alcohol-induced intestinal leakage mice (A) positive control group, (B) negative control group, (C) experimental group GKS6, (D) experimental group GKF3, (E) experimental group GKK2, (F) experiment Group GKLC1, (G) experimental group GKM3 intestinal tissue section staining results.

Figure 10 shows the statistical results of intestinal tissue slice lesions in the positive control group, negative control group, experimental group GKS6, experimental group GKF3, experimental group GKK2, experimental group GKLC1, and experimental group GKM3 in alcohol-induced intestinal leakage mice.

Figure 11 shows the cell survival rate of the control group and the experimental group treated with different concentrations of alcohol for different times.

Figure 12 shows the cell survival rate of the positive control group, the negative control group, and the experimental group treated with the freeze-dried powder of each single strain.

Figure 13 shows the cell survival rate of the positive control group, the negative control group, and the experimental group treated with the freeze-dried powder of different combinations of strains.

Lactobacillus rhamnosus GKLC1: BCRC 910828, February 12, 2017

Lactobacillus plantarum GKM3: BCRC 910787, July 14, 106

Bifidobacterium lactis GKK2: BCRC 910826, February 12, 2017

Lactobacillus paracasei GKS6: BCRC 910788, July 14, 106

<110> Grape King Biotechnology Co., Ltd.

<120> Lactobacillus rhamnosus GKLC1, composition and its use for improving alcoholic liver injury, stomach injury and/or intestinal injury

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<170> PatentIn version 3.5

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<212> DNA

<213> Artificial Sequence

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Claims (18)

A Lactobacillus rhamnosus GKLC1 is deposited with the Food Industry Development Institute under the deposit number BCRC910828. A composition for improving alcoholic liver injury, stomach injury and/or intestinal injury, comprising Lactobacillus rhamnosus GKLC1; wherein the Lactobacillus rhamnosus GKLC1 is deposited with the Food Industry Development Institute under the deposit number BCRC910828. A composition for improving alcoholic liver injury, stomach injury and/or intestinal injury, comprising: Lactobacillus rhamnosus GKLC1 and; selected from Lactobacillus embryo GKM3, Bifidobacterium lactis GKK2, Lactobacillus paracasei GKS6, and fermented lactic acid Any one of the group consisting of Bacillus GKF3; wherein the Lactobacillus rhamnosus GKLC1 is deposited with the Food Industry Development Institute under the deposit number BCRC910828. A composition for improving alcoholic liver injury, stomach injury and/or intestinal injury, comprising: an active substance of Lactobacillus rhamnosus GKLC1; wherein the Lactobacillus rhamnosus GKLC1 is deposited in the Food Industry Consortium under the deposit number BCRC910828 Development Research Institute; wherein the active substance is prepared by the following method: (a) a colony of a strain is inoculated into a solid medium for solid-state culture; and (b) a bacterial cell cultured in step (a) is inoculated into a liquid Medium for liquid culture. The composition according to claim 4, wherein the composition further comprises an activity selected from the group consisting of Lactobacillus embryonicum GKM3, Bifidobacterium lactis GKK2, Lactobacillus paracasei GKS6, and Lactobacillus fermentum GKF3 substance. The composition according to claim 4, wherein the method further comprises the following steps: (c) centrifuging the liquid medium containing bacterial cells in step (b) to obtain bacterial sludge; and (d) the step (c) obtained The sludge is freeze-dried as a freeze-dried powder. The composition according to claim 6, wherein the freeze-drying temperature is -196 to -40°C. The composition according to claim 4, wherein the method further comprises the following steps: (e) centrifuging the liquid medium containing the bacteria in step (b) to obtain a fermentation supernatant. The composition according to any one of claims 2 to 4, which comprises an additive selected from the following group: excipients, preservatives, diluents, fillers, absorption enhancers, sweeteners, or combinations thereof . The composition according to any one of claims 2 to 4, which is a medicine, feed, beverage, nutritional supplement, dairy product, food or health food. The composition according to any one of claims 2 to 4, in the form of powder, lozenge, granulation, suppository, microcapsule, ampoule, liquid spray or suppository. A use of the composition according to any one of claims 2 to 11 for preparing a pharmaceutical composition for improving alcoholic liver injury, stomach injury or intestinal injury. The use according to claim 12, wherein the liver damage is fatty liver, lipid degeneration, liver fibrosis, liver cirrhosis, elevated liver index, or Kühler cell activation. The use according to claim 12, wherein the liver damage is accumulation of lipids in liver cells. The use according to claim 12, wherein the gastric injury is gastroesophageal reflux, gastric ulcer or Marvel's syndrome. The use according to claim 12, wherein the gastric injury is necrosis or disease of gastric mucosal epithelial cells. The use according to claim 12, wherein the intestinal injury is intestinal leakage or endotoxemia. The use according to claim 12, wherein the intestinal injury is necrosis or disease of intestinal mucosal epithelial cells.

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