CN111269853B - Composite microbial inoculum with liver protection effect - Google Patents
Composite microbial inoculum with liver protection effect Download PDFInfo
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- CN111269853B CN111269853B CN202010096908.2A CN202010096908A CN111269853B CN 111269853 B CN111269853 B CN 111269853B CN 202010096908 A CN202010096908 A CN 202010096908A CN 111269853 B CN111269853 B CN 111269853B
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- bacterial liquid
- lactobacillus paracasei
- grx11
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
- A23G3/00—Sweetmeats; Confectionery; Marzipan; Coated or filled products
- A23G3/34—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof
- A23G3/36—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof characterised by the composition containing organic or inorganic compounds
- A23G3/364—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof characterised by the composition containing organic or inorganic compounds containing microorganisms or enzymes; containing paramedical or dietetical agents, e.g. vitamins
- A23G3/366—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof characterised by the composition containing organic or inorganic compounds containing microorganisms or enzymes; containing paramedical or dietetical agents, e.g. vitamins containing microorganisms, enzymes
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
- A23G3/00—Sweetmeats; Confectionery; Marzipan; Coated or filled products
- A23G3/34—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof
- A23G3/36—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof characterised by the composition containing organic or inorganic compounds
- A23G3/38—Sucrose-free products
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
- A23G3/00—Sweetmeats; Confectionery; Marzipan; Coated or filled products
- A23G3/34—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof
- A23G3/36—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof characterised by the composition containing organic or inorganic compounds
- A23G3/42—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof characterised by the composition containing organic or inorganic compounds characterised by the carbohydrates used, e.g. polysaccharides
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/165—Paracasei
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/21—Streptococcus, lactococcus
- A23V2400/249—Thermophilus
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Abstract
The invention discloses a composite microbial inoculum with liver protection effect, which comprises: streptococcus thermophilus grx11 and lactobacillus paracasei H9. The complex microbial inoculum can also further comprise trehalose. The streptococcus thermophilus grx11 and the lactobacillus paracasei H9 are food bacteria with high safety and the safety is guaranteed. Has obvious effect on alcoholic liver injury. The trehalose has the effects of neutralizing the effect of alcohol by cooperating with the mixed bacteria and protecting the liver function, and has good health-care activity.
Description
Technical Field
The invention relates to a composite microbial inoculum with a liver protection effect, belonging to the technical field of microorganisms.
Background
Alcoholic liver injury, which is the occurrence of liver injury due to long-term intake of large amounts of alcohol and beverages containing alcohol, is classified into species by the chinese medical society, namely, mild alcoholic liver injury, alcoholic fatty liver, alcoholic hepatitis, alcoholic liver fibrosis and alcoholic cirrhosis.
The consumption of alcohol is continuously increased due to rapid development of society, increasingly improved living standard, gradually increased working pressure and the like, the phenomenon is in a global development trend, and the problems of alcohol abuse, alcoholism, alcohol dependence and the like are more obvious in the current society. The number of people drinking wine in China is rapidly increased, and the harm of alcohol to human bodies is gradually highlighted.
Alcoholic liver disease is chemical liver injury caused by long-term drinking, and is often manifested as fatty liver at the initial stage, and further developed into liver diseases such as alcoholic hepatitis, alcoholic hepatic fibrosis and alcoholic cirrhosis. At present, the pathogenesis of alcoholic liver injury is not clear, and researches suggest that alcohol mainly disturbs nutrient metabolism in the metabolic process of the liver, generates injury and the like through oxidative stress, inflammation and immune reaction, and aggravates the progress of pathological injury of the liver by a plurality of factors such as local hypoxia, malnutrition and the superposition of viruses. The degree of liver damage caused by alcohol intake is related to the variation of polymorphism of alcohol dehydrogenase and acetaldehyde dehydrogenase in liver. Long-term heavy drinking is the basis for the onset of alcoholic liver disease. Metabolites such as ethanol and acetaldehyde directly or indirectly damage liver cells, so that the liver metabolism is unbalanced and the liver function is abnormal, and the liver cells generate steatosis, inflammatory reaction, fibrosis and the like. The pathological changes and pathogenesis of alcoholic liver injury at different stages are different, and the occurrence of alcoholic fatty liver is mainly related to lipid metabolism, apolipoprotein deficiency, fatty acid oxidation resistance and the like. The occurrence of alcoholic hepatitis is associated with monocytic cytotoxic effects, increased oxygen free radicals, deficient skin of valley in the liver, cytokine involvement and accumulation of inflammatory mediators. The occurrence of alcoholic liver fibrosis is related to repeated inflammation caused by acetaldehyde, increased collagen synthesis and reduced degradation.
Probiotics are a class of microorganisms that have essentially no toxic side effects on humans and lactating animals, and which produce beneficial effects on the body when administered to the body in an amount sufficient . The regulation of intestinal flora, the protection of intestinal barriers and the influence of probiotics on intestinal immune function have been widely reported. In the prior art, certain strains of lactobacillus rhamnosus are found, and lactobacillus plantarum can play a role in reducing inflammatory response of the liver and reducing liver lipid deposition by regulating bile acid metabolism and non-alcoholic fatty liver, but the research on alcoholic liver loss by probiotics is still less.
The invention aims to provide a composite microbial inoculum with a liver protection effect, which can effectively relieve alcoholism in vivo and remarkably alleviate liver injury, thereby achieving a good liver protection effect.
Disclosure of Invention
The invention aims to provide a composite microbial inoculum with a liver protection effect, which can effectively relieve alcoholism in vivo and remarkably alleviate liver injury, thereby achieving a good liver protection effect.
Chinese patent 201310308577 (Yangzhou university of the applicant) discloses Streptococcus thermophilus grx11 prepared from fermented milk and application thereof, wherein the preservation number of the Streptococcus thermophilus grx11 is CGMCC No: 7697. the strain has excellent curd effect and can be used for preparing yogurt with excellent flavor.
Chinese patent 2015106977109 (applicant's Chinese university of agriculture) discloses a Lactobacillus paracasei (Lactobacillus paracasei) H9. The lactobacillus paracasei strain is H9, is classified and named as Lactobacillus paracasei, and has a preservation number of CGMCC No.4780 in the China general microbiological culture Collection center. The Lactobacillus paracasei (Lactobacillus paracasei) H9 shows good ability of producing mucopolysaccharide, and the mucopolysaccharide is considered to have an anti-cancer function and increase the viscosity of the yoghurt; can effectively inhibit escherichia coli, salmonella and staphylococcus aureus; has good thermal stability, cholesterol lowering activity, blood pressure lowering activity and antioxidant activity.
The research team of the applicant finds that the streptococcus thermophilus grx11 or the lactobacillus paracasei H9 have no obvious effect of reducing the alcoholic liver injury when used alone, but have more excellent effect of reducing the alcoholic liver injury when used together. However, it was found in the study that it appeared that significant effects were only demonstrated if the drench was started at least one week before the alcoholic liver injury was modeled, and no significant effects were observed after modeling had taken (alcoholic liver injury occurred).
Trehalose is also called rhaponticum sugar, mushroom sugar, etc. Is a safe and reliable natural saccharide. Trehalose is widely present in edible animals, plants and microorganisms in nature, and for example, mushrooms, seaweed, beans, shrimps, bread, beer and yeast fermented food which are eaten by people in daily life have high content of trehalose. It can effectively increase the activity of the immune system in clinic.
The research and development team further studies, if trehalose is compounded in the complex microbial inoculum, the use time is not limited to the use before the alcoholic liver injury for a period of time, and the use after the liver injury also has a good effect of promoting the liver injury repair. The trehalose also has good health-care effect, so that the whole formula not only has the liver-protecting effect, but also has the effects of enhancing the immunity, regulating the immunity and the like.
The technical problem to be solved by the invention can be realized by the following technical scheme.
A complex microbial inoculum with liver protection effect, which comprises:
streptococcus thermophilus grx11 and lactobacillus paracasei H9.
The preservation number of the Streptococcus thermophilus (Streptococcus thermophilus) grx11 is CGMCC No: 7697.
the preservation number of the lactobacillus paracasei H is CGMCC No. 4780.
More preferably, the complex microbial inoculum further comprises trehalose.
The compound microbial inoculum can be made into tablet candy, powder, capsule and the like.
The compound microbial inoculum can also be used for preparing health-care food from fermented food.
The food product is preferably a fruit and dairy product.
The invention has the advantages that:
(1) the streptococcus thermophilus grx11 and the lactobacillus paracasei H9 are food bacteria with high safety and the safety is guaranteed.
(2) Has obvious effect on alcoholic liver injury.
(3) The trehalose has the effects of neutralizing the effect of alcohol by cooperating with the mixed bacteria and protecting the liver function, and has good health-care activity.
Detailed Description
The following examples of the present invention are described in detail, and are only for the purpose of illustrating the present invention and are not to be construed as limiting the present invention.
Specific examples of the present invention are described below. The streptococcus thermophilus grx11 and lactobacillus paracasei H9 used in the present invention were both obtained as gifts.
EXAMPLE 1 in vivo anti-alcoholic liver injury assay (Precaution group of Streptococcus thermophilus)
1. The activation culture of the bacterial liquid of streptococcus thermophilus grx11 and the bacterial liquid of lactobacillus paracasei H9 is carried out according to a conventional method. Adjusting the concentration of the bacterial liquid to 10 in the culture medium7cfu/ml. And preparing a group of mixing groups: activating the bacterial liquid of streptococcus thermophilus grx11 and lactobacillus paracasei H9, and mixing according to the concentration of the bacterial number of 1:1Adjusting the total concentration of the bacterial liquid to 107cfu/ml。
2. Healthy ICR male mice with the weight of 20-25g are selected, and after 1 week of breeding, formal experiments are started and randomly divided into 5 groups of 10 mice, and the weights of the mice in each group are not statistically different. Mice were divided into 5 groups. Each group started the gavage of S.thermophilus grx11 bacterial suspension (0.2ml/25g dose) daily at week 1 and each group started the gavage at week 2 as shown below.
Purified water or bacterial liquid (0.2ml/25g dosage) is used for the first intragastric administration of each group; half an hour later, the mice were given a second intragastric administration with purified water in group 1 and 40% ethanol in water (0.2ml/25g dose) in groups 2-5.
Group 1: purified water + purified water control group (negative control group);
group 2: purified water + 40% ethanol concentration treated group (positive control group);
group 3: a streptococcus thermophilus grx11 bacterial liquid and 40% ethanol concentration treatment group;
group 4: a lactobacillus paracasei H9 bacterial liquid and 40% ethanol concentration treatment group;
group 5: mixed bacteria liquid and 40% ethanol concentration treatment group.
Blood is collected from inner canthus of capillary eye twice in the second intragastric lavage (ethanol lavage) for 30min and 2h, and the blood is placed at room temperature for 120min, centrifuged at 2500rpm for 15min to obtain serum, and the alcohol concentration is measured by gas chromatography. The specific results are shown in Table 1-a.
TABLE 1-a serum alcohol concentration (mg/dl) after gastric lavage with ethanol
30min | 120min | |
Streptococcus thermophilus grx11 bacterial liquid | 314.35±25.241 | 133.65±13.531 |
Lactobacillus paracasei H9 bacterial liquid | 324.51±43.531 | 127.53±11.211 |
Mixed bacterial liquid | 284.35±41.631 | 117.84±9.721 |
Negative control | 0 | 0 |
Positive control | 315.31±59.96 | 139.81±11.47 |
Note: t test, l: p <0.05 (compared to negative control); 2: p <0.05 (compare with positive control group)
As a result, when only the S.thermophilus grx11 bacterial liquid was taken in advance at week 1, there was no statistical difference in serum alcohol concentration between the groups compared with the positive control group.
Detection of liver function:
ALT kit and AST kit (constructed organism) are adopted to measure ALT and AST activity in serum.
The results are shown in Table 1-b.
TABLE 1-b liver function assay in groups of mice after gastric lavage with ethanol
ALT | ALT | |
Streptococcus thermophilus grx11 bacterial liquid | 43.52±8.111 | 109.52±8.431 |
Lactobacillus paracasei H9 bacterial liquid | 42.73±6.321 | 106.25±11.371 |
Mixed bacterial liquid | 40.69±3.541 | 103.68±7.541 |
Negative control | 33.08±7.35 | 53.18±7.99 |
Positive control | 45.12±9.51 | 110.19±11.73 |
Note: t test, l: p <0.05 (compared to negative control); 2: p <0.05 (compare with positive control group)
As a result, when only the streptococcus thermophilus grx11 bacterial liquid is pre-taken at week 1, the groups have no statistical difference relative to the positive control group, and ALT and AST cannot be obviously reduced, which indicates that the liver function is not obviously protected.
EXAMPLE 2 in vivo anti-alcoholic liver injury assay (Pre-dose Lactobacillus paracasei test group)
1. The method of preparing the bacterial suspension was the same as in example 1.
2. Healthy ICR male mice with the weight of 20-25g are selected, and after 1 week of breeding, formal experiments are started and randomly divided into 5 groups of 10 mice, and the weights of the mice in each group are not statistically different. Mice were divided into 5 groups. Each group was started by daily gavage of Lactobacillus paracasei H9 strain (dose of 0.2ml/25 g) at week 1 and by gavage at week 2, as shown below.
Purified water or bacterial liquid (0.2ml/25g dosage) is used for the first intragastric administration of each group; half an hour later, the mice were given a second intragastric administration with purified water in group 1 and 40% ethanol in water (0.2ml/25g dose) in groups 2-5.
Group 1: purified water + purified water control group (negative control group);
group 2: purified water + 40% ethanol concentration treated group (positive control group);
group 3: a streptococcus thermophilus grx11 bacterial liquid and 40% ethanol concentration treatment group;
group 4: a lactobacillus paracasei H9 bacterial liquid and 40% ethanol concentration treatment group;
group 5: mixed bacteria liquid and 40% ethanol concentration treatment group.
Blood is collected from inner canthus of capillary eye twice in the second intragastric lavage (ethanol lavage) for 30min and 2h, and the blood is placed at room temperature for 120min, centrifuged at 2500rpm for 15min to obtain serum, and the alcohol concentration is measured by gas chromatography. The specific results are shown in Table 2-a.
TABLE 2-a serum alcohol concentration (mg/dl) after gastric lavage with ethanol
30min | 120min | |
Streptococcus thermophilus grx11 bacterial liquid | 310.53±36.541 | 130.08±8.461 |
Lactobacillus paracasei H9 bacterial liquid | 314.32±75.431 | 131.21±11.271 |
Mixed bacterial liquid | 310.87±60.951 | 124.50±11.261 |
Negative control | 0 | 0 |
Positive control | 329.14±54.65 | 134.84±22.33 |
Note: t test, l: p <0.05 (compared to negative control); 2: p <0.05 (compare with positive control group)
As a result, when only Lactobacillus paracasei was pre-administered at week 1, there was no statistical difference in serum alcohol concentration between the groups relative to the positive control group.
Detection of liver function:
ALT kit and AST kit (constructed organism) are adopted to measure ALT and AST activity in serum.
The results are shown in Table 2-b.
TABLE 2-b liver function assay in groups of mice after gastric gavage with ethanol
ALT | ALT | |
Streptococcus thermophilus grx11 bacterial liquid | 45.53±6.721 | 103.41±8.751 |
Lactobacillus paracasei H9 bacterial liquid | 44.75±5.321 | 102.30±7.051 |
Mixed bacterial liquid | 45.66±7.391 | 97.99±6.021 |
Negative control | 29.32±6.64 | 50.19±9.53 |
Positive control | 46.55±8.45 | 108.66±11.73 |
Note: t test, l: p <0.05 (compared to negative control); 2: p <0.05 (compare with positive control group)
As a result, when only lactobacillus paracasei bacterial liquid is taken in advance at week 1, the groups have no statistical difference relative to the positive control group, ALT and AST can not be obviously reduced, and no obvious protective effect on liver functions is shown.
Example 3 in vivo measurement of anti-alcoholic liver injury (Pre-oral Mixed bacterial liquid test group)
1. The method of preparing the bacterial suspension was the same as in example 1.
2. Healthy ICR male mice with the weight of 20-25g are selected, and after 1 week of breeding, formal experiments are started and randomly divided into 5 groups of 10 mice, and the weights of the mice in each group are not statistically different. Mice were divided into 5 groups. The mixed bacteria solution (0.2ml/25g dose) was administered daily to each group at week 1, and the gavage was initiated at week 2, as shown below.
Purified water or bacterial liquid (0.2ml/25g dosage) is used for the first intragastric administration of each group; half an hour later, the mice were given a second intragastric administration with purified water in group 1 and 40% ethanol in water (0.2ml/25g dose) in groups 2-5.
Group 1: purified water + purified water control group (negative control group);
group 2: purified water + 40% ethanol concentration treated group (positive control group);
group 3: a streptococcus thermophilus grx11 bacterial liquid and 40% ethanol concentration treatment group;
group 4: a lactobacillus paracasei H9 bacterial liquid and 40% ethanol concentration treatment group;
group 5: mixed bacteria liquid and 40% ethanol concentration treatment group.
Blood is collected from inner canthus of capillary eye twice in the second intragastric lavage (ethanol lavage) for 30min and 2h, and the blood is placed at room temperature for 120min, centrifuged at 2500rpm for 15min to obtain serum, and the alcohol concentration is measured by gas chromatography. The specific results are shown in Table 3-a.
TABLE 3-a serum alcohol concentration (mg/dl) after gastric lavage with ethanol
30min | 120min | |
Streptococcus thermophilus grx11 bacterial liquid | 304.62±28.071 | 118.43±14.691 |
Lactobacillus paracasei H9 bacterial liquid | 298.80±21.491 | 114.91±12.081 |
Mixed bacterial liquid | 164.66±15.4212 | 51.50±6.3212 |
Negative control | 0 | 0 |
Positive control | 337.27±40.08 | 127.86±14.15 |
Note: t test, l: p <0.05 (compared to negative control); 2: p <0.05 (compare with positive control group)
As a result, only the single bacteria solution group remained very high in alcohol concentration after the pre-administration of the mixed bacteria at week 1, while the mixed bacteria group had a statistical difference in serum alcohol concentration (P <0.05) with respect to the positive control group. Indicating that the tea has the function of synergy for relieving alcoholism.
Detection of liver function:
ALT kit and AST kit (constructed organism) are adopted to measure ALT and AST activity in serum.
The results are shown in Table 3-b.
TABLE 3-b liver function assay in groups of mice after gastric lavage with ethanol
ALT | ALT | |
Streptococcus thermophilus grx11 bacterial liquid | 41.33±7.171 | 94.57±8.841 |
Lactobacillus paracasei H9 bacterial liquid | 42.08±6.121 | 93.54±7.651 |
Mixed bacterial liquid | 32.07±6.2312 | 63.32±5.4612 |
Negative control | 31.86±7.43 | 54.84±4.43 |
Positive control | 45.88±8.64 | 108.43±9.29 |
Note: t test, l: p <0.05 (compared to negative control); 2: p <0.05 (compare with positive control group)
As a result, when the mixed bacteria solution was pre-taken at week 1, only the single bacteria solution was slightly decreased compared to the positive group, but there was no statistical difference. Compared with the positive group, the mixed bacteria liquid group can obviously reduce ALT and AST, has statistical difference (P is less than 0.05), and shows that 2 bacteria have synergistic protection effect and more obvious ALT and AST reduction.
Example 4 in vivo anti-alcoholic liver injury assay (general test group, not pre-gavage)
1. The activation culture of the bacterial liquid of streptococcus thermophilus grx11 and the bacterial liquid of lactobacillus paracasei H9 is carried out according to a conventional method. Adjusting the concentration of the bacterial liquid to 10 in the culture medium7cfu/ml. And preparing a group of mixing groups: activating the bacterial liquid of streptococcus thermophilus grx11 and lactobacillus paracasei H9, mixing according to the concentration of the bacterial quantity of 1:1, and adjusting the total concentration of the bacterial liquid to 107cfu/ml。
2. Healthy ICR male mice with the weight of 20-25g are selected, and after 1 week of breeding, formal experiments are started and randomly divided into 5 groups of 10 mice, and the weights of the mice in each group are not statistically different. Mice were divided into 5 groups. No bacterial liquid was perfused in advance for each test group, and the perfusion was performed from the first day of the experiment as follows.
Purified water or bacterial liquid (0.2ml/25g dosage) is used for the first intragastric administration of each group; half an hour later, the mice were given a second intragastric administration with purified water in group 1 and 40% ethanol in water (0.2ml/25g dose) in groups 2-5.
Group 1: purified water + purified water control group (negative control group);
group 2: purified water + 40% ethanol concentration treated group (positive control group);
group 3: a streptococcus thermophilus grx11 bacterial liquid and 40% ethanol concentration treatment group;
group 4: a lactobacillus paracasei H9 bacterial liquid and 40% ethanol concentration treatment group;
group 5: mixed bacteria liquid and 40% ethanol concentration treatment group.
Blood is collected from inner canthus of capillary eye twice in the second intragastric lavage (ethanol lavage) for 30min and 2h, and the blood is placed at room temperature for 120min, centrifuged at 2500rpm for 15min to obtain serum, and the alcohol concentration is measured by gas chromatography. The specific results are shown in Table 4-a.
TABLE 4-a serum alcohol concentration (mg/dl) after gastric lavage with ethanol
30min | 120min | |
Streptococcus thermophilus grx11 bacterial liquid | 312.32±32.991 | 128.33±12.691 |
Lactobacillus paracasei H9 bacterial liquid | 318.83±52.341 | 127.31±10.081 |
Mixed bacterial liquid | 304.64±40.531 | 112.64±7.921 |
Negative control | 0 | 0 |
Positive control | 330.22±38.94 | 135.64±9.95 |
Note: t test, l: p <0.05 (compared to negative control); 2: p <0.05 (compare with positive control group)
As a result, when the mixed bacterial liquid was not administered in advance for intragastric administration, there was no statistical difference in serum alcohol concentration between the groups compared with the positive control group.
Detection of liver function:
ALT kit and AST kit (constructed organism) are adopted to measure ALT and AST activity in serum.
The results are shown in Table 4-b.
TABLE 4-b liver function assay in groups of mice after gastric gavage with ethanol
ALT | ALT | |
Streptococcus thermophilus grx11 bacterial liquid | 46.54±5.921 | 102.32±8.691 |
Lactobacillus paracasei H9 bacterial liquid | 45.64±6.621 | 104.40±11.751 |
Mixed bacterial liquid | 45.31±7.391 | 99.87±8.781 |
Negative control | 31.21±6.73 | 53.46±8.18 |
Positive control | 47.33±6.22 | 109.755±9.34 |
Note: t test, l: p <0.05 (compared to negative control); 2: p <0.05 (compare with positive control group)
As a result, when the mixed bacterial liquid was not administered for intragastric administration in advance, the respective groups had no statistical significance relative to the positive control group, and ALT and AST could not be significantly reduced.
EXAMPLE 5 in vivo anti-alcoholic liver injury assay (general trehalose test group)
1. The activation culture of the bacterial liquid of streptococcus thermophilus grx11 and the bacterial liquid of lactobacillus paracasei H9 is carried out according to a conventional method. Adjusting the concentration of the bacterial liquid to 10 in the culture medium7cfu/ml. And preparing a group of mixing groups: activating the bacterial liquid of streptococcus thermophilus grx11 and lactobacillus paracasei H9, mixing according to the concentration of the bacterial quantity of 1:1, and adjusting the total concentration of the bacterial liquid to 107cfu/ml。
2. Preparing a trehalose solution: 0.5 mol/L.
3. 60 healthy ICR male mice with the weight of 20-25g are selected, and after 1 week of breeding, formal experiments are started and randomly divided into 6 groups of 10 mice, and the weights of the mice in each group are not statistically different. Mice were divided into 6 groups. No bacterial liquid was perfused in advance for each test group, and the perfusion was performed from the first day of the experiment as follows.
Purified water or bacterial liquid (0.2ml/25g dosage) is used for the first intragastric administration of each group; half an hour later, the mice were given a second intragastric administration with purified water in group 1, 40% ethanol in water (0.2ml/25g dose) in group 2, and 40% ethanol in water (0.2ml/25g dose) and trehalose in groups 3-6.
Group 1: purified water + purified water control group (negative control group);
group 2: purified water + 40% ethanol concentration treated group (positive control group);
group 3: the streptococcus thermophilus grx11 bacterial liquid + 40% ethanol concentration treatment group +0.05ml trehalose solution; group 4: lactobacillus paracasei H9 bacterial liquid, 40% ethanol concentration treatment group and 0.05ml trehalose solution; group 5: the mixed bacterial solution + 40% ethanol concentration treatment group +0.05ml trehalose solution.
Group 6: purified water + 40% ethanol concentration treatment group +0.05ml trehalose solution (trehalose control group).
Blood is collected from inner canthus of capillary eye twice in the second intragastric lavage (ethanol lavage) for 30min and 2h, and the blood is placed at room temperature for 120min, centrifuged at 2500rpm for 15min to obtain serum, and the alcohol concentration is measured by gas chromatography. The specific results are shown in Table 5-a.
TABLE 5-a serum alcohol concentration (mg/dl) after gastric lavage with ethanol
Note: t test, l: p <0.05 (compared to negative control); 2: p <0.05 (compare with positive control group)
As a result, although the mixed bacterial liquid and the mixed bacterial liquid were not administered for intragastric administration in advance, the mixed bacterial liquid group was administered with the trehalose solution, and the serum alcohol concentration was statistically different (P <0.05) from that of the positive control group. The result shows that the trehalose has the function of synergizing the mixed bacteria to relieve alcoholism, and the trehalose alone has no capacity of relieving alcoholism.
Detection of liver function:
ALT kit and AST kit (constructed organism) are adopted to measure ALT and AST activity in serum.
The results are shown in Table 5-b.
TABLE 5-b liver function assay in groups of mice after gastric lavage with ethanol
ALT | ALT | |
Streptococcus thermophilus grx11 bacterial liquid | 43.41±3.621 | 101.22±8.791 |
Lactobacillus paracasei H9 bacterial liquid | 45.54±5.921 | 105.45±7.331 |
Mixed bacterial liquid | 34.63±4.4912 | 69.32±4.5912 |
Negative control | 31.23±3.42 | 56.39±8.84 |
Positive control | 47.53±5.32 | 108.65±11.41 |
Trehalose control | 46.79±6.11 | 107.87±13.20 |
Note: t test, l: p <0.05 (compared to negative control); 2: p <0.05 (compare with positive control group)
As a result, although the mixed bacteria solution and the mixed bacteria solution are not used for intragastric administration in advance, the mixed bacteria solution group simultaneously gives the trehalose solution, compared with a positive control group (P <0.05), the ALT and the AST can be obviously reduced, the obvious protective effect on liver functions is shown, the trehalose has the effect of protecting the liver functions by cooperating with the mixed bacteria, but the trehalose alone does not have the obvious liver protection effect.
In conclusion, in the research and practice processes of the research team of the invention, the research team of the applicant finds that the streptococcus thermophilus grx11 or lactobacillus paracasei H9 has no obvious effect of reducing the alcoholic liver injury, but the combined use has a more excellent effect of reducing the alcoholic liver injury. However, it was found in the studies that it seems that the significant effect is only shown if the drench is started at least one week before the alcoholic liver injury is modeled, which is considered to be related to the mixed bacteria mobilizing the in vitro and in vivo anti-hangover capacity.
If the trehalose is compounded in the composite microbial inoculum, the use time is not limited to the use before the alcoholic liver injury for a period of time, and the use after the liver injury also has good effect of promoting the liver injury repair. Indicating that the trehalose has the function of protecting the liver by cooperating with the mixed bacteria.
The description of the terms "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
While embodiments of the invention have been shown and described, it will be understood by those of ordinary skill in the art that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.
Claims (3)
1. A composite microbial inoculum with liver protection effect is characterized in that: it consists of the following 2 strains:
streptococcus thermophilus (Streptococcus thermophilus) grx11 and lactobacillus paracasei (lactobacillus paracasei) H9;
the preservation number of the streptococcus thermophilus grx11 is CGMCC No: 7697;
the preservation number of the lactobacillus paracasei H9 is CGMCC No. 4780;
the preparation method of the complex microbial inoculum comprises the following steps: activating Streptococcus thermophilus grx11 and Lactobacillus paracasei H9, mixing according to the concentration of 1:1 of the number of bacteria, and adjusting the total concentration of the bacteria liquid to 107cfu/ml。
2. The complex microbial agent with a liver-protecting effect according to claim 1, wherein:
the compound microbial inoculum is prepared into tablet candy, powder and capsule.
3. The use of the complex microbial agent having a liver-protecting effect according to claim 1 as a health food.
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