CN109486715B - Selenium-enriched bifidobacterium longum and preparation method and application thereof - Google Patents

Selenium-enriched bifidobacterium longum and preparation method and application thereof Download PDF

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CN109486715B
CN109486715B CN201811491494.2A CN201811491494A CN109486715B CN 109486715 B CN109486715 B CN 109486715B CN 201811491494 A CN201811491494 A CN 201811491494A CN 109486715 B CN109486715 B CN 109486715B
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selenium
bifidobacterium longum
diabetes
enriched
preparation
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CN109486715A (en
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华子春
林艳
张燕
徐根兴
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Changzhou High-Tech Research Institute Of Nanjing University
Targetpharma Laboratories Jiangsu Co ltd
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Changzhou High-Tech Research Institute Of Nanjing University
Targetpharma Laboratories Jiangsu Co ltd
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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
    • 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
    • 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
    • A23L33/16—Inorganic salts, minerals or trace elements
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • 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
    • C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K2035/11—Medicinal preparations comprising living procariotic cells
    • A61K2035/115—Probiotics

Abstract

The invention belongs to the technical field of biology, and particularly discloses selenium-rich bifidobacterium longum and a preparation method and application thereof. The selenium-rich bifidobacterium longum prepared by the preparation method can be applied to preparing medicines or foods for delaying the progress of diabetes, and specifically comprises the following steps: the application of the medicine or food for improving physiological indexes and metabolic conditions of diabetes, improving insulin signal channels of a diabetes model, improving pathological changes of liver, pancreas and kidney of diabetes, improving serum creatinine and blood urea nitrogen levels of diabetes and preventing diabetic kidney complications or renal function damage is prepared.

Description

Selenium-enriched bifidobacterium longum and preparation method and application thereof
Technical Field
The invention relates to the technical field of biology, in particular to selenium-rich bifidobacterium longum, a preparation method thereof and application thereof in preparing medicines or foods for delaying the progress of diabetes.
Background
Bifidobacteria are a genus of bacteria that grow naturally in the human gastrointestinal tract and protect the host against viral infections, and belong to the gram-positive, non-motile, branched-chain anaerobes. Usually, bifidobacteria are used as probiotics to aid digestion. Scientific research has now found and demonstrated a new use of probiotics in metabolic disorders including diabetes.
Selenium is a basic trace element required for human and animal health, and is actively involved in animal physiology, mainly in the form of selenoprotein. Selenium agents have been reported to continuously improve the steady state metabolism of glucose, thereby regulating sugar metabolic processes such as glycolysis and glucose production. Thus, in diabetic patients, selenium supplementation can lower plasma glucose levels. The insulin and the selenium have a synergistic effect, and the synergistic treatment of the insulin and the selenium can control the blood sugar of diabetic rats. However, epidemiological data indicate a positive correlation between selenium status and diabetes risk. A great deal of research shows that organic selenium has lower toxicity and higher biological activity than inorganic selenium, and can be rapidly absorbed and utilized, so that it has been the focus of attention of researchers in recent years. The current research shows that bifidobacterium longum can enrich selenium and influence the immune function of tumor mice in the form of selenoprotein. Research shows that the dietary supplement containing various probiotics can prevent the fasting blood sugar of the diabetics from rising. Oral administration of Bifidobacterium longum mixture can lower serum glucose levels, enhance expression of related proteins in insulin signaling pathway, and improve fat content in diabetic mice. However, the effects of bifidobacterium longum rich in selenium on Streptozotocin (STZ) -induced diabetes models in mice and on renal function have not been reported in detail.
Disclosure of Invention
In order to solve the defects of poor improvement effect of diabetes and easy cause of poor kidney function in the prior art, the invention aims to provide selenium-rich bifidobacterium longum, a preparation method thereof and application thereof in preparing medicines or foods for delaying the progress of diabetes.
The invention is realized by the following technical method:
(1) dissolving sodium selenite in TPY culture medium to obtain selenium-containing TPY culture medium;
(2) and mixing the bifidobacterium longum bacterial liquid with the TPY culture medium containing selenium, then inoculating the mixture into a new TPY culture medium, culturing in an incubator, and collecting the cultured bacterial liquid to obtain the selenium-rich bifidobacterium longum.
Preferably, the concentration of sodium selenite in the selenium-containing TPY medium in the step (1) is 20-30 g/mL.
Preferably, the mixing ratio of the bifidobacterium longum bacterial liquid and the selenium-containing TPY culture medium in the step (2) is 1: 20-30; the culture conditions are as follows: under anaerobic conditions, the culture was carried out overnight in a 37 ℃ incubator until the OD600 reached 0.2.
Preferably, the selenium-enriched bifidobacterium longum is applied to the preparation of medicines or foods for delaying the progress of diabetes.
Preferably, the selenium-enriched bifidobacterium longum is applied to the preparation of medicines or foods for improving physiological indexes and metabolic conditions of diabetes, wherein the physiological indexes and metabolic conditions of diabetes are fasting blood sugar, body weight, serum insulin level, glucose tolerance test (I PTGG), food intake, water intake and urine volume.
Preferably, the selenium-enriched bifidobacterium longum is applied to the preparation of medicines or foods for improving insulin signal channels of diabetes models.
Preferably, the selenium-enriched bifidobacterium longum is applied to preparing medicines or foods for improving pathological changes of liver, pancreas and kidney of diabetes.
Preferably, the selenium-enriched bifidobacterium longum is applied to preparing medicines or foods for improving the serum creatinine (Scr) and Blood Urea Nitrogen (BUN) level of diabetes.
Preferably, the selenium-enriched bifidobacterium longum is applied to the preparation of medicines or foods for preventing diabetic kidney complications or renal function damage.
The beneficial effects of the invention are as follows:
(1) compared with the model group, the selenium-enriched bifidobacterium longum treatment group has the advantages that the blood sugar level is reduced, the weight of the mice is increased, the serum insulin level is reduced, and IPGTT, the food intake amount in 24 hours, the water intake amount and the urine volume are all obviously improved.
(2) H & E pathological results show that the selenium-rich bifidobacterium longum can relieve pathological changes of liver and pancreas.
(3) Pathological changes in the kidney are reduced in pathological sections, and the contents of Scr and BUN in serum are obviously reduced, which shows the protective effect of the selenium-enriched bifidobacterium longum on diabetic nephropathy. In the selenium-enriched bifidobacterium longum treatment group, the expression level of related proteins of the insulin signaling pathway is higher than that of the model group.
Drawings
FIG. 1A is a graph of blood glucose versus time before gastric lavage and after STZ injection in mice;
FIG. 1B is a graph of body weight versus time before gastric lavage and after STZ injection in mice;
FIG. 1C is a graph of the trend of serum insulin levels and time before and after gastric lavage of mice and post-STZ injection.
FIG. 2 is a graph of the relationship between time and blood glucose levels after glucose injection.
FIG. 3A is a bar graph of food intake for each group of mice at 1 week and 6 weeks after STZ injection;
FIG. 3B is a bar graph of urine volume in groups of mice at 1 week and 6 weeks after STZ injection;
wherein, 1-normal group (1 week); 2-model group (1 week); 3-low dose group (1 week); 4-medium dose group (1 week); 5-high dose group (1 week);
1' -normal group (6 weeks); 2' -model group (6 weeks); 3' -low dose group (6 weeks); 4' -medium dose group (6 weeks); 5' -high dose group (6 weeks).
Fig. 4 is a graph of urea nitrogen levels in mice in each experimental group.
Fig. 5 is a schematic diagram of the effect of selenium-enriched bifidobacterium longum on insulin signaling pathway.
Fig. 6 is a schematic diagram showing the effect of selenium-enriched bifidobacterium longum on pathological morphology of pancreas (a) and liver (B).
FIG. 7 is a comparison chart of kidney histological examination in each experimental group.
In the figure, the position of the first and second end faces,##represents the P value<0.01，###Represents the P value<0.001, n-8 (representing 8 samples), compared to the normal group; denotes the P value<0.05, denotes the P value<0.01, n is 8, compared to the model set.
Detailed Description
The present invention will be described in further detail with reference to the following embodiments.
Example 1
(1) Preparation of bacterial liquid
Bifidobacterium longum was grown anaerobically in TPY at 37 ℃. Dissolving sodium selenite in 200mL of TPY medium to a final concentration of 25g/mL (concentration of sodium selenite is tried to be 20, 22.5, 25, 27.5 and 30g/mL respectively, and the effect is similar); the bifidobacterium longum bacterial liquid is prepared by the following steps of: inoculating a selenium-containing culture medium to a new TPY culture medium, wherein the selenium-containing culture medium is 1:25 (the mass ratio of the bifidobacterium longum bacterial liquid to the selenium-containing culture medium is 1:20, 1:22.5, 1:25, 1:27.5 and 1:30 respectively, and the effects are similar); culturing overnight in a constant-temperature incubator at 37 ℃ under an anaerobic condition until OD600 reaches 0.2 or above, and collecting the cultured bacterial liquid; centrifuging at 3500rpm and 4 deg.C for 5min, and washing with 5% glucose physiological saline for 3 times. The collected bacterial suspension was resuspended in 0.1mL of 13% skim milk for use.
(2) Animal experiments
Mice grew at 21 ℃ in 12h alternating light-dark environment, and were free to ingest water and food. Normal group mice were injected with citrate 100. mu.L. The specific method for establishing the diabetes model group mice comprises the following steps: mice were fasted for 12h, allowed free water, STZ was dissolved in 0.1M cold citric acid buffer (pH 4.5) to a final concentration of 50mg/kg and mice were injected with 100 μ L STZ for 5 consecutive days. 1 week after the last STZ injection of the mice in the diabetes model group, the fasting blood glucose level of the mice is measured by a glucometer, and the success of molding is judged if the blood glucose level is more than or equal to 11.1 mM. Mice were monitored weekly for blood glucose levels and body weight 8 weeks after the last STZ injection.
50 male C57BL/6 mice 4-5 weeks old were randomly divided into 5 groups: grouping: normal group, model group, selenium-enriched bifidobacterium longum low dose group (0.75X 10)10One/kg), medium dose group (1.5 × 10)10One/kg), high dose group (3.0 × 10)10One/kg). In the treatment group, 100 mu L of selenium-enriched bifidobacterium longum is perfused into each mouse every day, and the lavage time is 4 weeks. The normal group was given 100 μ L of 13% skim milk per day. The mice are administered with the selenium-rich bifidobacterium longum on the 25 th day, the mice are fasted for 12 hours, and the intraperitoneal injection is carried out continuously for 5 daysAnd (4) shooting the STZ. Mice were sacrificed 8 weeks after the last STZ injection. Mice were monitored for blood glucose levels and body weight during weeks 1 to 8 following STZ injection (see figure 1). At weeks 1 and 6 after STZ injection, mice were placed in mouse metabolism cages and 24h food intake and urine volume of mice were collected (see FIGS. 3A-3B). Glucose tolerance experiments were performed at week 7 of STZ injection, and after mice were starved overnight, glucose (1.0g/kg) was intraperitoneally injected, blood was taken from the tail vein for 0, 15, 30, 60, 90min, and blood glucose content was measured with a glucometer (see fig. 2). At week 8 of STZ injection, serum insulin levels were measured using a mouse insulin ELISA kit and serum urea nitrogen levels were measured (see figure 4).
As can be seen from fig. 1, compared with the model group, the group administered with bifidobacterium longum rich in selenium (including the low dose group, the medium dose group and the high dose group) had a lower fasting blood glucose level than the model group and had a dose-dependent relationship. Body weight has the same tendency as blood glucose level.
As can be seen from FIG. 2, the level of blood glucose recovery was significantly reduced after the injection of glucose in the model group mice, and the results showed that the glucose-removing ability in vivo was improved after the administration of the selenium-enriched Bifidobacterium longum.
As can be seen from FIGS. 3A-3B, at weeks 1 and 6 after STZ injection, mice were placed in metabolism cages and 24-hour food intake and urine volume of the mice were recorded; compared with the model group, the administration group of the selenium-rich bifidobacterium longum obviously reduces the metabolic condition of the mice; the intake of the selenium-enriched bifidobacterium longum administration group water also changes in the same way.
As can be seen from fig. 4, the serum urea nitrogen level of the mice was measured, and as a result, it was found that the level of the selenium-enriched bifidobacterium longum-treated group was decreased as compared with the model group mice; the same changes in the levels were also observed when serum creatinine was measured in mice. The pathological results and serum measurement results show that the selenium-enriched bifidobacterium longum can improve the kidney function damaged by diabetes.
(3) Western blotting experiment
Homogenizing the liver tissue of the mouse in a cell lysate, after cracking the liver tissue on ice for 30min, centrifuging the liver tissue at the rotating speed of 12000r/min for 10min at the temperature of 4 ℃, collecting the supernatant, detecting the protein concentration by using a BCA kit, adjusting the protein concentration to be consistent, adding a 5 × loading buffer, boiling the sample in a boiling water bath for 5min, taking 30g of total protein, carrying out SDS-PAGE electrophoresis, and transferring the total protein to a PVDF membrane. The membrane was washed with PBST after 5% skim milk for 1.5h at room temperature. Incubate with the following anti-dilutions of antibodies anti-GSK-3 β, anti-pGSK-3 β, anti-AKT, anti-pAKT and GAPDH (1: 1000) overnight at 4 ℃. After PBST membrane washing, the membrane is incubated for 0.5-1h at room temperature again by using a secondary antibody diluent (1: 5000), and after PBST membrane washing, ECL color development liquid is used for color development, and an automatic chemiluminescence analyzer (Tanon) is used for imaging (as shown in figure 5).
As can be seen from fig. 5, compared with the model group, the expression of pAKT in liver was significantly increased, and the expression of pGSK-3 β was significantly decreased; the increased expression level of the insulin signaling pathway-associated protein and the activation of the insulin signaling pathway indicate an increase in the sensitivity of hepatic insulin signaling following administration.
(4) H & E staining experiments
To observe morphological changes in liver, pancreas and kidney, the H & E staining procedure was as follows: liver, pancreas and kidney tissues were fixed in 4% paraformaldehyde overnight; routine paraffin embedding and 5-micron section; the slices were dewaxed conventionally with xylene and washed with various grades of ethanol to water. And (3) transparency: soaking xylene I and xylene II for 5min respectively, and dehydrating: anhydrous ethanol I, anhydrous ethanol II, 95% ethanol, 80% ethanol, and 75% ethanol, each for 5 min; after the hematoxylin is counterstained for 2min, the mixture is washed by running water and turned to blue, and is soaked in tap water for 15min or warm water (about 50 ℃) for 5 min; placing in eosin solution for 2min, and washing with tap water. And (3) dehydrating: 75% alcohol, 80% alcohol, 95% alcohol, anhydrous ethanol I, and anhydrous ethanol II each for 5 min. And (3) transparency: soaking xylene I and xylene II in xylene I and xylene II for 5min, and air drying for 3 min. Sealing: and (3) dropwise adding a neutral resin mounting sheet on the tissue sheet, then taking a clean cover glass, carefully adding the cover glass on the mounting medium, and slowly flattening to ensure that the position of the cover glass is moderate. Observed under a microscope, photographed and stored for a long period of time (see fig. 6 and 7).
As can be seen from FIG. 6, pathological changes of the selenium-enriched Bifidobacterium longum and the model group are significantly different as shown by pathological analysis of H & E staining results of the liver and the pancreas. As shown in fig. 6A, no pathological changes were evident in the normal group of mice; the islet cells of the model group had inflammatory cell infiltration; the low dose group was observed to contain a large amount of fat vacuoles in the pancreatic tissue, and as the dose of bifidobacterium longum rich in selenium was increased, the number of fat vacuoles in the pancreatic tissue was decreased. As shown in fig. 6B, there was a large amount of inflammatory cell infiltration of hepatocytes in the model group, and the degree of inflammatory cell infiltration gradually decreased with increasing dose of bifidobacterium longum rich in selenium. In conclusion, after administration of selenium-enriched bifidobacterium longum, the progression of pathological lesions of the liver and pancreas is slowed down.
As can be seen from FIG. 7, renal tissue examination showed that the glomerular cystic space in the kidney tissue of the model group disappeared, the glomerulus was adhered to the cystic wall, massive coagulative necrosis occurred in the glomerulus, and the red deposits in the glomerulus gradually decreased with the increase of the dose of Bifidobacterium longum rich in selenium.
The invention also tries to apply the conventional medicament or food preparation method and the medicament or food preparation method of the bifidobacterium, which are reported in the literature, to the selenium-rich bifidobacterium longum, and has the effects of better delaying the progress of diabetes, improving the physiological indexes and metabolic conditions of the diabetes, improving the insulin signal path of the diabetes, improving the pathological changes of the liver, pancreas and kidney of the diabetes, preventing diabetic kidney complications or renal function damage and improving the serum creatinine and the blood urea nitrogen of the diabetes; the invention also tries to combine the selenium-rich bifidobacterium longum with the conventional diabetes treatment or prevention medicine or food and the treatment method like the conventional diabetes treatment or prevention medicine or food, so that the diabetes treatment or prevention effect can be further improved.
The above-mentioned embodiments, objects, technical solutions and advantages of the present invention are further described in detail, it should be understood that the above-mentioned embodiments are only examples of the present invention, and should not be construed as limiting the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (6)

1. The application of selenium-enriched bifidobacterium longum is characterized by being applied to the preparation of a medicament for delaying the progress of diabetes; the diabetes delaying process is to improve physiological indexes and metabolic conditions of diabetes, wherein the physiological indexes and metabolic conditions of diabetes are fasting blood sugar, body weight, serum insulin level, glucose tolerance test, food intake, water intake and urine volume;
the preparation method of the selenium-enriched bifidobacterium longum comprises the following steps:
(1) dissolving sodium selenite in TPY culture medium to obtain selenium-containing TPY culture medium;
(2) mixing the bifidobacterium longum bacterial liquid with the TPY culture medium containing selenium, then inoculating the mixture into a new TPY culture medium, culturing the mixture in an incubator, and collecting the cultured bacterial liquid to obtain the selenium-rich bifidobacterium longum;
the concentration of sodium selenite in the selenium-containing TPY culture medium in the step (1) is 20-30 g/mL; in the step (2), the mixing ratio of the bifidobacterium longum bacterial liquid to the selenium-containing TPY culture medium is 1: 20-30; the culture conditions are as follows: under anaerobic conditions, the culture was carried out overnight in a 37 ℃ incubator until the OD600 reached 0.2.
2. The use of Bifidobacterium longum enriched in selenium as claimed in claim 1, in the manufacture of a medicament for improving insulin signaling pathway in diabetes models.
3. Use of selenium-enriched bifidobacterium longum according to claim 1 in the preparation of a medicament for ameliorating pathological changes in the liver, pancreas and kidneys of diabetes.
4. The use of Bifidobacterium longum enriched in selenium as claimed in claim 1 in the manufacture of a medicament for improving serum creatinine and blood urea nitrogen levels in diabetes.
5. The use of Bifidobacterium longum enriched in selenium according to claim 1 for the preparation of a medicament for the prevention of diabetic renal complications or renal impairment.
6. The use of Bifidobacterium longum enriched in selenium as claimed in claim 1, in the manufacture of a medicament for delaying the progression of diabetes for use in combination with conventional diabetes treating or preventing medicaments.
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