CN113425750B - Preparation and application of microecological preparation with liver injury protection effect - Google Patents

Preparation and application of microecological preparation with liver injury protection effect Download PDF

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CN113425750B
CN113425750B CN202110700296.8A CN202110700296A CN113425750B CN 113425750 B CN113425750 B CN 113425750B CN 202110700296 A CN202110700296 A CN 202110700296A CN 113425750 B CN113425750 B CN 113425750B
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parts
preparation
fermentation
bacterial liquid
liver
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CN113425750A (en
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刘国文
金波
杜希良
李心慰
沈泰钰
房志愿
宋玉祥
王哲
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Jilin University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/062—Ascomycota
    • A61K36/064—Saccharomycetales, e.g. baker's yeast
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    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
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    • 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
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C12N1/18—Baker's yeast; Brewer's yeast
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    • 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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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/16—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing two or more hetero rings
    • C12P17/162—Heterorings having oxygen atoms as the only ring heteroatoms, e.g. Lasalocid
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    • C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously

Abstract

The invention discloses a preparation and application of a microecological preparation with a liver injury protection effect, which is prepared by fermenting grape seed procyanidin powder, a fermentation culture medium and a compound bacterial liquid; the composite bacterial liquid is prepared by mixing saccharomyces cerevisiae H2 and lactobacillus fermentum O, and the adding ratio of the saccharomyces cerevisiae H2 to the lactobacillus fermentum O is 2: 3; the fermentation medium consists of the following components: 26-27 parts of glucose, 10-11 parts of peptone, 4-5 parts of yeast extract powder, 1-2 parts of dipotassium hydrogen phosphate, 1-2 parts of ammonium citrate, 0.1-0.3 part of magnesium sulfate and 1000 parts of water 950-doped sodium bicarbonate. The preparation and application of the microecological preparation with the liver injury protection effect are characterized in that the coarse extraction commercial grape seed procyanidin powder with low cost is selected, the impurities and the polymerization degree of the grape seed procyanidin powder are high, the average polymerization degree of the procyanidin can be reduced and stably dissolved in the compound bacterial liquid through combined fermentation, and the preparation components can be absorbed and utilized by organisms so as to prevent and treat the liver injury.

Description

Preparation and application of microecological preparation with liver injury protection effect
Technical Field
The invention relates to the technical field of biological preparations, in particular to preparation and application of a microecological preparation with a liver injury protection effect.
Background
The grape yield of China is at the world leading level, and with the rapid development of the grape processing industry, the grape waste is generated more and more. At present, the waste is partially used for producing low-value products such as fertilizers, activated carbon, biogas raw materials and the like, and most of the waste is directly treated as garbage. However, grape seeds not only contain abundant nutrients such as vitamins, amino acids and minerals, but also contain a large amount of procyanidine; and the procyanidine extracted from the grape seeds has strong oxidation resistance, can efficiently remove free radicals, and has the capacity of 20 times of that of the vitamin C. Therefore, the reasonable extraction and utilization of the procyanidine in the grape seeds have important economic value and practical significance.
Procyanidin (PC) is a flavonoid with different polymerization degrees formed by polymerizing flavan-3-ol monomers. The procyanidine extracted from the crude plant is mostly a high polymer with the polymerization degree of more than 5. At present, the grape seed extract is the most main source for industrially preparing procyanidine, and most of PC in grape exists in the outer integument of grape seed and is connected with protein, cellulose, hemicellulose, lignin and the like in cell matrix. The content of Procyanidin Polymer (PPC) with polymerization degree of >5 in Grape Seed Procyanidin (GSPC) is 50%, and the content of procyanidin Monomer (MPC) and dimer etc. is less than 10%. PPC is difficult to permeate biological membranes, the bioavailability is low, and a large amount of PPC accumulated in the intestinal tract can stimulate the intestinal tract, induce the inflammatory reaction of the intestinal epithelium and damage the health of organisms. Therefore, the procyanidin with low polymerization degree is beneficial to improving the utilization rate in the organism and enhancing the oxidation resistance.
Oxidative stress refers to the excessive production of highly reactive molecules such as Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS) in the body when the body is subjected to various harmful stimuli, the degree of oxidation exceeds the clearance of oxides, and the oxidative system and the antioxidant system are unbalanced, thereby causing tissue damage. In the oxidative stress process, various substances constituting cell tissues, such as lipids, saccharides, proteins, and all macromolecular substances such as deoxyribonucleic acid (DNA), undergo various degrees of oxidative reactions due to the oxidative stress of free radicals, causing oxidative damage such as denaturation, cross-linking, and fragmentation, which in turn leads to the destruction of cell structures and functions, and the damage of body tissues and the pathological changes of organs. Research shows that the oxidative stress of the liver is a common feature of diseases such as fatty liver, liver cirrhosis, liver cancer and the like, and relieving the oxidative stress is helpful for recovering the diseases. Therefore, the search for high-efficiency antioxidant preparations is of great significance for preventing and treating liver injury diseases caused by oxidative stress.
Disclosure of Invention
Technical problem to be solved
Aiming at the defects of the prior art, the invention provides the preparation and the application of the microecological preparation with the liver injury protection effect, has the advantages of preventing and treating liver injury diseases caused by oxidative stress, and effectively relieves the problem that the organism is injured due to the oxidative stress.
(II) technical scheme
In order to achieve the purpose, the invention provides the following technical scheme: a microecological preparation with liver injury protecting effect is prepared by fermenting grape seed procyanidin powder, fermentation culture medium and compound bacterial liquid;
the composite bacterial liquid is prepared by mixing saccharomyces cerevisiae H2 and lactobacillus fermentum O, and the adding ratio of the saccharomyces cerevisiae H2 to the lactobacillus fermentum O is 2: 3;
the fermentation medium consists of the following components: 26-27 parts of glucose, 10-11 parts of peptone, 4-5 parts of yeast extract powder, 1-2 parts of dipotassium hydrogen phosphate, 1-2 parts of ammonium citrate, 0.1-0.3 part of magnesium sulfate and 1000 parts of water 950-doped materials;
further comprising the steps of:
s1, preparing a composite bacterial liquid: respectively carrying out recovery and rejuvenation culture on strains forming the composite bacterial liquid, respectively adjusting the bacterial liquid to the turbidity of a 1 McLeeb tube by using normal saline, and then mixing saccharomycetes and lactic acid bacteria according to the inoculation ratio of 2:3 in volume to obtain the composite bacterial liquid;
s2, mixed fermentation: mixing grape seed procyanidin, a compound bacterial liquid and a fermentation culture medium, adding into a fermentation tank for fermentation, wherein the addition amount of the grape seed procyanidin is 40-60 parts by weight, the inoculation amount of the compound bacterial liquid is 20-30 parts by volume, the fermentation temperature is 38-40 ℃, the stirring speed is 110-130r/min, the pH is 5.5-6.5, the fermentation time is 18-22h, and the fermentation finished product is the microecological preparation.
Preferably, in step S1, before the fermentation culture, the activation and rejuvenation of the strain is performed, and the rejuvenation medium comprises the following components: 18-22 parts of glucose, 10-12 parts of peptone, 4-6 parts of yeast extract powder, 6-8 parts of beef extract powder, 2-4 parts of dipotassium phosphate, 2-4 parts of ammonium citrate, 3-5 parts of sodium acetate, 0.02-0.04 part of manganese sulfate, 0.2-0.4 part of magnesium sulfate, 801-2 parts of tween and 1000 parts of secondary distilled water 950-.
Preferably, the grape seed procyanidin has the purity of 80-90% and the average polymerization degree of 6.5-8.8.
(III) advantageous effects
Compared with the prior art, the invention provides the preparation and the application of the microecological preparation with the liver injury protection effect, and the microecological preparation has the following beneficial effects:
1. the preparation and application of the microecological preparation with the liver injury protection effect are characterized in that the coarse extraction commercial grape seed procyanidin powder with low cost is selected, the impurities and the polymerization degree of the grape seed procyanidin powder are high, the average polymerization degree of the procyanidin can be reduced and stably dissolved in the compound bacterial liquid through combined fermentation, and the preparation components can be absorbed and utilized by organisms so as to prevent and treat the liver injury.
2. The preparation and application of the microecological preparation with the liver injury protection effect improve the growth rate of saccharomyces cerevisiae H2 and lactobacillus fermentum O in the compound bacterial liquid by adopting the combined fermentation of the procyanidine and the compound bacterial liquid, shorten the time for the two bacteria to enter the growth stabilization period, accelerate the preparation efficiency of the preparation, and meanwhile, the procyanidine has an inhibition effect on common pathogenic bacteria of the gastrointestinal tract, can help the compound probiotics to colonize and grow in the gastrointestinal tract, maintain the stable flora of the gastrointestinal tract, promote nutrient digestion and absorption, and enhance the immunity and disease resistance of organisms.
Drawings
FIG. 1 is a graph showing the growth of Saccharomyces cerevisiae H2 in procyanidin-supplemented liquid medium (Saccharomyces cerevisiae H2+) and in procyanidin-unsupplemented liquid medium (Saccharomyces cerevisiae H2) in accordance with the present invention;
FIG. 2 is a graph showing the growth of Lactobacillus fermentum O in the liquid medium supplemented with proanthocyanidins (Lactobacillus fermentum O +) and in the liquid medium without proanthocyanidins (Lactobacillus fermentum O) according to the present invention;
FIG. 3 is a graph showing the DPPH radical clearance (%) of the blank medium group, the compound bacterial liquid group, the procyanidin medium group and the microecologics in the present invention;
FIG. 4 is a graph showing ABTS free radical scavenging rate (%) of the blank medium group, the compound bacterial liquid group, the procyanidin medium group and the microecologics in the present invention;
FIG. 5 is an apparent image of a liver biopsy of a mouse in an acute liver injury prevention test of the microecological preparation of the present invention;
FIG. 6 shows the blank control group, CCl, of the present invention4Model group, Microecological Agents the activity of SOD and GSH-Px in liver tissue (unit U/mgprot represents the activity of enzyme in 1mg tissue protein) of mice in the prevention group;
FIG. 7 shows the HE staining result of liver slices in the experiment of preventing acute liver injury with the microecological preparation of the present invention;
FIG. 8 is an appearance diagram of a liver biopsy of a mouse in an acute liver injury test treated by the microecological preparation of the present invention;
FIG. 9 is the enzyme activity diagrams of mouse liver SOD and GSH-Px in the experiment of treating acute liver injury by the microecological agent of the present invention;
FIG. 10 shows the HE staining result of mouse liver slices in the experiment of treating acute liver injury with the microecological preparation of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides preparation and application of a microecological preparation with a liver injury protection effect, which is prepared by fermenting grape seed procyanidin powder, a fermentation culture medium and a compound bacterial liquid;
the composite bacterial liquid is prepared by mixing saccharomyces cerevisiae H2 and lactobacillus fermentum O, and the adding ratio of the saccharomyces cerevisiae H2 to the lactobacillus fermentum O is 2: 3;
the fermentation medium consists of the following components: 26-27 parts of glucose, 10-11 parts of peptone, 4-5 parts of yeast extract powder, 1-2 parts of dipotassium hydrogen phosphate, 1-2 parts of ammonium citrate, 0.1-0.3 part of magnesium sulfate and 1000 parts of water 950-doped materials;
further comprising the steps of:
s1, preparing a composite bacterial liquid: respectively recovering and rejuvenating the strains forming the composite bacterial liquid, respectively adjusting the bacterial liquid to the specific turbidity of a 1 McLeod turbidimetric tube by using normal saline, and then mixing the saccharomycetes and the lactic acid bacteria according to the inoculation ratio of 2:3 in volume to obtain the composite bacterial liquid;
s2, mixed fermentation: mixing grape seed procyanidin, a compound bacterial liquid and a fermentation culture medium, adding into a fermentation tank for fermentation, wherein the addition amount of the grape seed procyanidin is 40-60 parts by weight, the inoculation amount of the compound bacterial liquid is 20-30 parts by volume, the fermentation temperature is 38-40 ℃, the stirring speed is 110-130r/min, the pH is 5.5-6.5, the fermentation time is 18-22h, and the fermentation finished product is the microecological preparation.
The examples are as follows:
first step recovery and passage of bacterial species
CGMCC sequencing is used for identifying the preferred saccharomyces cerevisiae and lactobacillus fermentum, and rejuvenation culture medium is used for recovery passage to recover the activity of the saccharomyces cerevisiae and lactobacillus fermentum. The rejuvenation culture medium comprises the following components: 18-22 parts of glucose, 10-12 parts of peptone, 4-6 parts of yeast extract powder, 6-8 parts of beef extract powder, 2-4 parts of dipotassium phosphate, 2-4 parts of ammonium citrate, 3-5 parts of sodium acetate, 0.02-0.04 part of manganese sulfate, 0.2-0.4 part of magnesium sulfate, 801-2 parts of tween and 1000 parts of secondary distilled water 950-.
Second step optimization of fermentation Medium composition and fermentation conditions
The optimized culture medium comprises 26-27 parts by weight of glucose, 10-11 parts by weight of peptone, 4-5 parts by weight of yeast extract powder, 1-2 parts by weight of dipotassium hydrogen phosphate, 1-2 parts by weight of ammonium citrate, 0.1-0.3 part by weight of magnesium sulfate and 1000 parts by weight of water 950-.
The third step of composite fermentation
Adding 40-60 parts by weight of grape seed procyanidin, inoculating 20-30 parts by volume of the composite bacterial liquid, fermenting at 38-40 ℃, stirring at the rotation speed of 110-.
Fourth step of safety test
The micro-ecological preparation provided by the invention is adopted to carry out acute toxicity test on Kunming mice, 12 males and females are selected in the test, the weight of the Kunming mice is 20-25g, the Kunming mice are randomly divided into two groups, the fasting is carried out for 16h before gavage, a control group is subjected to oral gavage for 50 mu l/g of normal saline once, an experimental group is subjected to oral gavage for 50 mu l/g of the micro-ecological preparation once, then the normal diet observation is carried out for 14 days, the poisoning symptoms and survival conditions of the mice are recorded, the eyeballs of the mice are removed after 14 days, blood serum biochemical indexes including blood sugar (GLU), glutamic-oxalacetic transaminase (AST), glutamic-pyruvic transaminase (ALT), creatinine (Cr) and Lactate Dehydrogenase (LDH) are measured, and the dissection examination and the weighing of the liver weight ratio are carried out after the neck is cut.
In order to coordinate the above experiments, the following data support is now provided:
data one:
TABLE 1 body condition index and blood biochemical index of acute toxicity test mice
Figure RE-GDA0003214214610000061
Results are expressed as mean ± sem (number of samples ═ 6).
In the process of the oral acute toxicity test of the mice, the tested mice have no toxic symptoms during observation, the mice die, and the observation of all organs of the mice subjected to autopsy has no obvious change, as can be seen from table 1, the weight, the liver weight and the liver weight ratio of the mice in an experimental group are not affected, the growth and development of animals are normal, and the index difference of GLU, AST, ALT, Cr and LDH in the serum of the mice has no statistical significance (p is more than 0.05), so that the invention has no toxic or side effect on normal mice.
Data II:
the method comprises the steps of carrying out a subchronic toxicity test on Kunming mice by adopting the microecological preparation provided by the invention, selecting 24 Kunming mice of which the male part and the female part are half respectively, weighing 20-25g, randomly dividing into 4 groups, namely a control group (drenched with 12.5 mu l/g of physiological saline), a microecological preparation drenched with low (12.5 mu l/g), medium (25 mu l/g) and high (37.5 mu l/g) dose test group, drenching each group of mice every day during the test period, continuously carrying out 28d observation on the activity state, physical signs, food intake, drinking water and excrement conditions of the mice every day, after 28d, fasting the mice for 12h, carrying out weighing and removing, carrying out blood sampling on eyeballs of the mice, carrying out measurement on serum biochemical indexes including GLU, AST, ALT, Cr and LDH, and carrying out cesarean section examination and liver weighing after neck breaking treatment.
TABLE 2 body condition index and blood biochemical index of subchronic toxic mice
Figure RE-GDA0003214214610000071
Results are expressed as mean ± sem (sample number 6), shoulder · indicates comparison of control and experimental groups, p < 0.05.
In the whole process of the mouse undergoing the subchronic toxicity test, the tested mouse has no toxic symptom during observation, the mouse dies, and the observation of organs of the mice subjected to the autopsy has no obvious change, as can be seen from table 2, the weight of the mice in an experimental group is obviously increased after 28 days of gastric lavage (p is less than 0.05), the ratio of the liver weight to the liver weight is not influenced, the growth and development of animals are normal, and the index difference of GLU, AST, ALT, Cr and LDH in the serum of the mice has no statistical significance (p is more than 0.05), which indicates that the preparation has good safety.
Fifth step of effectiveness test
The microecologics were tested for two free radical scavenging abilities, 1-diphenyl-2-trinitrophenylhydrazine (DPPH), 2' -biazoyl-bis-3-ethylbenzothiazoline-6-sulfonic Acid (ABTS).
In the free radical scavenging experiment, in order to eliminate concentration errors caused by crude extract impurities of grape seed procyanidin, analytically pure PC is used for verifying the free radical scavenging capacity during preparation of the preparation, and two experiments are divided into four groups: 1) Control group containing medium only; 2) a liquid medium group added with analytically pure PC (the addition amount of analytically pure PC is equal to the PC content of the microecologics group); 3) a composite bacterium liquid group only containing saccharomyces cerevisiae and lactobacillus fermentum; 4) the results of experiments performed on the microecological preparation group using the DPPH and ABTS free radical scavenging assay kits are shown in FIGS. 3 and 4.
In vitro free radical scavenging experiments show that the free radical scavenging capacity of the microecological preparation group is remarkably higher than that of the compound bacterium liquid group and the procyanidin group added independently (p is less than 0.05), which indicates that the preparation can remarkably improve the scavenging capacity of the procyanidin on free radicals.
Data three:
prevention effect of microecologics on liver injury of mice
This experiment utilized CCl4As an inducer for liver injury, 18 Kunming mice with male and female halves and a Kunming mouse with the weight of 20-25g are selected in the test and randomly divided into a blank control group and CCl4Model group, microecological preparation prevention group, each group of mice is half male and female, the microecological preparation prevention group is continuously drenched with microecological preparation for 7 days in a volume of 20 μ l/g, blank control group and CCl4The model group was continuously administered with physiological saline at a volume of 20 μ l/g for 7 days, and after completion of the intragastric administration, the blank control group was administered with 10 μ l/g volume of physiological saline, CCl4Model group and microecological preparation prevention group are injected with CCl with the volume of 10 mul/g and the concentration of 0.2 percent in abdominal cavity4And (3) taking eyeballs and blood from soybean oil solution in a fasting state for 16h without water prohibition, standing for 1h, centrifuging at a rotating speed of 3000 r/min for 10min, collecting serum, measuring biochemical indexes of the serum including GLU, AST, ALT, Cr and LDH, performing neck amputation treatment, and weighing liver weight.
Performing a dissecting examination on the killed blood-sampled mice after neck fracture, wherein FIG. 5 is a dissecting examination picture of the apparent morphology of the liver of the test mice, (a) a blank control group; (b) CCl4A model group; (c) the microecological preparation prevention group and the blank control group have normal liver appearance and no pathological change, CCl4The model group showed a large number of necrotic foci on the surface of liver, and CCl4Compared with the model group, the microecological preparation prevents the mice in the group from reducing the number of liver necrotic foci, only sporadic necrotic foci appear along the edge of liver lobes, and after counting, CCl4The number of the liver surface necrotic foci of the model group mice is 10.67 +/-2.34 (n is 6), and the number of the liver surface necrotic foci of the mice in the microecological preparation prevention group is 6.72 +/-1.06 (n is 6), and compared with CCl, the quantity of the liver surface necrotic foci of the mice in the microecological preparation prevention group4Significant reduction in model group (p)<0.05)。
TABLE 3 Microecological preparation for preventing signs and blood biochemical index of mice in acute liver injury test
Figure RE-GDA0003214214610000091
Results are expressed as mean ± sem (sample number ═ 6), identical shoulders in each row indicate that the data was not significantly different between the two groups, p < 0.05.
As can be seen from Table 3, CCl was compared with the blank control group4The liver weight and the weight ratio of the liver weight of the model group mice are obviously increased (p)<0.05), ALT, AST and LDH activities were significantly elevated (p)<0.05), indicating that the liver of the mouse has acute liver injury; cr level is remarkably increased (p)<0.05), indicating impaired renal function, the probiotic-protected group compared to CCl4The AST, ALT, Cr and LDH indexes in the serum of the model group are all obviously reduced (p)<0.05), indicating that the probiotic is capable of preventing CCl4Induced acute liver injury and impaired renal function.
0.2g of blank control group and CCl were respectively taken4The liver tissues of the mice in the model group and the probiotic protected group were subjected to the measurement of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD), and the measurement results are shown in FIG. 6.
From the results of the detection in FIG. 6, CCl is shown4The enzyme activity of GSH-Px and SOD in the liver tissue of the model group is obviously reduced (p) compared with that of the blank control group<0.01), Microecological agent prevention group vs CCl4The enzyme activities of SOD and GSH-Px in the liver of the model group mice are obviously improved (p)<0.01) shows that the preparation can improve the activity of liver antioxidase, enhance the liver antioxidant ability, and prevent CCl4Resulting in oxidative stress of the liver.
Taking blank control group, CCl4The livers of the mice in the model group and the microecological agent prevention group were fixed with 4% paraformaldehyde, and HE-stained, and liver tissue sections were observed as shown in fig. 8, wherein (a) the blank control group; (b) CCl4A model group; (c) a probiotic prevention group; (d) the area ratio (%) of each group of necrotic liver tissues to the area of liver tissues in the visual field.
From the results in FIG. 7, it can be seen that the liver lobules of the blank control group mice were intact, the liver cells were normal in morphology, and there was no abnormality in the zone of the sink; CCl4The mouse liver lobule structure of the model group is disordered, and a large area of necrotic foci surround the central vein; the liver lobule structure of the liver of the mice in the micro-ecological preparation prevention group is complete, the necrotic area around the central vein is reduced, and the proportion of the liver injury area to the whole liver tissue is obviously reduced (p)<0.01)。
The above results illustrate the formulation for CCl4Can prevent acute liver injury of mice.
And (4) data four:
the microecological preparation has therapeutic effect on liver injury of mice.
This experiment utilized CCl4As an inducer for liver injury, 18 Kunming mice with male and female halves and a Kunming mouse with the weight of 20-25g are selected in the test and randomly divided into a blank control group and CCl4Model group, microecological agent treatment group, each group of mice male and female half, CCl4Model group and microecological preparation treatment group are injected with CCl with the volume of 10 mul/g and the concentration of 0.2 percent in abdominal cavity4Soybean oil solution, the blank control group is injected with normal saline with the volume of 10 mul/g, after the injection, the microecological preparation treatment group is continuously infused with the microecological preparation for 7 days with the volume of 20 mul/g, the blank control group and CCl4The model group is continuously infused with physiological saline for 7 days in a volume of 20 mul/g, fasted and water-forbidden for 16h, the eyeballs are picked up and blood is taken, the model group is kept stand for 1h, the model group is centrifuged for 10min at a rotating speed of 3000 r/min, serum is collected, the biochemical indexes of the serum including GLU, AST, ALT, Cr and LDH are measured, and the dissection and the weighing of liver weight are carried out after the neck amputation treatment.
FIG. 8 is an apparent morphology of the liver of a test mouse, wherein (a) is a blank control group; (b) CCl4A model group; (c) microecological agent treatment group, see CCl4The surface of the model group is rough and dull, and a large number of necrotic foci are arranged on the surface of the liver; and CCl4Compared with the model group, the mouse treated by the microecologics has the advantages that the number of necrotic foci on the surface of the liver is reduced, and part of the surface isNecrotic foci are separated from liver shedding, counted, CCl4The number of liver surface necrotic foci of each mouse in the model group is 10.09 +/-1.22 (n is 6), the number of liver surface necrotic foci of each mouse in the microecological preparation treatment group is 6.92 +/-1.86 (n is 6), and the number of liver surface necrotic foci of the microecological preparation treatment group is compared with that of CCl4Significant reduction of model group (p)<0.05)。
TABLE 4 Microecological preparation for treating signs and blood biochemical index of mice in acute liver injury test
Figure RE-GDA0003214214610000111
Results are expressed as mean ± sem (sample number ═ 6), with identical shoulders on each row indicating that the data were not significantly different between the two groups, p < 0.05.
As can be seen from Table 4, CCl was compared with the control group4The liver-to-body weight ratio of the model group mice is obviously increased (p)<0.05), ALT, AST and LDH activities were significantly elevated (p)<0.05), indicating that the liver of the mouse has acute liver injury; cr level is remarkably increased (p)<0.05), indicating impaired renal function, the probiotic treated group compared to CCl4The AST, ALT, Cr and LDH indexes in the serum of the model group are all obviously reduced (p)<0.05), indicating that the probiotic is capable of treating CCl4Induced acute liver injury and impaired renal function.
Collecting 0.2g blank control group, CCl4The liver tissues of the mice in the model group and the microecological preparation treatment group were subjected to measurement of SOD and GSH-Px. The results are shown in FIG. 10, in which (a) the blank control group and CCl4The enzyme activity of SOD in the liver tissue protein of the mouse in the model group and the microecological preparation treatment group; (b) blank control group, CCl4Model group, microecological preparation treatment group the enzyme activity of GSH-Px in mouse liver tissue protein (unit U/mgprot represents the activity of the enzyme in 1mg tissue protein):
as can be seen from the results of the detection in FIG. 9, CCl was compared with the blank control group4The enzyme activities of SOD and GSH-Px in the liver tissue of the model group are obviously reduced (p)<0.01), the activity of antioxidant enzymes SOD and GSH-Px of liver of the microecological preparation treatment group is obviously improved (p)<0.01)，The preparation can enhance the activity of the antioxidant enzyme of the liver, improve the capacity of the liver to remove free radicals and relieve the oxidative stress of the liver.
Taking blank control group, CCl4After the livers of the mice in the model group and the microecological agent-treated group were fixed with 4% paraformaldehyde, HE staining was performed, and liver tissue sections were observed, as shown in fig. 10, in which (a) a blank control group; (b) CCl4A model group; (c) a probiotic treatment group; (d) the area ratio (%) of each group of necrotic liver tissues to the area of liver tissues in the visual field.
From the results in fig. 10, it can be seen that the liver lobules of the blank control group mice were intact, the liver cells were normal in morphology, and there was no abnormality in the zone of the sink; CCl4The mouse liver lobule structure of the model group is disordered, and a large area of necrotic foci surround the central vein; in the liver of mice in the microecological preparation treatment group, the necrotic area surrounding the central vein is reduced, and the proportion of the damaged liver area to the whole liver tissue is obviously reduced (p)<0.01)。
In conclusion, the preparation has good safety, improves the capacity of the procyanidine crude extract for removing free radicals through combined fermentation with probiotics, and can prevent and treat acute liver injury of mice.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims (3)

1. The preparation method of the microecological preparation with the liver injury protection effect is characterized by comprising the following steps: prepared by fermenting grape seed procyanidin powder, a fermentation medium and a compound bacterial liquid;
the composite bacterial liquid is prepared by mixing saccharomyces cerevisiae H2 and lactobacillus fermentum O, and the adding ratio of the saccharomyces cerevisiae H2 to the lactobacillus fermentum O is 2: 3;
the fermentation medium consists of the following components: 26-27 parts of glucose, 10-11 parts of peptone, 4-5 parts of yeast extract powder, 1-2 parts of dipotassium hydrogen phosphate, 1-2 parts of ammonium citrate, 0.1-0.3 part of magnesium sulfate and 1000 parts of water 950-doped materials;
further comprising the steps of:
s1, preparing a composite bacterial liquid: respectively recovering and rejuvenating the strains forming the composite bacterial liquid, respectively adjusting the bacterial liquid to the turbidity of a 1 McLeeb by using normal saline, and then mixing Saccharomyces cerevisiae H2 and lactobacillus fermentum O according to the inoculation ratio of 2:3 by volume to obtain the composite bacterial liquid, wherein the Saccharomyces cerevisiae H2 is bovine-derived Saccharomyces cerevisiae H2 identified by sequencing, and the lactobacillus fermentum O has the number of CGMCC 1.3223;
s2, mixed fermentation: mixing grape seed procyanidin, a compound bacterial liquid and a fermentation culture medium, adding into a fermentation tank for fermentation, wherein the addition amount of the grape seed procyanidin is 40-60 parts by weight, the inoculation amount of the compound bacterial liquid is 20-30 parts by volume, the fermentation temperature is 38-40 ℃, the stirring speed is 110-130r/min, the pH is 5.5-6.5, the fermentation time is 18-22h, and the fermentation finished product is the microecological preparation.
2. The preparation of the microecological preparation having liver injury protecting effect according to claim 1, wherein: in the step S1, before fermentation culture, activation and rejuvenation of the strain are carried out, wherein the rejuvenation culture medium comprises the following components: 18-22 parts of glucose, 10-12 parts of peptone, 4-6 parts of yeast extract powder, 6-8 parts of beef extract powder, 2-4 parts of dipotassium phosphate, 2-4 parts of ammonium citrate, 3-5 parts of sodium acetate, 0.02-0.04 part of manganese sulfate, 0.2-0.4 part of magnesium sulfate, 801-2 parts of tween and 1000 parts of secondary distilled water 950-.
3. The preparation of the microecological preparation having liver injury protecting effect according to claim 1, wherein: the grape seed procyanidin has the purity of 80-90% and the average polymerization degree of 6.5-8.8.
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