CN115125163B - Lactobacillus casei and application thereof - Google Patents
Lactobacillus casei and application thereof Download PDFInfo
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- CN115125163B CN115125163B CN202210553148.2A CN202210553148A CN115125163B CN 115125163 B CN115125163 B CN 115125163B CN 202210553148 A CN202210553148 A CN 202210553148A CN 115125163 B CN115125163 B CN 115125163B
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Abstract
The invention relates to the technical field of microorganisms, and discloses lactobacillus casei and application thereof. The lactobacillus casei provided by the invention has the characteristics of inhibiting the activity of alpha-glucosidase, resisting bile salts and gastrointestinal digestive enzymes, and still maintaining higher survival rate under acidic conditions. The lactobacillus casei has the effects of improving glucose tolerance, controlling liver index, controlling blood fat, reducing insulin resistance and the like, so that the lactobacillus casei, the microbial inoculum or the culture supernatant thereof can be used as a medicament for preventing and/or treating metabolic syndrome.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to lactobacillus casei and application thereof.
Background
With the improvement of living standard and the exploration and love of people on food, people currently ingest a large amount of high-sugar high-fat high-oil food, and long-term unhealthy dietary structure and dietary method can also cause the appearance of metabolic syndrome, and main diseases of the metabolic syndrome are obesity, hyperlipidemia, impaired glucose tolerance and the like.
Cardiovascular and cerebrovascular diseases, tumors, and diabetes are recognized as three fatal conditions worldwide. There are many obvious disadvantages of the current methods for diagnosing blood sugar, such as hyperglycemia, drug resistance, allergy, etc. caused by insulin therapy, and biguanide hypoglycemic agents and sulfonylurea hypoglycemic agents are not suitable for patients with severe liver and kidney failure and pregnant women. The transplantation of human islet cells and organs forms a huge rejection reaction, but genetic therapy cannot be applied due to the factors of high cost, limitation of technical means and the like.
The high cholesterol content in serum is considered to be the most important cause of severe hypertension, severe coronary heart disease and other cardiovascular diseases. Statistics indicate that cardiovascular disease is still an important factor in mortality, and the morbidity and mortality rate are still increasing. Therefore, reducing the serum cholesterol technical level creates a direct hazard to the health of people. At present, most of the diagnosis of cardiovascular and cerebrovascular diseases is carried out through drug diagnosis. However, drug therapy is expensive and has a great influence on side effects. Studies have shown that lowering blood lipid levels can significantly prevent death from cardiovascular disease.
At present, many studies report that probiotics have been used to improve metabolic diseases. Lactobacillus is the most predominant probiotic in humans, and thus there is still a need for more intensive research into the prevention and treatment of metabolic syndrome caused by lactobacillus.
Disclosure of Invention
The invention aims to overcome the problems in the prior art and provide lactobacillus casei and application thereof, wherein the lactobacillus casei has higher and stable inhibition rate on alpha-glucosidase and potential glucose control capability.
In order to achieve the above object, in one aspect, the present invention provides lactobacillus casei (Lactobacillus casei) with a preservation number of CGMCC No.24050.
In a second aspect the invention provides a microbial agent comprising lactobacillus casei as described above.
In a third aspect the invention provides the use of lactobacillus casei as described above or a bacterial agent as described above or a culture supernatant thereof in the preparation of a food additive.
In a fourth aspect the invention provides the use of lactobacillus casei as described above or a bacterial agent as described above or a culture supernatant thereof in the manufacture of a medicament for the prevention and/or treatment of metabolic syndrome.
In a fifth aspect the invention provides the use of lactobacillus casei as described above or a bacterial agent as described above or a culture supernatant thereof in the manufacture of a medicament for improving glucose tolerance;
and/or use of lactobacillus casei as described above or a bacterial agent as described above or a culture supernatant thereof in the manufacture of a medicament for controlling body weight;
and/or use of lactobacillus casei as described above or a bacterial agent as described above or a culture supernatant thereof in the manufacture of a medicament for controlling liver index;
and/or use of lactobacillus casei as described above or a microbial agent as described above or a culture supernatant thereof in the manufacture of a medicament for controlling epididymal fat weight;
and/or use of lactobacillus casei as described above or a bacterial agent as described above or a culture supernatant thereof in the manufacture of a medicament for controlling blood lipid;
and/or use of lactobacillus casei as described above or a bacterial agent as described above or a culture supernatant thereof for the manufacture of a medicament for controlling the hormone content in serum;
and/or use of lactobacillus casei as described hereinbefore or a bacterial agent as described hereinbefore or a culture supernatant thereof in the manufacture of a medicament for reducing insulin resistance and/or leptin resistance;
and/or use of lactobacillus casei as described hereinbefore or a bacterial agent as described hereinbefore or a culture supernatant thereof in the preparation of a medicament for reducing pancreatic tissue damage and/or liver tissue damage;
and/or use of lactobacillus casei as described hereinbefore or a bacterial agent as described hereinbefore or a culture supernatant thereof in the manufacture of a medicament for reducing fat cavitation in liver tissue;
and/or use of lactobacillus casei as hereinbefore described or a bacterial agent as hereinbefore described or a culture supernatant thereof in the manufacture of a medicament for reducing lipid accumulation in liver tissue;
and/or use of lactobacillus casei as described above or a bacterial agent as described above or a culture supernatant thereof in the manufacture of a medicament for promoting the production of short chain fatty acids;
and/or use of lactobacillus casei as described above or a bacterial agent as described above or a culture supernatant thereof for the manufacture of a medicament for modulating intestinal flora;
and/or use of lactobacillus casei as described above or a bacterial agent as described above or a culture supernatant thereof for the manufacture of a medicament for inhibiting alpha-glucosidase activity.
The lactobacillus casei provided by the invention has the characteristics of inhibiting the activity of alpha-glucosidase, resisting bile salts and gastrointestinal digestive enzymes, and still maintaining higher survival rate under acidic conditions. The lactobacillus casei has the effects of improving glucose tolerance, controlling liver index, controlling blood fat, reducing insulin resistance and the like, so that the lactobacillus casei, the microbial inoculum or the culture supernatant thereof can be used as a medicament for preventing and/or treating metabolic syndrome.
Drawings
FIG. 1 is a photograph of immunofluorescence staining of pancreatic tissue of each group of treated mice, blue labeled nuclei, green fluorescence labeled insulin, red fluorescence labeled glucagon.
FIG. 2 shows HE staining of liver tissue of mice treated in each group.
FIG. 3 is a graph of colony histogram at portal level of intestinal content of mice treated in each group, wherein CK is a blank control group, moxing is a model group, jun161 is a live bacterial group 161, shangqing161 is a supernatant group 161.
FIG. 4 is a thermal map of the Spearman-related classification of the genus level of intestinal contents of mice treated in each group, where CK is a blank group, moxing is a model group, jun161 is a live bacterial group No. 161, shangqing161 is a supernatant group No. 161.
FIG. 5 is a PIRUSt predicted three-level functional layer heat map of intestinal contents of mice treated in each group, wherein CK is a blank group, moxing is a model group, jun161 is a live bacterial group No. 161, shangqing161 is a supernatant group No. 161.
Preservation of organisms
The strain lactobacillus casei (Lactobacillus casei) is preserved in China general microbiological culture Collection center (address: national institute of microbiology: 100101) (CGMCC, abbreviation of preservation unit), with a preservation number of CGMCC No.24050, and abbreviation of 161, of China general microbiological culture Collection center (China general microbiological culture Collection center, address: north Chen West Lu No.1, beijing, chachiensis, korea, and the like) at 12 months 07 of 2021.
Detailed Description
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
The first aspect of the invention provides lactobacillus casei (Lactobacillus casei) with the preservation number of CGMCC No.24050.
The lactobacillus casei is obtained by collecting milk samples, water samples, soil and feed samples of Ningxia dairy plants, separating and screening.
The method for culturing lactobacillus casei provided by the invention is not particularly limited as long as the lactobacillus casei can be proliferated, and for example, the lactobacillus casei viable cells can be inoculated into a lactobacillus culture medium in an inoculum size of 1 to 5vol.% and cultured at a temperature of 30 to 40 ℃ for 9 to 24 hours to obtain a culture solution. The lactic acid bacteria culture medium may be various culture media suitable for lactobacillus casei culture, which are known in the art, and for example, may be MRS culture medium.
The method of the present invention is not particularly limited as long as the method is capable of enriching the cells from the culture solution, and the method may be carried out, for example, by centrifugation and/or filtration, and the conditions of the centrifugation and the filtration may be known conditions, and the present invention is not described in detail herein.
In a second aspect the invention provides a microbial agent comprising lactobacillus casei as described above.
In the present invention, the form of the microbial inoculum may be a form of microbial inoculum conventional in the art, for example, may be a solid, liquid or semisolid form.
In some embodiments of the invention, the bacterial agent contains viable cells of the lactobacillus casei.
The number of living cells in the microbial inoculum can be selected within a wide range, so long as the requirements of the relevant standards are met, for example, the number can be 10 9 cfu/g microbial inoculum above.
The preparation method of the microbial inoculum can refer to the conventional preparation method in the field, and is not described herein.
In a third aspect the invention provides the use of lactobacillus casei as described above or a bacterial agent as described above or a culture supernatant thereof in the preparation of a food additive.
In the present invention, the preparation method of the culture supernatant may be selected conventionally in the art, and may be, for example: inoculating the strain or microbial inoculum into a lactobacillus culture medium for culturing to obtain fermentation liquor, centrifuging the fermentation liquor to obtain fermentation supernatant, adjusting the pH value of the fermentation supernatant to 3-8, and filtering with a filter membrane to obtain culture supernatant. Wherein the filter membrane has a diameter of 0.20-0.50 μm, preferably 0.22 μm.
In a fourth aspect the invention provides the use of lactobacillus casei as described above or a bacterial agent as described above or a culture supernatant thereof in the manufacture of a medicament for the prevention and/or treatment of metabolic syndrome.
In the present invention, the preparation method of the culture supernatant may be selected conventionally in the art, and for example, may be the preparation method according to the third aspect of the present invention.
In some embodiments of the invention, the metabolic syndrome comprises at least one of obesity, diabetes, lipid metabolism disorder.
In a fifth aspect the invention provides the use of lactobacillus casei as described above or a bacterial agent as described above or a culture supernatant thereof in the manufacture of a medicament for improving glucose tolerance;
and/or use of lactobacillus casei as described above or a bacterial agent as described above or a culture supernatant thereof in the manufacture of a medicament for controlling body weight;
and/or use of lactobacillus casei as described above or a bacterial agent as described above or a culture supernatant thereof in the manufacture of a medicament for controlling liver index;
and/or use of lactobacillus casei as described above or a microbial agent as described above or a culture supernatant thereof in the manufacture of a medicament for controlling epididymal fat weight;
and/or use of lactobacillus casei as described above or a bacterial agent as described above or a culture supernatant thereof in the manufacture of a medicament for controlling blood lipid;
and/or use of lactobacillus casei as described above or a bacterial agent as described above or a culture supernatant thereof for the manufacture of a medicament for controlling the hormone content in serum;
and/or use of lactobacillus casei as described hereinbefore or a bacterial agent as described hereinbefore or a culture supernatant thereof in the manufacture of a medicament for reducing insulin resistance and/or leptin resistance;
and/or use of lactobacillus casei as described hereinbefore or a bacterial agent as described hereinbefore or a culture supernatant thereof in the preparation of a medicament for reducing pancreatic tissue damage and/or liver tissue damage;
and/or use of lactobacillus casei as described hereinbefore or a bacterial agent as described hereinbefore or a culture supernatant thereof in the manufacture of a medicament for reducing fat cavitation in liver tissue;
and/or use of lactobacillus casei as hereinbefore described or a bacterial agent as hereinbefore described or a culture supernatant thereof in the manufacture of a medicament for reducing lipid accumulation in liver tissue;
and/or use of lactobacillus casei as described above or a bacterial agent as described above or a culture supernatant thereof in the manufacture of a medicament for promoting the production of short chain fatty acids;
and/or use of lactobacillus casei as described above or a bacterial agent as described above or a culture supernatant thereof for the manufacture of a medicament for modulating intestinal flora;
and/or use of lactobacillus casei as described above or a bacterial agent as described above or a culture supernatant thereof for the manufacture of a medicament for inhibiting alpha-glucosidase activity.
In the present invention, the preparation method of the culture supernatant may be selected conventionally in the art, and for example, may be the preparation method according to the third aspect of the present invention.
In some embodiments of the invention, the use is in the manufacture of a medicament for the prevention and/or treatment of type II diabetes.
In some embodiments of the invention, the short chain fatty acid is at least one of acetic acid, propionic acid, butyric acid, isobutyric acid. Preferably, the short chain fatty acid is at least one of propionic acid and isobutyric acid.
In some embodiments of the invention, the hormone is at least one of insulin, leptin, adiponectin, glucagon-like peptide-1. In some embodiments of the invention, when the hormone in the serum is insulin and/or leptin, the use is in the manufacture of a medicament for reducing the amount of insulin and/or leptin in the serum. When the hormone in the serum is adiponectin and/or glucagon-like peptide-1, the use is in the manufacture of a medicament for increasing adiponectin and/or glucagon-like peptide-1 in the serum.
In some embodiments of the invention, the blood lipid is serum triglyceride and/or serum cholesterol.
Insulin resistance refers to the fact that the host's islet beta cells can normally secrete insulin, but because the immune receptor of the target organ is progressively less sensitive to insulin signaling molecules, this results in insulin not producing a normal acting effect, while insulin in the plasma continues to accumulate, i.e. hyperinsulinemia occurs. The adipocytes of the host are also capable of producing two important hormones associated with lipid metabolism, one of which is the leptin receptor, which interacts with the meal-inhibiting signaling molecule in the hypothalamus, thereby promoting reduced uptake and thus reduced lipid level reserves. Similarly to insulin resistance, host cells may also develop resistance to leptin receptors, but also appear as insensitivity of the recipient cells to signal molecules, allowing further accumulation of leptin receptors in the plasma, which may cause hyperleptinemia.
Studies have shown that after frequent ingestion of lactic acid bacteria, lactic acid bacteria and part of the dietary organic fibers and insoluble carbohydrates combine with fermentation in the large colon to produce short chain fatty acids. Short chain fatty acids are capable of not only or directly supplying energy (e.g., butyrate) to host cells, but also regulating metabolism (e.g., glucose homeostasis and dyslipidemia).
The present invention will be described in detail by examples. In the following examples, unless otherwise indicated, the methods are conventional in the art and the reagents are commercially available. MRS solid Medium (1L) containing calcium carbonate: 10g of peptone, 10g of beef extract, 5g of yeast extract and KH 2 PO 4 2g, trisodium citrate 2g, sodium acetate 2g, glucose 20g, tween 80 (Tween 80) 1mL, mgSO 4 ·7H 2 O 0.58g、MnSO 4 ·4H 2 0.25g of O and 20g of calcium carbonate. Adjusting pH to 6.5, and sterilizing at 121deg.C for 15min.
MRS liquid medium: 10g of peptone, 10g of beef extract, 5g of yeast extract and KH 2 PO 4 2g, trisodium citrate 2g, sodium acetate 2g, glucose 20g, tween 80 1mL, mgSO 4 ·7H 2 O 0.58g、MnSO 4 ·4H 2 O0.25 g. Adjusting pH to 6.5, and sterilizing at 121deg.C for 15min.
EDTA solution (containing 2g/100mL NaHCO) 3 ): 0.372g EDTA was dissolved in 1L deionized water, the pH of the solution was adjusted to 8.0, and NaHCO was added at 2g/100mL 3 。
PBS solution (1L): na (Na) 2 HPO 4 1.44g、KH 2 PO 4 0.24g、KCl 0.2g、NaCl 8g。
α -glucosidase, 4-nitrophenyl-D-glucopyranoside (PNPG): sigma in the united states.
Ox gall salt (product number: BCBX 0128), pepsin (product number: cat. No. P8160), trypsin (product number: cat. No. T8150).
Enzyme-linked immunosorbent assay kit for insulin (H203-1-2) and leptin (H174-1-2): nanjing builds the institute of bioengineering.
4% neutral paraformaldehyde: beijing Chang Hua Zhicheng technologies Co.
RNA Later: 700g of ammonium sulfate, 20mL of EDTA (0.5M, pH 7.0), 25mL of sodium citrate (1M) and diluted sulfuric acid after magnetic stirring and dissolution are used for adjusting the pH to 5.2.
955mL of ultrapure water, and magnetically stirring and dissolving the mixture, and adjusting the pH to 5.2 with dilute sulfuric acid.
High fat diet D12492, maintenance diet D12450B: research diabetes Inc. in the United states.
Streptozocin (STZ): sigma Co., USA.
Experimental strains: lactobacillus casei (Lactobacillus casei) is preserved in China general microbiological culture Collection center (address: north Silu No.1, 3 of the area of Chaoyang in Beijing, postal code: 100101) at about 12 months and 7 days in 2021.year, with a preservation number of CGMCC No.24050, 161.
Reference strain: bifidobacterium animalis A12 (collection number CGMCC No.17308, disclosed in Chinese patent CN 110604749A).
Bifidobacterium lactis BB-12 (commercial strain).
The data processing method comprises the following steps: statistical analysis of the test results was performed using SPSS software version 18.0, where the data comparison between groups used a one-way analysis of variance, where p <0.05 was expressed as significant difference between the data between groups.
Example 1
This example is used to demonstrate the in vitro α -glucosidase inhibitory effect of lactobacillus casei.
Preparation of culture supernatant: the strain was inoculated in an inoculum size of 2vol.% in MRS liquid medium, cultured at 37℃for 12 hours, three generations were activated, 8000g was centrifuged at 4℃for 10 minutes, the supernatant was collected, and NaOH (0.1 mmol/L) was adjusted to pH6.5,0.22 μm and filtered through a filter membrane.
The inhibition rate of the culture supernatant of Lactobacillus casei No. 161 on alpha-glucosidase was measured using Bifidobacterium animalis A12 (CGMCC No. 17308) as a positive control. Measurement method the alpha-glucosidase inhibition ratio of the culture supernatant was measured by the method of Li Tong (Li Tong. Prevention of mouse glycolipid metabolism disorder by breast-fed bifidobacterium infantis [ D ]. Beijing: beijing institute of agriculture, 2020.).
As a result, it was found that the inhibition ratio of alpha glucosidase of Lactobacillus casei No. 161 was 31.75.+ -. 1.22 (%).
Example 2
This example is used to illustrate the in vitro tolerability evaluation of Lactobacillus casei.
The in vitro tolerance evaluation of the strain was performed by the method of reference Li Tong (Li Tong. Prevention effect of bifidobacterium infantis on mouse glycolipid metabolism disorder [ D ]. Beijing: beijing institute of agriculture, 2020.). The results are shown in Table 1.
Table 1 Lactobacillus casei in vitro tolerating viable count (10) 9 CFU/mL)
Note that: the same letters indicate no significance under the same treatment conditions (p > 0.05)
As can be seen from table 1, lactobacillus casei No. 161 is resistant to bile salts and gastrointestinal digestive enzymes; in addition, the survival rate of the Lactobacillus casei No. 161 in the environment of pH2.5 after 2 hours is 72%, the survival rate of the Lactobacillus casei after 4 hours is 45%, but the viable count is still kept at 7.4X10 8 CFU/mL。
Example 3
This example is used to illustrate the mouse treatment, the level of glucose tolerance of Lactobacillus casei to mice, the effect of Lactobacillus casei on the weight of mice, and the test sample treatment.
1. Preparation of viable bacteria and supernatant
Lactobacillus casei No. 161 and BB-12 frozen at-80deg.C are activated in MRS liquid medium at 37deg.C for 12 hr, and subjected to three passages, and centrifuged at 8000g at 4deg.C for 10min to respectively retain thallus and fermentation supernatant.
Preparation of viable bacteria: the centrifuged thalli are washed three times by sterile PBS buffer solution,resuspension, adjusting cell concentration to 10 10 CFU/mL。
Preparation of supernatant (i.e., culture supernatant): the fermentation supernatant of the corresponding strain was adjusted to pH6.5 with 0.1mmol/LNaOH and filtered through a 0.22 μm filter.
2. Treatment of mice
SPF class C57/6J male mice 72, 3-5 weeks old purchased from Beijing Veitz Lihua laboratory animal technologies Co., ltd, were kept in Beijing academy of agricultural clean laboratory at 22 ℃ + -2 ℃ with 50% + -10% humidity and irradiated for 12h. All procedures followed the rules of the ethical committee of animals at the Beijing academy of agriculture. During the first week of the experiment, mice were acclimatized to eat and were free to drink.
Mice were randomly divided into 6 groups (n=12 per group) according to body weight, each group being treated as follows:
(1) Blank control group: feeding maintenance feed and 0.1mLPBS buffer solution for intragastric administration every day.
(2) Model group (mouse model of glycolipid metabolic disorder): high fat feed + daily lavage of 0.1ml fbs buffer was fed.
(3) 161 viable bacteria group: viable bacteria (10) of 0.1mL of Lactobacillus casei per day fed with high-fat feed and lavage 10 CFU/mL)。
(4) 161 supernatant group: feeding high-fat feed + 0.1mL of lactobacillus casei supernatant infused daily.
(5) Positive control group (BB-12 group): feeding high-fat feed and 0.1mL BB-12 viable bacteria (10) 10 CFU/mL)。
(6) Positive control drug group (acarbose drug group): high fat feed + acarbose solution at 20mg/kg/d per day of lavage.
At the 5 th, 6 th and 7 th weeks of the experiment, mice in other groups except the blank group were all injected with STZ intraperitoneally four times at a dose of 40mg/Kg, the blank group mice were injected with the corresponding buffer, no water was inhibited for 12 hours before the injection of STZ, the weight of the mice was weighed after the fasting, and the injected dose of each mouse was calculated at 40 mg/Kg. Mice were timely supplemented with glucose within two hours after STZ injection, and the glucose solution was changed to drinking water after two hours. At week 8 of the experiment, the mice of each group except the blank group were intraperitoneally injected with STZ at a dose of 80 mg/kg.
4. Blood glucose and oral glucose tolerance measurements
At the 13 th week of the experiment, the blood glucose value of the tail vein of the mouse is measured by adopting a blood glucose meter of a Qiangsheng company and matched blood glucose test paper according to an electrochemical principle. Oral glucose tolerance (Oral glucose tolerance test, OGTT): each group of mice is fasted without water control for 12 hours, the glucose solution is infused in 2.5g/kg body weight, the blood sugar level of the mice is measured at 0-2 hours after the glucose infusion, after blood is taken, the tail is wiped by alcohol to prevent infection, and experimental data are characterized by calculating the Area under the peak curve (AUC) by the glucose tolerance change curve.
The results showed that the glucose stimulation curve of the mice in the model group (i.e., AUC) was about 2400, the glucose stimulation curve of the mice in the blank group was about 900, the glucose stimulation curve of the live bacteria group 161 was about 1900, the glucose stimulation curve of the supernatant group 161 was about 1600, the glucose stimulation curve of the positive control BB-12 group was about 2250, and the glucose stimulation curve of the positive control drug group was about 1850. The test results show that the upper and lower range values of the glucose stimulation curves of the mice in the model group are significantly higher than those of the mice in other test groups (P < 0.05), which represents that the glucose tolerance capacity is significantly reduced, and the glucose tolerance capacity of the mice with live lactobacillus casei and lactobacillus casei supernatant is significantly improved.
4. Measurement of body weight and energy intake of mice
The mental state of the mice was observed periodically during the experiment, and the body weight and food intake were measured and recorded.
The weight gain of the model group is obviously higher than that of the other groups when the model group takes the high-fat feed for a long period, and the weight gain of each mouse is 40% of the weight of the mice when the model group feeds the mice for 12 weeks with high-fat feed. The weight gain of mice in the blank control group was 30%, the weight gain of mice in the positive control group was 30.5%, and the weight gain of mice in the positive drug control group was 33%. The weight gain of mice in the 161 live bacteria group and the 161 supernatant group respectively reaches 31 percent and 28 percent of the weight of the mice, wherein the weight gain of the mice in the 161 supernatant group is significantly lower than that of the mice in the model group (P < 0.05); co-intake of 4851kcal energy was significantly increased over the 12 week period of the high fat diet fed model group compared to the placebo group (P < 0.05), but there was no significant difference compared to the other experimental groups (P > 0.05).
5. Blood and tissue processing
After the experiment is finished at week 13, the mice are fasted for 12 hours before night, blood is taken from the anesthetized eyeballs, the blood is centrifuged for 10 minutes at 4 ℃ and 4000g, the upper transparent liquid is collected as serum, and the serum is frozen at-80 ℃ for later use.
The mice were sacrificed by cervical removal, and the liver, pancreas, epididymal fat, small intestine and colon tissues were rapidly removed, rinsed in normal saline, drained and weighed.
Fixing liver and pancreas tissue with 4% neutral paraformaldehyde solution, and preserving at normal temperature for pathological analysis; the rest tissues are quickly frozen and stored in liquid nitrogen to prevent degradation of RNA; the colon contents were collected into a stool collection tube, and the tube was packed with 0.3g of glass beads (diameter=0.3 cm) and 2mL of RNA later-protecting solution, and the tube was temporarily stored at 4℃to extract DNA.
Example 4
This example is used to illustrate the effect of Lactobacillus casei on the liver index and epididymal fat weight of mice.
Liver index was calculated from the liver weight obtained in example 3. The liver index of the mice in the model group is 0.049, the liver index of the mice in the blank control group is 0.031, the liver index of the positive control group is 0.047, and the liver index of the positive drug control group is 0.037; the model group showed significantly increased (P < 0.05) compared to the placebo group, indicating that liver damage may occur in mice with type two diabetes (model group) resulting in increased liver weight. The liver index of the 161 live bacteria group was 0.042, and the liver index of the 161 supernatant group was 0.048, wherein the liver index of the 161 live bacteria group was significantly reduced (P < 0.05) compared with the model group, and there was no significant difference from the blank group, which indicates that the 161 live bacteria group alleviated liver injury.
Analysis was performed based on epididymal fat weight obtained in example 3. Type II diabetes causes fat accumulation in the body, the weight of epididymal fat in a model group is 0.70g, the weight of epididymal fat in a blank group is 0.52g, the weight of epididymal fat in a positive control group is 0.45g, and the weight of epididymal fat in a positive medicine control group is 0.60g; the model group had significantly increased (P < 0.05) compared to the placebo group. The epididymal fat weights of the 161 live bacteria group and the 161 supernatant group are respectively 0.43g and 0.46g, which are obviously reduced compared with the model group (P < 0.05) and have no obvious difference from the blank group (P > 0.05), thus indicating that 161 lactobacillus casei relieves fat accumulation in mice.
Example 5
This example is used to illustrate the effect of Lactobacillus casei on biochemical indicators in mice.
The serum sample obtained in example 3 was measured using an ELISA kit for Nanjing's institute of biological engineering, and the detection procedure was performed according to the kit instructions.
(1) Triglyceride content
The triglyceride content of the model group is 1.00mol/L, the triglyceride content of the blank control group is 0.85mol/L, the triglyceride content of the positive control group is 0.48mol/L, the triglyceride content of the positive medicine control group is 0.65mol/L, the triglyceride content of the 161 # living bacteria group and the 161 # supernatant group are respectively 0.79mol/L and 0.56mol/L; triglyceride content of the 161 live bacteria group and 161 supernatant group was significantly reduced (P < 0.05) compared with the model group.
(2) Insulin content, leptin content
The insulin content in the serum of the mice in the model group is 2.05mIu/L, the insulin content in the serum of the mice in the blank control group is 0.62mIu/L, the insulin content in the serum of the mice in the positive control group is 0.67mIu/L, the insulin content in the serum of the mice in the positive medicine control group is 0.71mIu/L, and the insulin content in the serum of the mice in the 161 # supernatant group is 0.51mIu/L; the insulin content of the 161 # supernatant group was significantly reduced compared to the model group (P < 0.05).
The leptin content in the serum of the mice in the model group is 2.57ng/mL, the leptin content in the serum of the mice in the blank group is 1.29ng/mL, the leptin content in the serum of the mice in the positive control group is 1.51ng/mL, the leptin content in the serum of the mice in the positive drug control group is 1.22ng/mL, the leptin contents in the live bacteria group 161 and the supernatant group 161 are respectively 1.13ng/mL and 1.50ng/mL, and compared with the serum of the mice in the model group, the leptin content is obviously reduced (P < 0.05).
Example 7
This example is used to illustrate the effect of Lactobacillus casei on pancreatic tissue.
The pancreatic tissue sample sections obtained in example 3 were dewaxed, antigen repaired, serum blocked, primary antibody incubated overnight at 4 ℃, PBS washed three times, 5min each time, secondary antibody incubated at normal temperature for 50min each time, PBS washed three times, 5min each time, dapi nuclear staining for 8min, sudan black blocked for 5min, running water for 10min, anti-fluorescence quenching capper, observed under a fluorescence microscope and photographed. The results are shown in FIG. 1.
As shown in fig. 1, green fluorescence labeled insulin, red fluorescence labeled glucagon, blue label nuclei. In fig. 1, the nuclei (blue), insulin (green), glucagon (red) and fusion results are shown in order from left to right. Compared with the blank control group, islets in the model group were smaller, while islets in the 161 # supernatant group were larger, and the boundaries were clear.
Example 8
This example is used to illustrate the effect of Lactobacillus casei on liver tissue.
Liver tissue obtained in example 3 was completely immersed in 4% neutral paraformaldehyde solution for fixation overnight, paraffin-embedded, sectioned, HE stained, and tissue sections were observed under a 400-fold light microscope. The results are shown in FIG. 2.
As shown in fig. 2, the liver cells of mice in the blank group have no abnormal changes such as steatosis, the liver histological structure is clear and neat, the liver lobule structure is normal, the cell boundary is obvious, and the cell nucleus is positioned in the middle. Abnormal accumulation of lipids, vesicular lipid degeneration of liver tissues, increased lipid concentration of liver tissues, and generation of a large number of fatty vacuoles occurred in the liver of mice in the model group. After 161 viable bacteria and 161 supernatant intervention, the degree of hepatic tissue steatosis is obviously reduced compared with that of a model group, and the hepatic cell fat vacuoles are smaller and less, and the morphology of the hepatic cell fat vacuoles is similar to that of a blank control group. In summary, lactobacillus casei No. 161 can prevent the formation of fatty liver and the impairment of liver function in mice.
Example 9
This example is intended to illustrate the effect of Lactobacillus casei on short chain fatty acid synthesis.
Short chain fatty acids in the colon content (i.e., stool) of the mice of example 3 were extracted and measured by reference to the experimental procedure of Goossens et al (Chen P, zhang Q, dang H, et al screening for potential new probiotic based on probiotic properties and a-glucosidase inhibitory activity [ J ]. Food Control,2014, 35:65-72.).
The result shows that the propionic acid content in the mouse faeces of the model group is 75ng/mg, the propionic acid content in the mouse faeces of the blank control group is 45ng/mg, the propionic acid content in the mouse faeces of the positive control group is 160ng/mg, the propionic acid content in the mouse faeces of the positive medicine control group is 145ng/mg, and the propionic acid content in the mouse faeces of the 161 # supernatant group is 131ng/mg, wherein the propionic acid content in the mouse faeces of the 161 # supernatant group is obviously higher than that of the model group (p < 0.05);
the content of isobutyric acid in the mouse feces of the model group is 6ng/mg, the content of isobutyric acid in the mouse feces of the blank control group is 16.5ng/mg, the content of isobutyric acid in the mouse feces of the positive control group is 22ng/mg, the content of isobutyric acid in the mouse feces of the positive drug control group is 22.5ng/mg, the content of isobutyric acid in the mouse feces of the 161 # supernatant group is 17ng/mg, wherein the content of isobutyric acid in the mouse feces of the 161 # supernatant group is significantly higher than that of the model group (p < 0.05);
example 10
This example is intended to illustrate the effect of Lactobacillus casei on the intestinal flora of mice
The intestinal contents (i.e. colon contents) of the mice obtained in the example 3 were subjected to high-throughput sequencing by the Meji biological company, and the 16SrDNA sequence was divided into a conserved region and a variable region, wherein the conserved region is common to all bacteria, and the bacteria have no difference, so that the relationship among the bacteria can be reflected; the variable region has genus or species specificity, has certain difference along with the difference of the genetic relationship among bacteria, can reveal the characteristic nucleic acid sequence of biological species, and can be used as an index for classifying and identifying bacteria.
The metagenomic method was used to analyze changes between bacterial composition and diversity in mouse feces.
Colony pillars on the gate level are shown in fig. 3, and the gates with the content of 1% or more on the gate level in each experimental group are respectively: bacterioides (bacteroides), firmicutes (Firmicutes), proteobacteria (proteobacteria), desulfobacteria (Desulfobacteria), actinomycetes (actinomycetes), verrucom microbiota (wart microbiota). Wherein the dominant bacterial groups are bacteria and Firmicutes. In recent years, researches show that short-chain fatty acids mainly comprise acetic acid, propionic acid and butyric acid, and account for more than 90% of the total amount of the short-chain fatty acids, and have very important effects on human health. Propionic acid is the main product of the fermentation of bacterioides, butyric acid is produced mainly by firmates metabolism. There was a tendency for the specific gravity of bacterioides and firmics in the 161 # supernatant group to decrease compared to the model group. Scientific studies have demonstrated that an increase in the ratio of bacteroides to firmicutes is related to energy extraction from food, which also suggests that these two major mycorrhizas are critical in regulating obesity. Therefore, the Lactobacillus casei 161 can play a remarkable role in reducing fat by adjusting the ratio between the bacteroides and the firmicutes in the small intestine so as to reduce the intake of dietary energy.
To study the relationship between changes in the intestinal flora, a Spearman related class of thermal profile was constructed at a subordinate level (fig. 4), and it was found that the colony structure of the intestinal bacteria in the 161 live bacterial group and the 161 supernatant group was significantly changed compared to the blank group, and that the colony structures of these 2 groups had a certain similarity.
To study the function of the dominant microbial flora in the viable and supernatant groups of lactobacillus casei No. 161, PICRUSt function prediction and analysis were performed on the high throughput sequencing results. The results show (figure 5) that during the modeling process, the flora regulating and controlling the beta-galactosidase, the DNA polymerase, the deoxyribonuclease, the helicase and the cellobiase are more active, compared with the model group, obvious changes occur, the abundance of the enzymes is increased, and the synthesis pathways of galactose metabolism, other polysaccharide degradation, starch and sucrose metabolism, metabolic pathways and secondary metabolites are regulated and controlled.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, a number of simple variants of the technical solution of the invention are possible, including combinations of the individual technical features in any other suitable way, which simple variants and combinations should likewise be regarded as being disclosed by the invention, all falling within the scope of protection of the invention.
Claims (7)
1. Lactobacillus casei (Lactobacillus casei), which is characterized in that the preservation number of the lactobacillus casei is CGMCC No.24050.
2. A microbial inoculum comprising the Lactobacillus casei as claimed in claim 1.
3. The microbial inoculum according to claim 2, wherein the microbial inoculum contains viable cells of the lactobacillus casei.
4. Use of the lactobacillus casei of claim 1 or the microbial agent of claim 2 or 3 in the preparation of a food additive.
5. Use of lactobacillus casei as claimed in claim 1 or a microbial agent as claimed in claim 2 or 3 in the manufacture of a medicament for the prevention and/or treatment of metabolic syndrome selected from at least one of obesity, diabetes, lipid metabolism disorders.
6. Use of the lactobacillus casei of claim 1 or the microbial agent of claim 2 or 3 in the manufacture of a medicament for improving glucose tolerance;
and/or the use of the lactobacillus casei as claimed in claim 1 or the microbial agent as claimed in claim 2 or 3 in the manufacture of a medicament for controlling body weight;
and/or the use of the lactobacillus casei as claimed in claim 1 or the microbial agent as claimed in claim 2 or 3 in the manufacture of a medicament for controlling liver index;
and/or the use of lactobacillus casei as claimed in claim 1 or a microbial agent as claimed in claim 2 or 3 in the manufacture of a medicament for controlling epididymal fat weight;
and/or the use of the lactobacillus casei as claimed in claim 1 or the microbial inoculum as claimed in claim 2 or 3 in the manufacture of a medicament for controlling blood lipid;
and/or the use of lactobacillus casei as claimed in claim 1 or a microbial agent as claimed in claim 2 or 3 in the manufacture of a medicament for controlling the amount of a hormone in serum, said hormone being insulin and/or leptin;
and/or the use of the lactobacillus casei as claimed in claim 1 or the microbial agent as claimed in claim 2 or 3 in the manufacture of a medicament for reducing insulin resistance and/or leptin resistance;
and/or use of the lactobacillus casei as claimed in claim 1 or the microbial agent as claimed in claim 2 or 3 in the manufacture of a medicament for reducing pancreatic tissue damage and/or liver tissue damage;
and/or use of the lactobacillus casei as claimed in claim 1 or the microbial agent as claimed in claim 2 or 3 in the manufacture of a medicament for reducing fat cavitation in liver tissue;
and/or use of the lactobacillus casei as claimed in claim 1 or the microbial agent as claimed in claim 2 or 3 in the manufacture of a medicament for reducing lipid accumulation in liver tissue;
and/or the use of the lactobacillus casei as claimed in claim 1 or the microbial agent as claimed in claim 2 or 3 in the manufacture of a medicament for promoting the production of short chain fatty acids, which are propionic acid and/or isobutyric acid;
and/or the use of the lactobacillus casei as claimed in claim 1 or the microbial agent as claimed in claim 2 or 3 in the manufacture of a medicament for modulating intestinal flora;
and/or the use of the lactobacillus casei as claimed in claim 1 or the microbial agent as claimed in claim 2 or 3 in the manufacture of a medicament for inhibiting alpha-glucosidase activity.
7. The use according to claim 6, wherein the use is in the manufacture of a medicament for the prevention and/or treatment of type two diabetes.
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