NL2035211B1 - Lactobacillus paracasei and applications of lactobacillus paracasei in preparation for alleviating alcohol-induced visceral injury - Google Patents
Lactobacillus paracasei and applications of lactobacillus paracasei in preparation for alleviating alcohol-induced visceral injury Download PDFInfo
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Abstract
The present application belongs to the technical field of microorganism applications, and specifically, relates to a lactobacillus paracasei and applications of the lactobacillus paracasei in a preparation for alleviating alcohol-induced visceral injury. The strain is 10 deposited with the China General Microbiological Culture Collection Center (CGMCC), and is assigned with the accession number of CGMCC No. 24630. The lactobacillus paracasei can significantly improve the antioxidant capability of the liver of mice with alcohol-induced liver injury, prevent liver injury caused by alcohol, recover a liver tissue structure, remarkably improve the antioxidant capability of gastric tissue, prevent an 15 inflammatory response of the gastric tissue in the mice caused by the alcohol and recover the morphology of the gastric mucosa, such that the purpose of alleviating alcohol-induced visceral injury is achieved, and the lactobacillus paracasei has no side effects and high safety.
Description
LACTOBACILLUS PARACASEI AND APPLICATIONS OF
LACTOBACILLUS PARACASEI IN PREPARATION FOR
ALLEVIATING ALCOHOL-INDUCED VISCERAL INJURY
The present application belongs to the technical field of microorganism applications, and specifically, relates to a lactobacillus paracasei and applications of the lactobacillus paracasei in a preparation for alleviating alcohol-induced visceral injury.
Ethanol is a main ingredient of Chinese liquor and alcoholic beverages, and a liver is the main organ responsible for metabolizing the ethanol. The majority of alcohol is decomposed and metabolized in the liver, accelerating the formation of Triglyceride (TG), such that a lipid peroxidation reaction is triggered due to the synthesis of a large number of oxygen radicals and fat deposition, and the lipid peroxidation reaction is an important cause of liver injury. In addition, the main pathway of ethanol metabolism is the conversion to acetaldehyde. When excessive alcohol is drunk, the ethanol may also cause oxidative stress damage to hepatocytes by activating Cytochrome P450 2E1 (CYP2E1) and being oxidized by the CYP2ZEI so as to generate large amounts of Reactive Oxygen
Species (ROS); and acetaldehyde and the ROS, which are the metabolites of the ethanol, are highly toxic, and can react with biomacromolecules, resulting in cell injury. If the cells died, macrophages and neutrophils may also be activated, inducing inflammation, thus further damaging the liver.
Furthermore, due to the lipid solubility of the ethanol, gastric injury is easily caused, such that alcohol abuse may lead to severe gastric mucosal erosion and even cause gastric injury and atrophic gastritis. The Chinese liquor with high concentration may even directly erode the gastric mucosa. In addition to this, during metabolism of the ethanol in the human body, the release of the oxygen radicals by the neutrophils leads to endothelial cell injury. Such environment suppresses the repairing of gastric injury. By means of respiration, the neutrophils continue to generate large amounts of oxygen radicals, to form a vicious circle, leading to rupture of a blood vessel wall, thereby causing gastric ulcer and gastric bleeding. As a result, excessive drinking or improper drinking will cause the metabolism of ethanol in the human body to produce many harmful substances and large amounts of free radicals, causing great harm to the human body, especially alcoholic-induced liver injury and gastric injury.
At present, the main methods of prevention and treatment of alcoholic-induced liver injury and gastric injury are alcohol abstinence, chemical synthesis-assisted treatment and natural extract-assisted treatment. However, some of the drugs available on the market for the prevention and treatment of alcoholic-induced liver injury and gastric injury, especially chemical synthetic drugs, are effective, but the drugs are also accompanied by different side effects that are detrimental to human health. Therefore, a product with high safety for the prevention and treatment of alcoholic-induced liver injury and gastric injury is necessary.
In view of the above problems, one of the objectives of the present application is to provide a lactobacillus paracasei, which may be used or applied to a product with high safety for the prevention and treatment of alcohol-induced visceral injury. The lactobacillus paracasei is obtained by being separated from yak yogurt from Tibet lhasa, and can significantly improve the antioxidant capability of the liver of mice with alcohol-induced liver injury, prevent liver injury caused by alcohol, recover a liver tissue structure, may also remarkably improve the antioxidant capability of gastric tissue, prevent an inflammatory response of the gastric tissue in the mice caused by the alcohol and recover the morphology of the gastric mucosa, such that alcohol-induced visceral injury is alleviated.
In order to achieve the above purpose, the present application may use the following technical solutions.
One aspect of the present application provides a lactobacillus paracasei. The lactobacillus paracasei is deposited with the China General Microbiological Culture
Collection Center (CGMCC) and is assigned with the accession number of CGMCC No. 24630.
Another aspect of the present application provides a composition. The composition includes one or a combination of a plurality of the following substances: (a) the lactobacillus paracasei; (b) lysate of the lactobacillus paracasei; (c) a culture of the lactobacillus paracasei; and (d) a fermentation broth of the lactobacillus paracasei.
Still another aspect of the present application provides a preparation. The preparation includes the lactobacillus paracasei or the composition, and a carrier. The carrier is a medicinal carrier or an edible carrier.
Still another aspect of the present application provides applications of the lactobacillus paracasei or the composition in preparation of preparations used for alleviating alcohol-induced visceral injury.
Still another aspect of the present application provides applications of the lactobacillus paracasei or the composition in preparation of preparations used for preventing drunkenness and/or alleviating a hangover.
The collection information of the lactobacillus paracasei in the present application includes the following. The lactobacillus paracasei is deposited with the CGMCC at No. 3,
Yard 1, BeiChen West Road, Chaoyang District, Beijing on April 1, 2022 and is assigned with the accession number of CGMCC No. 24630, with the classification name being lactobacillus paracasei.
The beneficial effects of the present application include the following. (1) The lactobacillus paracasei provided in the present application can significantly improve the antioxidant capability of the liver of the mice with alcohol-induced liver injury, prevent liver injury caused by alcohol, recover a liver tissue structure, remarkably improve the antioxidant capability of gastric tissue, prevent an inflammatory response of the gastric tissue in the mice caused by the alcohol and recover the morphology of the gastric mucosa, such that the purpose of alleviating alcohol-induced visceral injury is achieved; and the lactobacillus paracasei has no side effects and high safety. (2) The lactobacillus paracasei provided in the present application can significantly prolong drunkenness time of the human body and shorten sober-up time, such that the purpose of preventing drunkenness and alleviating a hangover is effectively achieved.
Fig. 1 shows colonial morphology of TY-W09.
Fig. 2 shows a gram staining result of TY-W09.
Fig. 3 is a cell adhesion map of TY-W09.
Fig. 4 shows the impact of TY-W09 on drunkenness time of mice.
Fig. 5 shows the impact of TY-WO09 on sober-up time of mice.
Fig. 6 shows the impact of TY-W09 on liver coefficients of mice.
Fig. 7 shows HE staining of liver tissue of mice.
Fig. 8 shows the impact of TY-W09 on Alanine Transaminase (ALT) activity in the serum of mice.
Fig. 9 shows the impact of TY-WO09 on Aspartate aminotransferase (AST) activity in the serum of mice.
Fig. 10 shows the impact of TY-WO09 on Triglyceride (TG) content in the serum of mice.
Fig. 11 shows the impact of TY-WO09 on Total Cholesterol (TC) content in the serum of mice.
Fig. 12 shows the impact of TY-W09 on Glutathione (GSH) content in the liver of mice.
Fig. 13 shows the impact of TY-W09 on Superoxide Dismutase (SOD) activity in the liver of mice.
Fig. 14 shows the impact of TY-W09 on lipid hydroperoxide Malondialdehyde (MDA) content in the liver of mice.
Fig. 15 shows the impact of TY-WO09 on mRNA relative expression of Nuclear Factor
E2-Related Factor 2 (NRF2) in the liver of mice.
Fig. 16 shows the impact of TY-W09 on mRNA relative expression of Heme
Oxygenase 1 (HO-1) in the liver of mice.
Fig. 17 shows the impact of TY-W09 on mRNA relative expression of Cytochrome
P450 2E1 (CYP2E1) in the liver of mice.
Fig. 18 is a protein ribbon diagram.
Fig. 19 shows the impact of TY-WO09 on protein relative expression of NRF2 in the liver of mice.
Fig. 20 shows the impact of TY-WO09 on protein relative expression of HO-1 in the liver of mice.
Fig. 21 shows the impact of TY-WO09 on protein relative expression of CYP2E1 in the liver of mice.
Fig. 22 is an appearance observation diagram of gastric tissue of mice.
Fig. 23 shows gastric mucosa injury indexes of mice.
Fig. 24 shows the impact of TY-W09 on GSH content in gastric tissue of mice.
Fig. 25 shows the impact of TY-W09 on SOD activity in gastric tissue of mice.
Fig. 26 shows the impact of TY-WO09 on IL-6 content in gastric tissue of mice.
Fig. 27 shows the impact of TY-W09 on IL-1 content in gastric tissue of mice.
Fig. 28 shows the impact of TY-W09 on TNF-a content in gastric tissue of mice.
In the drawings: "*" represents that there is a statistical difference (p < 0.05) between two groups; "**" represents that there is a statistical difference (p < 0.01) between two 5 groups; "***" represents that there is a statistical difference (p < 0.001) between two groups; and "****" represents that there is a statistical difference (p < 0.0001) between two groups.
The embodiments are given to better describe the present application, but the content of the present application is not limited only to the embodiments given. Therefore, non-essential improvements and adjustments to the embodiments made by a person skilled in the art in accordance with the content of the above present application still fall within the scope of protection of the present application.
The terms used herein are only intended to describe specific embodiments and are not intended to limit the present disclosure. Expressions in the singular form include those in the plural form unless the expressions have a distinctly different meaning in the context. As used herein, it is to be understood that terms such as "include", "have", "contain", and the like are intended to indicate the presence of features, figures, operations, components, parts, elements, materials, or combinations. The terms of the present application are disclosed in the specification and are not intended to exclude the possibility that one or more other features, figures, operations, components, parts, elements, materials, or combinations thereof may exist or may be added. As used here, "/" may be interpreted as "and" or "or", as appropriate.
In the present application, a lactobacillus paracasei is also called lactobacillus paracasei TY-W09 or TY-W09.
In the present application, the term "preparation" does not only refer to a pharmaceutical preparation for medicinal purposes, but also includes an edible food preparation or a health care product preparation.
An embodiment of the present application provides a lactobacillus paracasei. The lactobacillus paracasei is deposited with the CGMCC and is assigned with the accession number of CGMCC No. 24630.
It is to be noted that, the lactobacillus paracasei 1s derived from yak yogurt from Tibet lhasa. After being detected by means of gram staining results, the lactobacillus fermentum is found to be in a rod-like shape, and is determined as gram-positive bacteria (G*). In addition, by means of PCR amplification of a 16SrDNA sequence, the I6SrDNA sequence is detected to include a sequence shown as SEQIDNO. 1, and by means of homology analysis, it indicates that the strain is the lactobacillus paracasei. In addition, the lactobacillus fermentum has strong gastric juice tolerance and bile salt tolerance; the survival rate of the lactobacillus fermentum in gastric juice may reach 111.76%; and the growth efficiency of the lactobacillus fermentum in bile salt may reach 25.63%. The number of TY-WO09 adhered by 100 cells is 1852, and it indicates that the lactobacillus fermentum has desirable digestive tract resistance and good intestinal tract colonization capabilities.
Another embodiment of the present application provides a composition. The composition includes one or a combination of a plurality of the following substances: (a) the lactobacillus paracasei; (b) lysate of the lactobacillus paracasei; (c) a culture of the lactobacillus paracasei; and (d) a fermentation broth of the lactobacillus paracasei.
It is to be noted that, in the composition, as described above, the lactobacillus paracasei TY-WO09 has desirable digestive tract resistance and good intestinal tract colonization capabilities, and may be prepared into an edible or medicinal composition. In addition, when the lactobacillus paracasei TY-W09 is prepared into the composition, the lactobacillus paracasei may achieve an effect by being directly introduced into the composition in the form of viable bacteria, may achieve an effect by being introduced into the composition in the form of inactivated bacteria after inactivation by means of the existing technology, may achieve an effect by introducing the lysate of the lactobacillus paracasei into the composition, may achieve an effect by introducing products such as proteins, peptides, secretions or metabolites obtained from the culture of the lactobacillus paracasei into the composition, or may achieve an effect by introducing the fermentation broth from the fermentation of the lactobacillus paracasei into the composition. In a specific use process, different forms of the lactobacillus paracasei may be selected for the preparation of the composition according to specific requirements.
In some specific embodiments, the composition further includes one or a combination of probiotics, prebiotics, dietary fiber and traditional Chinese drugs.
It is to be noted that, the lactobacillus paracasei TY-WO9 and different forms thereof can also be used in combination with one or the combination of probiotics, dietary fiber and a pharmacologically active compound. For example, the lactobacillus paracasei
TY-W09 can be used in combination with bacillus subtilis, bi-fidobacterium or lactobacillus, so as to cause the composition to simultaneously have the effects of the lactobacillus paracasei TY-WO09 and other probiotics. For another example, the lactobacillus paracasei TY-W09 can be used in combination with prebiotics, and the prebiotics can provide an energy source for the lactobacillus paracasei TY-W09, such that the effect of the lactobacillus paracasei TY-W09 can be improved. For another example, the lactobacillus paracasei TY-WO09 can be used in combination with the dietary fiber, and the dietary fiber can assist in colonization of the lactobacillus paracasei TY-W09, such that the effect of the lactobacillus paracasei TY-WO09 can be improved. For another example, the lactobacillus paracasei TY-WO09 can be used in combination with the Chinese patent drugs to form the composition, such that the effects of the lactobacillus paracasei TY-W09 and the Chinese patent drugs can be simultaneously achieved.
Still another embodiment of the present application provides a preparation. The preparation includes the lactobacillus paracasei or the composition, and a carrier. The carrier is a medicinal carrier or an edible carrier.
It is to be noted that, drugs or edible food or health care products may be prepared by adding the medicinal carrier or the edible carrier to the composition including the lactobacillus paracasei TY-WO09 and different forms thereof. The medicinal carrier or the edible carrier 1s known in the art and may be selected according to the dosage form as needed. For example, the preparation of tablets mainly uses a diluent (such as starch, dextrin, sucrose or sugar), an absorbent (such as calcium sulfate, calcium hydrogen phosphate or light magnesium oxide), an adhesive (such as povidone, syrup or hydroxypropyl methylcellulose), a wetting agent (such as water), or a disintegrating agent (such as dry starch, sodium hydroxymethyl starch or cross-linked povidone). For example, the preparation of the liquid preparation mainly uses a bulking agent, a suspending agent, an emulsifying agent, a colorant, or the like.
In some specific embodiments, the preparation is tablets, pills, capsules, powder, gel, granules or a liquid preparation.
It is to be noted that, solid dosage forms such as the tablets, the pills, the granules or the capsules may be product forms such as probiotic tablets, probiotic sugar pills, probiotic powder or probiotic capsules. The liquid preparation may be a product form such as a probiotic beverage. The gel may be product forms such as probiotic jelly, probiotic milk foam or solidified yogurt.
Still another embodiment of the present application provides applications of the lactobacillus paracasei or the composition in preparation of preparations used for alleviating alcohol-induced visceral injury.
Still another embodiment of the present application provides applications of the lactobacillus paracasei or the composition in preparation of preparations used for preventing drunkenness and/or alleviating a hangover. It is to be noted that, the lactobacillus paracasei TY-WO09 in the present application may not only alleviate alcohol-induced visceral injury, but also have the effect of alleviating a hangover and sobering up. In some specific embodiments, compared with the drunkenness time of a mouse alcohol intragastric administration model that does not use the lactobacillus paracasei TY-WO09, the drunkenness time of a mouse alcohol intragastric administration model that uses the lactobacillus paracasei TY-WO09 in the present application is prolonged by approximately 200%, and the sober-up time is shortened by approximately 40%, such that the lactobacillus paracasei TY-WO09 in the present application has a significant effect of preventing drunkenness and alleviating a hangover.
In some specific embodiments, the applications include applications of one or a combination of a plurality of the following: (a) an application of the lactobacillus paracasei or the composition in preparation of preparations used for improving the antioxidant capability of liver tissue; and (b) an application of the lactobacillus paracasei or the composition in preparation of preparations used for improving the antioxidant capability of gastric tissue.
In some specific embodiments, the application (a) includes: an application of the lactobacillus paracasei or the composition in preparation of preparations used for reducing a liver coefficient; and/or an application in preparation of preparations used for reducing
ALT activity and/or AST activity and/or a TG level and/or a TC; and/or an application in preparation of preparations used for increasing GSH activity and/or SOD activity in liver tissue and/or reducing MDA; and/or an application in preparations for improving the mRNA and protein expression level of NRF2 and/or HO-1 and/or reducing the mRNA and protein expression level of CYP2E1; and the application (b) includes an application of the lactobacillus paracasei or the composition in preparation of preparations used for increasing GSH activity and/or SOD activity in the gastric tissue; and/or an application in preparations for reducing the content of an inflammatory factor TNF-a and/or IL-1B and/or
IL-6 in the gastric tissue.
It is to be noted that, the lactobacillus paracasei TY-WO9 in the present application may improve the antioxidant capability of the liver of mice with alcohol-induced liver injury, prevent liver injury caused by alcohol and recover a liver tissue structure by means of reducing the liver coefficient of the mice, reducing the activity of liver function indicators ALT and AST and the content of TG and TC in blood fat, increasing the content of GSH having an antioxidant function and the activity of SOD in liver tissue, reducing the content of MDA, significantly improving the mRNA and protein expression level of NRF2 or HO-1 and reducing the mRNA and protein expression level of CYP2E1.
It is further to be noted that, the TY-WO09 in the present application can also improve the antioxidant capability of the gastric tissue, prevent an inflammatory response of the gastric tissue in the mice caused by the alcohol and recover the morphology of the gastric mucosa by means of increasing the GSH content and SOD activity in the gastric tissue, and reducing the content of inflammatory factors TNF-a, 1L-1B and IL-6 in the gastric tissue.
In order to better understand the present application, the content of the present application is further described below with reference to specific embodiments, but is not only limited to the following examples.
Embodiment 1 Separation, purification and identification of TY-W09 (1) Experimental material
Yak yogurt from Tibet lhasa: homemade yak yogurt from a herdsman was taken by using a sterile spoon, and was put into a 15 mL sterile centrifuge tube containing a proper amount of sterile calcium carbonate and soluble starch; and the mixture is uniformly stirred, then transported, in a cold storage manner, back to a laboratory for immediate purification and separation of lactobacillus. (2) Separation and purification of TY-W09 1 mL of a sample was pipetted to 9 mL of sterile saline, so as to obtain 10° sample diluent; then 10-fold gradient dilution was successively performed on the diluent to 107, the diluent of 10%, 10% and 107 was coated on an MRS solid culture medium, and the culture medium was inverted and cultured for 48h at 37°C; and after culture was finished, colonial morphology was observed; raised and circular single colonies with medium sizes and neat edges were selected for strain purification by using a streak plate method, and the steps were repeated until purified strains were obtained. (3) Morphological structure observation
The colonial morphology of the purified strains was shown in Fig. 1. The colonies of the purified strains were consistent in morphology, smooth in surface, neat in edge, hemispherical in shape, and white in color.
Gram staining was performed on the purified strains; then purple cell morphology was observed under a 10x100-fold magnifier. Results were shown in Fig. 2 (grey processing was performed in Fig. 2, and an original drawing was purple), and the rod-like shaped strains were determined as gram-positive bacteria (G+). (4) PCR amplification of 16SrDNA sequence
PCR amplification was performed by using a 25 pL of a reaction system, including 1 pL of a template, 1 pL of an upstream primer (10 pM), 1 pL of a downstream primer (10 uM), and 12.5 pL of 2xTaq PCR Master Mix, and making up to 25 pL with sterile ultrapure water. Primer sequences included an upstream primer sequence
AGAGTTTGATCCTGGCTCAG (SEQIDNO.2) and a downstream primer sequence
TACGACTTAACCCCAATCGC (SEQIDNO.3). PCR amplification conditions included: performing pre-denaturation at 94°C for 5 min, performing denaturation at 94°C for 30 s, performing annealing at 55°C for 30 s, and performing extension at 72°C for 1 min, where there was a total of 35 cycles; and performing end extension at 72°C for 10 min. After sequence amplification, Sangon Biotech (Shanghai) Co., Ltd. was entrusted to sequence qualified PCR amplification products, and the obtained sequence was shown as
SEQIDNo.1; and searching and similarity comparison were performed in GeneBank by using BLAST(http://www.ncbi.nlm.nih. gov/BLAST). Results showed that the strain in the present application is lactobacillus paracasei.
Embodiment 2 Measurement of digestive tract resistance and cell adhesion capability of TY-W09 (1) Experimental material
Lactobacillus paracasei TY-WO09: separated from yak yogurt from Tibet lhasa, deposited with the CGMCC, and is assigned with the accession number of CGMCC No. 24630. (2) Measurement of TY-WO09 tolerant to artificial gastric juice with pH being 3.0
The strains were inoculated in the MRS liquid culture medium by 2% of the inoculation amount and cultured at 37°C for 18h; centrifugation was performed at 4000 r/min for 10 min to collect a bacterial precipitate, and the bacterial precipitate was resuspended in the isochoric sterile saline, so as to obtain bacterial suspension. 1 ml of the bacterial suspension and 9 ml of the artificial gastric juice (0.2% NaCl:0.35% pepsin being 1:0000, 1 mol/L of HCI being used to adjust pH to be 3, the mixture being filtered and sterilized for later use) were mixed, then placed in a thermostatic oscillator after being well mixed, and cultured at 37°C and 100 r/min for 3h; and the viable counts at Oh and 3h were respectively measured by using a pour plate method.
Survival rate/% 3h viable Count(CrU/mL) 100 © Ohviablecount{CFU/mL)«
Before the probiotics entered the human intestinal tract to exert functions, the probiotics needed to pass through the stomach (the pH value of the gastric juice was about 3.0, and the retention time was 1h - 3h), but the strongly acidic stomach environment was not conducive to the survival of the probiotics. Therefore, the probiotics had certain survival capabilities in the strongly acidic environment. An experimental result showed that, the survival rate of the TY-W09 cultured in the simulated gastric juice with pH=3.0 for 3h was 111.76%, such that the TY-WO09 has very strong tolerance to the gastric juice. (3) Measurement of TY-W09 tolerant to 0.3% bile salt
The strains were inoculated in the MRS liquid culture medium by 2% of the inoculation amount and cultured at 37°C for 18h, then inoculated in MRS-THIO culture media respectively containing 0.0 and 0.3% bile salt (0.2% sodium mercapto-acetate being added to the MRS liquid culture medium) by 2% of the inoculation amount, and after well mixing, placed in the constant temperature oscillator to culture for 24h at 37°C and 100r/min; and growth efficiency was calculated. 0.3% bile salt containing culture medium ODsoosn - blank control ODeso: growth EE er —————— zt {JE efficiency: 0.0% bile salt containing culture medium ODesoo- - blank control ODso0a
After entering the intestinal tract, the probiotics were suppressed by the bile salt in the small intestine; and the concentration of the bile salt in the human intestinal tract environment was constantly changing, and a fluctuation range was 0.03%-0.3%. From the above experimental results, it showed that the growth efficiency of the TY-WO09 in the bile salt with the concentration being 0.3% was 25.63%, such that the TY-WO09 has desirable bile salt tolerance.
(4) Measurement of cell adhesion capability of TY-W09
HT-29 cells were normally cultured and passaged; well-cultured cells were collected, and resuspended by using a McCoy's 5A culture medium (adding penicillin-streptomycin and fetal bovine serum); the number of cells was adjusted to 2x105 cell/mL by means of a hemocytometer counting method; a 6-well culture plate was placed into cover glass which has been subjected to acid pickling and sterilization, and 2 mL of cell suspension was respectively added to each well; overnight, washing was performed for 3 times by using sterile PBS; the bacterial suspension (the concentration of bacterial fluid being 108CFU/mL) was added; incubation was performed for 2h, and then washing was performed for 5 times by using the sterile PBS; 2 mL of methanol was added in each well to cure for lh at room temperature; and the methanol was discarded, and then a gram staining solution was added. The total number, that is, an adhesion value, of TY-W09 adhered by 100 cells was recorded under the microscope (20 views being randomly selected).
After the human body ingested the probiotics, under the digestion of gastric acid and bile, the growth and reproduction of the probiotics were affected to a certain extent, and it was impossible to show a better growth trend in a short period of time. If the probiotics had desirable adhesion capabilities, the retention time of the probiotics in the human intestinal tract could be prolonged, facilitating the growth and reproduction of the strains. If the number of strains adhered to 100 cells was less than 40, it indicated that the strains had no adhesion capability; if the number of strains adhered to 100 cells was between 41 and 100, it indicated that the strains were general in adhesion capability; and if the number of strains adhered to 100 cells exceeded 100, it indicated that the strains were strong in adhesion capability. The experimental result of the cell adhesion capability was shown in Fig. 3, the number of TY-W09 adhered by 100 cells was 1852, such that the TY-W09 had strong cell adhesion capability.
Embodiment 3 Effect of TY-W09 on alleviating a hangover, protecting the liver and nourishing the stomach (1) Experimental material
Lactobacillus paracasei TY-WO09: separated from yak yogurt from Tibet lhasa, deposited with the CGMCC, and 1s assigned with the accession number of CGMCC No. 24630.
Mouse model: mice were randomly grouped into 3 groups (n=10) according to weights, that is, Group Blank, Group Alcohol and Group TY-W09. The mice in Group
TY-W09 were intra-gastrically administered with the 1.0x10° CFU/kg TY-WO09 bacterial fluid daily for 28 consecutive days, and the mice in Group Blank and the Group Alcohol were intra-gastrically administered with equivalent amount of normal saline; on Day 29, the mice in each group were fasted for 12 hours before model building; then the mice in
Group Alcohol and Group TY-W09 were intra-gastrically administered with an alcohol solution (12 ml/kg) with volume fraction of 50% at one time; the mice in the Group Blank were given equal amount of normal saline; after intragastric administration, the mice were all fasted but could drink water, and were sacrificed after 10 hours.
Mouse sample collection and treatment: after the mice were sacrificed, eyeball blood was extracted; standing was performed for 1h at 4°C; centrifugation was performed for 15 min at 3000r/min, and upper serum was collected; the livers and gastric tissue of the mice were simultaneously collected; the livers of the dissected mice were weighed after being rinsed in the cold normal saline and dried, and a complete section of left lobe was taken for use; the gastric tissue was taken and cut along greater curvature of the stomach; stomach contents were washed; and the gastric mucosa was unfolded, and was photographed by a digital camera. (2) Recording of drunkenness time and sober-up time in the mice
After intragastric administration of alcohol, righting reflex disappearance time and recovery time of the mice in each group were recorded, and the drunkenness time and sober-up time of the mice were calculated; the drunkenness time: the time for righting reflex from disappearance to recovery; and righting reflex: the mice were turned sideways with their backs downward at a certain time after being intra-gastrically administrated with the alcohol, and it was considered that righting reflex disappears if the mice were held in a back-down position for more than 30s.
Drunkenness is caused by the high concentration of ethanol in the blood entering the brain in a short period of time, causing the brain to gradually shift from an excited state to an inhibited state, and the excitation of the subcortical center of the brain to lose control, thereby causing a series of pathological changes such as cognition, judgment and motor impairment. The most intuitive behavioral expression after the mice were drunk was the disappearance and recovery of the righting reflex; and by means of observing the disappearance time and recovery time of the righting reflex after drunkenness, the effect of the TY-WO09 on alleviating a hangover was determined, such that whether the TY-W09 has the effect of alleviating a hangover was preliminarily determined. Results were shown in
Fig. 4 and Fig. 5. Compared with Group Alcohol, in Group TY-W09, the drunkenness time of the mice might be significantly prolonged (by approximately 200%) (p<0.0001), and the sober-up time of the mice was shortened (by approximately 30%) (p<0.01). The result showed that the TY-W09 had the effect of preventing drunkenness and alleviating a hangover. (3) Measurement of liver coefficients of the mice
The liver was rapidly dissected after the blood was drawn, and was washed with cold normal saline. The wet weight of the liver was weighted after moisture is absorbed with a filter paper, to calculate the liver coefficient (the liver coefficient reflected the health condition of the liver to a certain extent), where liver coefficient = (liver mass/total body mass) x 100%.
A calculation result was shown in Fig. 6. Compared with Group Alcohol, the liver coefficient of Group TY-W09 was significantly reduced (by approximately 9.5%) (p<0.001). The result showed that the TY-W09 could effectively suppress the increasing of the liver coefficient of the mice caused by alcohol. (4) Histopathological observation on the liver of the mice
Complete tissue from the left lobe of the liver of the mice was taken, HE staining was performed after curing was performed by using a paraformaldehyde solution with the mass fraction being 10%, and then histopathological changes are observed under a microscope (400x).
Observation results were shown in Fig. 7. The hepatocellular structure of Group
Blank was normal, a nuclear membrane was intact, and no lipid droplets or inflammatory cell infiltration were seen in the hepatocyte cytoplasm; and after alcohol intervention, the hepatocytes of the mice were swollen and showed obvious steatosis, and cytoplasmic vacuolation, inflammatory cell infiltration and necrosis were seen. However, the steatosis of the liver of the mice in Group TY-W09 was obviously improved compared with that in
Group Alcohol, the morphology of the hepatocytes was basically recovered, and the tissue structure was normalized. The result showed that the TY-W09 could effectively prevent the alcohol-induced liver injury of the mice. (5) Measurement of serum biochemical indexes in the mice
The ALT, AST, TG and TC in the serum were all common biomarkers of liver injury; and the ALT activity, AST activity, TG content and TC content in the serum of the mice were measured by using a kit. Detection results were shown in Fig. 8, Fig. 9, Fig. 10 and
Fig. 11. Compared with Group Blank, the levels of the ALT activity, AST activity, TG content and TC content in the serum of Group Alcohol were significantly improved.
However, the pre-processing of the TY-WO09 significantly prevents the increasing of the levels of the ALT activity (p<0.0001}, AST activity (p<0.0001), TG content (p<0.001) and
TC content (P<0.01). The result showed that the TY-WO09 could effectively prevent the alcohol-induced liver injury and elevated blood lipid levels in the mice. (6) Measurement of antioxidant active substances of the liver in the mice (I) SOD activity, GSH content and MDA content of antioxidant substances
A BCA kit (purchased from Shanghai Beyotime Biotechnology Co., Ltd) was first used to test a protein concentration of liver homogenate; then the kit was used to test the activity of the liver antioxidant substance SOD, GSH content and MDA content in the homogenate. Test results were shown in Fig. 12, Fig. 13 and Fig. 14. Compared with
Group Alcohol, the SOD activity (p<0.0001) and GSH content of the antioxidant substances in the liver tissue homogenate of the mice in Group TY-W09 were both increased (p<0.0001), and the content of the lipid peroxidation product MDA caused by oxidative stress is reduced (p<0.001). (2) Measurement of mRNA expression levels of NRF2, HO-1 and CYP2E1
It is to be noted that, as a transcription factor, the NRF2 might alleviate oxidative stress by means of improving the transcription of a series of cell protective genes. When the alcohol was metabolized by the human body, a signal path mediated by the NRF2 is a key signal path for antioxidation. The NRF2 played a key role in cellular antioxidant defense by regulating several major antioxidant response element-dependent antioxidant mechanisms, including the NQO1 and the HO-1. The loss of the NRF2 leaded to a significant reduction in the detoxification capability of acetaldehyde and hepatic steatosis; the mRNA and protein expression of the HO-1 might be upregulated following oxidative stress and cellular injury; and activation of the CYP2E1 was associated with increased lipid peroxidation, mitochondrial dysfunction and hepatotoxicity.
Therefore, in the embodiments of the present application, a TRIzol reagent was also used to extract the total RNA of the liver tissue, and the total RNA was subjected to reverse transcription to cDNA by using a reverse transcription kit; and the mRNA expression levels of target genes NRF2, HO-1 and CYP2E1 were detected by means of a fluorescence quantitative PCR instrument. A primer sequence was shown in Table 1, and the mRNA relative expression levels of the target genes were calculated by using a 224“ method.
Table 1 Primer sequence
The detection calculation results of the mRNA relative expression levels of the NRF2, the HO-1 and the CYP2EI in the livers of the mice were shown in Fig. 15, Fig. 16 and Fig. 17. Compared with Group Blank, the mRNA relative expression levels of the NRF2 and the HO-1 in Group Alcohol were significantly reduced, and the mRNA relative expression level of the CYP2EI was significantly increased. After TY-WO09 intervention, the mRNA expression of the NRF2 (p<0.01), the HO-1 (p<0.05) and the CYP2El (p<0.05) was remained at a normal level. (3) Measurement of protein expression levels of NRF2, HO-1 and CYP2E1
A Western-blot method was used to measure the protein expression level of the liver tissue of the mice; proper amount of mouse liver was taken, an RIPA lysis solution was added, and the mixture was fully ground and cracked on ice; and the lysis solution was centrifuged for 10 min at 4°C and 12000g, and a small amount of clear supernatant was taken. According to the measured protein concentration, all samples were unified to the same concentration, an SDS-PAGE electrophoretic sample-loading buffer solution was added, then the mixture was boiled for 5 min at 98°C, so as to denature the proteins; an electrophoresis experiment was performed on the well-prepared protein samples, and electrophoresis conditions were 70V for 30 min and 110V for 1.5h; and after electrophoresis was completed, membrane transferring was performed in a membrane-transferring tank at 300mA for 2h. A PVDF membrane was taken and sealed in 5% defatted milk powder for 2h at room temperature, and washing was performed with
TBST. B-actin, NRF2, HO-1 and CYP2E1 primary antibodies were incubated overnight at 4°C. Removing was performed at next day, and membrane washing was performed, and then secondary antibodies were incubated on a horizontal shaker for lh at room temperature; membrane washing was performed, and color development was performed with ECL; and a Bio-Rad imaging system was used for color development imaging, and results were shown in Fig. 18.
Image] software was used to calculate the gray value of each band, and the protein relative expression of the NRF2, the HO-1 and the CYP2E1 was calculated. Results were shown in Fig. 19, Fig. 20 and Fig. 21. Compared with Group Blank, the protein relative expression levels of the NRF2 and the HO-1 in Group Alcohol was significantly reduced, and the protein relative expression level of the CYP2E1 is significantly increased. After
TY-WO09 intervention, the protein relative expression of the NRF2 (p<0.01), the HO-1 (p<0.05) and the CYP2E1 (p<0.05) was remained at a normal level.
The above results showed that, the TY-WO09 could improve the antioxidant capability of the liver of the mice with alcohol-induced liver injury, and improve alcohol-induced liver oxidative damage of the mice, such that alcohol-induced liver injury is protected. (7) Measurement of gastric mucosa injury indexes of the mice
Gastric mucosa injury of the mice was observed with naked eyes, and the index of gastric mucosa injury was measured by using a straight scale (the index of gastric mucosa injury was calculated by using a Guth method: every three punctate ulcers (of which width of hemorrhagic erosive dots or mucosa defect is less than 1 mm) were recorded as 1 point; stripe hemorrhage: the maximum length diameter of the ulcer and the maximum width diameter perpendicular to the maximum length diameter were measured, and the product of the maximum length diameter and the maximum width diameter was an ulcer index; and the one with the width being 1 mm was 1 point per millimeter length, the one with the width being 2 mm was 2 points per millimeter length, the one with the width being 3 mm was 3 points per millimeter length, and so on).
The gastric mucosa injury of the mice in each group was shown in Fig. 22. The gastric mucosa layer of the mice induced with the alcohol developed hemorrhagic erosion.
Compared with Group Blank, the gastric mucosa of the mice in Group Alcohol showed obvious bleeding and erosion; and compared with Group Alcohol, the bleeding and erosion of the gastric mucosa of the mice in Group TY-W09 were obviously improved.
The indexes of gastric mucosa injury of the mice in each group were shown in Fig. 23.
Compared with Group TY-W09 and Group Alcohol, the indexes (p<0.01) of gastric mucosa injury of the mice were significantly reduced.
The above results showed that, the TY-W09 could prevent the alcohol-induced gastric mucosa injury of the mice. (8) Measurement of an oxidation index of gastric tissue in the mice
It is to be noted that, the gastric mucosa generated large amounts of oxygen radicals under the action of ethanol, causing lipid peroxidation of mucosal cells, resulting in gastric mucosa blood flow disorder, thereby triggering gastric mucosa injury. The antioxidant substances such as GSH and SOD in the gastric tissue could maintain the balance of a human body antioxidant defense system.
In the embodiments of the present application, the BCA kit (purchased from Shanghai
Beyotime Biotechnology Co., Ltd.) was first used to test the protein concentration of gastric homogenate; then the kit was used to test the activity of the liver antioxidant substance SOD and GSH content in the gastric homogenate. Test results were shown in Fig. 24 and Fig. 25. Compared with Group Alcohol, the SOD activity (p<0.01) and GSH content (p<0.01) of Group TY-WO09 were both significantly increased. The above results showed that, the TY-W09 could improve the antioxidant capability of the gastric tissue. (9) Measurement of an inflammatory factor level of gastric tissue of the mice
It is to be noted that, IL-6 and IL-1p play an important role in the inflammatory response caused by injury and were associated with various physio pathological processes such as inflammatory response and immune regulation in the human body. The levels of the IL-6 and the IL-1B might the degree of the inflammatory response, and were related to an anti-inflammatory treatment effect. The TNF-a was a type of pro-inflammatory cytokine, which could be produced by the body after various stimuli in internal and external environments, and might improve the effects of other inflammatory cytokines during the inflammatory response.
In the embodiments of the present application, enzyme-linked immunosorbent assay was used; and the expression levels of the inflammatory factors IL-6, IL-1 and TNF-a in the gastric tissue of the mice were detected according to related operating instructions of corresponding ELISA kits (which are all purchased from Shanghai Enzyme-linked
Biotechnology Co., Ltd.). Detection results were shown in Fig. 26, Fig. 27 and Fig. 28.
Compared with Group Blank, the levels of the IL-6 (p<0.001), IL-1B (p<0.0001) and
TNF-a (p<0.01) in the gastric tissue of the mice in Group Alcohol were significantly increased. However, after TY-W09 intervention, in the gastric tissue of the mice, the IL-6 (p<0.0001) and the IL-1B (p<0.001) with high levels and the level of the TNF-a (p<0.01) were all significantly reduced, and recovered to the normal level. The above results showed that, the TY-WO09 could effectively prevent the alcohol-induced inflammatory response of the gastric tissue of the mice.
To sum up, the lactobacillus paracasei TY-WO09 in the present application has desirable digestive tract resistance and good intestinal tract colonization capabilities, and may significantly prolong the drunkenness time of the mice and shorten the sober-up time, such that the lactobacillus paracasei has the effect of preventing drunkenness and alleviating a hangover. The lactobacillus paracaset TY-W09 may also improve the antioxidant capability of the liver, improve alcohol-induced liver oxidative damage of the mice and recover the liver tissue structure by means of simultaneously reducing the liver coefficient, reducing the activities of the liver function indicators ALT and AST in the serum and the content of the TG and TC in blood fat, increasing the content of GSH having the antioxidant function and the activity of SOD in the liver tissue, reducing the content of the lipid peroxide MDA, significantly improving the mRNA and protein expression level of the NRF2 or the HO-1 and significantly reducing the mRNA and protein expression level of the CYP2E1. In addition, the lactobacillus paracasei TY-WO09 may also improve the antioxidant capability of the gastric tissue, prevent the alcohol-induced inflammatory response of the gastric tissue in the mice and recover the morphology of the gastric mucosa by means of simultaneously increasing the GSH content and SOD activity in the gastric tissue, and reducing the content of the inflammatory factors (TNF-a, IL-16 and IL-6) in the gastric tissue.
It is finally to be noted that, the above embodiments are merely for describing and not intended to limit the technical solutions of the present application. Although the present application is described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present application can be modified or equivalently replaced without departing from the purpose and scope of the technical solutions of the present application, and shall all fall within the scope defined by the claims of the present application.
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| CN108203702A (en) * | 2016-12-20 | 2018-06-26 | 北京中科星冠生物技术股份有限公司 | A kind of compound probiotic alcohol-decomposing beverage and its specific complex probiotics |
| CN111662850B (en) * | 2020-07-10 | 2022-03-15 | 江南大学 | Lactobacillus paracasei capable of relieving alcoholic intestinal injury and application thereof |
| KR20220011937A (en) * | 2020-07-22 | 2022-02-03 | (주)지에프씨생명과학 | Lactobacillus paracasei GFC_GFV5 for removing hangover and food composition comprising the same as an effective ingredient |
| CN111944725B (en) * | 2020-08-24 | 2021-12-07 | 汤臣倍健股份有限公司 | Lactobacillus paracasei 207-27 and application thereof |
| CN113337430B (en) * | 2021-06-04 | 2022-03-11 | 青岛诺森生物技术有限责任公司 | Lactobacillus paracasei NSL0201 and application thereof |
-
2023
- 2023-01-05 CN CN202310011354.5A patent/CN118291298A/en active Pending
- 2023-06-28 NL NL2035211A patent/NL2035211B1/en active
Also Published As
| Publication number | Publication date |
|---|---|
| NL2035211A (en) | 2023-07-27 |
| CN118291298A (en) | 2024-07-05 |
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