CN118291334A - Lactobacillus johnsonii for relieving hypercholesterolemia and application thereof - Google Patents
Lactobacillus johnsonii for relieving hypercholesterolemia and application thereof Download PDFInfo
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- CN118291334A CN118291334A CN202410489363.XA CN202410489363A CN118291334A CN 118291334 A CN118291334 A CN 118291334A CN 202410489363 A CN202410489363 A CN 202410489363A CN 118291334 A CN118291334 A CN 118291334A
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- 238000002525 ultrasonication Methods 0.000 description 1
- RUDATBOHQWOJDD-UZVSRGJWSA-N ursodeoxycholic acid Chemical compound C([C@H]1C[C@@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)CC1 RUDATBOHQWOJDD-UZVSRGJWSA-N 0.000 description 1
- 235000019786 weight gain Nutrition 0.000 description 1
- 235000013618 yogurt Nutrition 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention discloses lactobacillus johnsonii for relieving hypercholesterolemia and application thereof, and belongs to the technical field of microorganisms. The invention screens out a lactobacillus johnsonii CCFM1376, and the lactobacillus johnsonii CCFM1376 has the effect of relieving hypercholesterolemia and is specifically characterized in that: has high BSH activity; lowering serum TC, LDL-C concentration; increasing serum HDL-C concentration; lowering liver TC concentration; improving pathological conditions of the liver; regulating intestinal flora; regulating fecal bile acid composition; regulate the expression of key genes for cholesterol metabolism.
Description
Technical Field
The invention relates to lactobacillus johnsonii for relieving hypercholesterolemia and application thereof, and belongs to the technical field of microorganisms.
Background
Hypercholesterolemia (Hypercholesterolemia) is a complex metabolic disorder characterized by abnormal cholesterol levels in blood cells and plasma. Elevated blood cholesterol levels are closely related to increased risk of atherosclerosis and cardiovascular disease. Cardiovascular disease is a major cause of death in many developed and industrialized countries. Extensive random experiments with respect to reducing blood lipids have found that even a reduction of 1% of total serum cholesterol levels reduces the incidence of coronary artery disease by 2-3%. To date, drug therapy (e.g., statins) is the most widely used treatment for hypercholesterolemia. However, some of these drugs are expensive and have adverse side effects. Thus, probiotics should be studied as a safe and cost-effective alternative against cholesterol-related diseases.
The intestinal flora is closely related to hypercholesterolemia, and it is reported that disturbances in the composition and function of intestinal microorganisms play an extremely important role in the development of metabolic-change-related diseases. The probiotics can regulate intestinal flora, so that the probiotics are potentially applicable to relieving hypercholesterolemia.
Lactobacillus johnsonii is one of the genus lactobacillus. Lactobacillus has more than 150 species and subspecies composition, most of which are facultative aerobacteria, and only about 20% of the species are obligate aerobacteria. Wherein the lactobacillus includes Lactobacillus mucosa, lactobacillus delbrueckii, lactobacillus gasseri, lactobacillus fermentum, lactobacillus plantarum, lactobacillus rhamnosus, lactobacillus acidophilus, lactobacillus casei, lactobacillus crispatus, lactobacillus jensenii, lactobacillus salivarius, lactobacillus brevis, etc.
Because of the wide variety of lactobacillus, the same species of lactobacillus have obvious differences in aspects of morphology, physiology, metabolism, physiological functions and the like, and no patent has been found to be capable of relieving hypercholesterolemia by animal experiments so far. There is no study to identify the cause or mechanism of the discrepancy.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, and the lactobacillus johnsonii CCFM1376 for relieving hypercholesterolemia is obtained through in vivo animal experiment researches, has higher bile salt hydrolase activity, and has the effects of reducing serum TC and LDL-C concentration, increasing serum HDL-C concentration, reducing liver TC concentration, improving pathological conditions of liver, regulating intestinal flora, regulating bile acid composition, regulating expression of key genes of cholesterol metabolism and the like.
In order to achieve the above object, the present invention provides a lactobacillus johnsonii (Lactobacillus johnsonii) CCFM1376, which has been deposited with the collection of microorganism strains in the cantonese province at 2/2024 under the accession number GDMCC No:64367.
The strain of the invention is subjected to species identification by adopting the steps of bacterial genome DNA extraction, 16S rDNA specific primer PCR amplification, amplification product purification, DNA sequencing, sequence comparison and the like, is identified as lactobacillus johnsonii and is named as lactobacillus johnsonii CCFM1376.
In one embodiment, the lactobacillus johnsonii CCFM1376 has the following biological characteristics:
characteristics of the cells: is in a milky yellow color; colony characteristics: bacterial colonies on MRS solid plates are provided with milky yellow bulges, regular edges and gram positive bacteria; growth characteristics: culturing in MRS medium at 37deg.C under aerobic condition for about 16 hr to end of logarithm.
The invention also provides a method for preparing a medicine for preventing and/or treating hypercholesterolemia, which is to use the lactobacillus johnsonii CCFM1376.
In one embodiment of the present invention, the viable count of the Lactobacillus johnsonii CCFM1376 is not less than 10 9 CFU/g.
In one embodiment of the invention, the pharmaceutical product comprises lactobacillus johnsonii CCFM1376, a pharmaceutical carrier and/or a pharmaceutical adjuvant as described above.
The invention also provides a product containing the lactobacillus johnsonii CCFM 1376.
In one embodiment of the invention, the viable count of the lactobacillus johnsonii CCFM1376 in the product is not less than 10 9 CFU/g.
In one embodiment of the invention, the product comprises a food, pharmaceutical or nutraceutical.
In one embodiment of the invention, the food product comprises a health food product comprising the above lactobacillus johnsonii CCFM 1376; or the food product comprises a dairy product, a soy product, a meat product or a fruit and vegetable product produced using a starter culture comprising the lactobacillus johnsonii CCFM1376 described above.
In one embodiment of the invention, the preparation method of the starter comprises inoculating the lactobacillus johnsonii CCFM1376 into a culture medium according to an inoculum size accounting for 1-5% of the total mass of the culture medium, and culturing for 18h at 37 ℃ to obtain a culture solution; centrifuging the culture solution to obtain thalli; the thalli are resuspended by normal saline to obtain the ferment.
In one embodiment of the invention, the medium is an MRS medium.
The invention also provides a medicine for preventing and/or treating hypercholesterolemia, which contains the lactobacillus johnsonii CCFM1376.
In one embodiment of the present invention, the viable count of the Lactobacillus johnsonii CCFM1376 is not less than 10 9 CFU/g.
In one embodiment of the invention, the pharmaceutical product comprises lactobacillus johnsonii CCFM1376, a pharmaceutical carrier and/or a pharmaceutical adjuvant as described above.
In one embodiment of the invention, the preparation method of the starter comprises inoculating the lactobacillus johnsonii CCFM1376 into a culture medium according to an inoculum size accounting for 1-5% of the total mass of the culture medium, and culturing for 18h at 37 ℃ to obtain a culture solution; centrifuging the culture solution to obtain thalli; the thalli are resuspended by normal saline to obtain the ferment.
In one embodiment of the invention, the medium is an MRS medium.
Furthermore, the present invention provides the use of lactobacillus johnsonii CCFM1376 in a food additive, in particular as a food starter.
The invention also provides a method for degrading the combined bile acid, which comprises the step of adding the lactobacillus johnsonii CCFM1376 or the microbial agent into a system containing the combined bile acid for fermentation.
The beneficial effects are that:
The invention proves that lactobacillus johnsonii CCFM1376 can effectively reduce the total cholesterol and the low-density lipoprotein cholesterol content in serum of a mouse with hypercholesterolemia by 30.9 percent and 35.7 percent respectively compared with a molding; the high-density lipoprotein cholesterol content in serum is improved by 36.7 percent compared with a modeling module; reducing total cholesterol and low density lipoprotein cholesterol levels in the liver by 30.4% and 27.3% respectively compared to the molding; improving the diversity of the flora. The composition shows better treatment or prevention effect on the hypercholesterolemia, so that the composition can be used for preparing probiotic foods, health products and medicines for preventing and treating the hypercholesterolemia, and has very wide application prospects.
Preservation of biological materials
Lactobacillus johnsonii (Lactobacillus johnsonii) CCFM1376, taxonomic designation Lactobacillus johnsonii, deposited at the cantonese collection of microorganisms and cell cultures, month 2, 2024, accession number: building 5, accession number GDMCC No, from Guangzhou city martyr, university 100, no. 59: 64367.
Drawings
FIG. 1 shows total enzyme activities of bile salt hydrolase (Bile Salt Hydrolase, BSH) of each strain.
Figure 2 shows the body weight gain of each group of mice; in the figure: the letter differences above the histogram represent significant differences (p < 0.05).
FIG. 3 shows total cholesterol (total cholesterol, TC) concentrations in each group of sera; in the figure: the letter differences above the histogram represent significant differences (p < 0.05).
FIG. 4 shows the concentration of low density lipoprotein cholesterol (low density lipoproteins cholesterol, LDL-C) in each serum group; in the figure: the letter differences above the histogram represent significant differences (p < 0.05).
FIG. 5 shows the concentration of high density lipoprotein cholesterol (HIGH DENSITY lipoproteins cholesterol, HDL-C) in each serum group; in the figure: the letter differences above the histogram represent significant differences (p < 0.05).
FIG. 6 shows TC concentrations in each group of livers; in the figure: the letter differences above the histogram represent significant differences (p < 0.05).
FIG. 7 shows LDL-C concentration in each group of livers; in the figure: the letter differences above the histogram represent significant differences (p < 0.05).
FIG. 8 shows the H & E stained sections of each group of livers and scoring according to the SAF scoring system; in the figure: the letter differences above the histogram represent significant differences (p < 0.05).
FIG. 9 shows the variation of the Chao1 index of alpha diversity in the 16S rDNA sequencing of rat feces of each group; in the figure: the letter differences above the histogram represent significant differences (p < 0.05).
FIG. 10 shows the ratio of each group of conjugated bile acid to free bile acid.
FIG. 11 shows the expression level of the farnesoid X receptor (farnesoid X receptor, FXR) gene in each group relative to that in the blank group; in the figure: the letter differences above the histogram represent significant differences (p < 0.05).
FIG. 12 shows the expression levels of the respective constitutive fibroblast growth factor-15 (fibroblast growth factor, FGF 15) genes relative to the blank; in the figure: the letter differences above the histogram represent significant differences (p < 0.05).
FIG. 13 shows the expression levels of cholesterol 7α -hydroxylase (cholesterol 7-alpha hydroxylase, CYP7A 1) genes in each group relative to the blank group; in the figure: the letter differences above the histogram represent significant differences (p < 0.05).
Detailed Description
The invention will be better understood by the following examples.
In the present invention, "%" or percentage used to describe concentration or ratio is weight percent unless otherwise indicated.
The invention relates to the following media:
MRS liquid medium: 10g of tryptone, 10g of beef extract, 5g of yeast powder, 20g of glucose, 2g of diammonium hydrogen citrate, 5g of sodium acetate, 2g of dipotassium hydrogen phosphate, 0.5g of magnesium sulfate heptahydrate, 0.25g of manganese sulfate monohydrate, 1mL of Tween 80 and 1000mL of water.
MRS solid medium was obtained by adding 1.5% agar based on the total weight of the liquid medium.
Example 1: screening and cultivation of Lactobacillus johnsonii CCFM1376
1. Screening and identification of Lactobacillus johnsonii CCFM1376
Taking 1g of adult female feces sample obtained from Jin Changshi Yongchang county of Gansu province, coating the sample on an MRS solid culture medium after gradient dilution, culturing the sample in an aerobic environment at 37 ℃ for 72 hours, observing and recording colony morphology, picking out colony for streak purification, culturing the sample in an MRS liquid culture medium at 37 ℃ for 48 hours, carrying out gram staining on the obtained colony, recording strain morphology, discarding gram-negative strains and gram-positive cocci in the colony, selecting to obtain gram-positive bacillus, discarding the catalase-positive strain after catalase analysis, retaining the catalase-negative strain, detecting the strain by fructose-6-phosphate kinase to discard the negative strain, and identifying the obtained strain as about lactobacillus through 16SrDNA sequencing and named CCFM1376. Performing subculture on the lactobacillus johnsonii, collecting thalli, placing the thalli in a centrifuge tube, centrifuging at 3000rpm for 10min, washing, repeating for 3 times, adding the obtained thalli into a matrix protective agent, performing cryopreservation, and after 16SrDNA identification, preserving the strain CCFM1376 in the Guangdong province microorganism strain preservation center with the preservation number of GDMCC No:64367. another strain of Lactobacillus johnsonii screened in the same batch was designated QJSWX M160M 2.
16SrDNA amplification conditions: 95 ℃ for 5min;35 cycles (95 ℃ 30s,55 ℃ 30s,72 ℃ 2 min); 72 ℃ for 10min; amplification primers: 27F (5'-AGAGTTTGATCCTGGCTCAG-3'), 1492R (5'-TACGGCTACCTTGTTACGACTT-3') amplification product purification and sequence alignment were performed according to the methods described in document (Turroni F et al.Exploring the Diversity of the Bifidobacterial Population in the Human Intestinal Tract[J].Appl Environ Microb.2009;75(6):1534–45).
2. Lactobacillus johnsonii CCFM1376 has high bile salt hydrolase activity
Lactobacillus johnsonii CCFM1376 culture method
(1) And (3) activating and culturing:
and adopting MRS liquid culture medium, and standing and culturing in a common incubator at 37 ℃.
Culturing the target: the frozen and preserved thalli are selected, single colony is selected in liquid MRS liquid culture medium, and is subjected to static culture in a common incubator at 37 ℃ for about 24 hours, and lactobacillus johnsonii CCFM1376 is activated.
(2) Primary culture:
and adopting MRS liquid culture medium, and standing and culturing in a common incubator at 37 ℃.
Culturing the target: the activated lactobacillus johnsonii CCFM1376 was transferred to MRS broth at 2% inoculum size by volume of the broth for two generations.
(3) Secondary culture:
culture medium and culture conditions are the same-stage culture.
The first-order cultured Lactobacillus johnsonii CCFM1376 was transferred to 1L MRS liquid medium at an inoculum size of 2% by volume of the medium, and the culture was allowed to stand in a normal incubator at 37℃for about 24 hours to collect the cells. Washing with physiological saline twice, then re-suspending in 30% glycerol solution, and centrifuging to remove glycerol when the viable count is 1×10 10 CFU/mL and the subsequent test is used, washing with physiological saline once, and re-suspending in physiological saline.
The bacterial liquid activated to the secondary culture is inoculated into 20mL MRS culture medium according to the inoculation amount of 1 percent, and the bacterial liquid is subjected to static culture at 37 ℃ until the bacterial liquid reaches a stationary phase. The fermentation broth was subjected to centrifugation for 10 minutes at 10000 Xg at 4℃to collect cells. After that, the cells were washed and centrifuged twice with 0.1M phosphate buffer (pH 7.0). The bacterial concentration was adjusted so that the absorbance of the bacterial solution diluted 10 times was 1 at 600 nm. 1mL of the undiluted cell suspension was subjected to ultrasonication for 3 minutes (working time: batch time: 2:3). Then, the centrifugation was carried out again for 10 minutes at 10000 Xg at 4℃to remove cell debris, to obtain a cell-free extract.
0.1ML of the supernatant was added to 1.8mL of 0.1M phosphate buffer (pH 6.0) and 0.1mL of bound bile salt (200 mM), and the mixture was mixed and incubated at 37℃for 30 minutes at a final concentration of bound bile salt of 0.5 mmol/L. After 30 minutes, 0.5mL of the reaction solution was taken and added with 0.5mL of 15% triacetic acid solution (w/t) to terminate the reaction, and after uniform mixing, the mixture was centrifuged for 10 minutes at a maximum rotation speed of 4℃and the supernatant was taken. 0.1mL of the supernatant was mixed with 1.9mL of ninhydrin color solution, and the mixture was stirred and stirred evenly, and then the mixture was placed in a boiling water bath and boiled for 15 minutes. After cooling for 3 minutes, the absorbance at 570nm was determined.
The preparation method of the ninhydrin color solution comprises the following steps: 1.9mL of ninhydrin color solution included 0.5mL of 1% ninhydrin (dissolved in 0.5M citrate buffer (pH 5.5)), 1.2mL of glycerol, and 0.2mL of 0.5M citrate buffer (pH 5.5). Standard curves were made with glycine and taurine, respectively. Definition of total enzyme activity of BSH: the total enzyme activity (TA) of BSH is defined as: the amount of amino acid-producing substance per unit time, unit μmol (min mL) -1, per unit volume of crude enzyme hydrolyzes the bound bile salts. The calculation formula is as follows: wherein Caa represents the amino acid concentration.
TABLE 1 amino acid standard curve
BSH converts conjugated bile acids, taurodeoxycholic acid and glycodeoxycholic acid into free bile acids, changing the composition of bile acids, which are important substances in cholesterol metabolism. Thus BSH activity is an important criterion for screening probiotics for their ability to modulate cholesterol metabolism.
FIG. 1 shows that Lactobacillus johnsonii CCFM1376 has better BSH activity with total enzyme activities of 0.28. Mu. Mol mL -1min-1 and 0.30. Mu. Mol mL -1min-1 higher than 0.21. Mu. Mol mL -1min-1 and 0.29. Mu. Mol mL -1min-1 of Lactobacillus plantarum ST-III, respectively, using taurodeoxycholic acid and glycodeoxycholic acid as substrates.
Example 2: lactobacillus johnsonii CCFM1376 improves hypercholesterolemia mouse body weight
1. Experimental animal
Male SPF grade C57BL/6J mice were purchased from Experimental animal technologies Inc. of Beijing velari, china. Mice were housed in IVC cages, 4 mice per cage, food and water were contained in the cages, the temperature (22 ℃) was controlled, the relative humidity (50% + -10%), water was freely consumed, all mice were fed laboratory mouse growth propagation feed (Jiangsu province collaborative medical bioengineering Co., ltd., 20200215) during the adaptation period, and after the end of the adaptation period, the blank groups were fed with standard control feed, and the model building block and the experimental group were fed with Paigen high cholesterol model feed to the end of the experiment. Standard control feed (TP 23302), paigen feed (TP 28600, 15% fat, 1.25% cholesterol and 0.5% cholate) was purchased from south-pass terlafei feed technologies.
2. Experimental method
(1) Establishment of hypercholesterolemia mouse model
Male C57BL/6J mice at 4 weeks of age were randomly grouped and model hypercholesterolemia using Paigen feed after one week of adaptation.
(2) Experimental grouping and administration
Acquisition of lactobacillus johnsonii CCFM1376 bacterial suspension: the strain culture was performed by the method of example 1 to obtain a secondary culture broth. Bacteria were collected by centrifugation at 6000 Xg for 3min before use in lavage and then resuspended in an equal amount of sterile physiological saline.
Blank, model, lactobacillus johnsonii CCFM1376 treated and lactobacillus johnsonii QJSWX M2 groups of 8 mice each. Starting to irrigate the stomach from the end of the adaptation period, and irrigating the stomach once a day, wherein the gastric lavage dosage is 2x 10 9 CFU/day; the blank and model groups were perfused with the same volume of saline. All groups were sacrificed after gavage to 12 weeks of age and mouse blood and tissue were collected for detection.
Figure 2 shows that the model group significantly increased body weight (p < 0.05), increased by 7.08g, and the lactobacillus johnsonii CCFM1376 group mice increased by 4.24g compared to the blank group during the modeling period, significantly reduced hypercholesterolemia mice body weight (p < 0.05), whereas lactobacillus johnsonii QJSWX M2 had no effect.
Example 3: lactobacillus johnsonii CCFM1376 improves serum cholesterol in hypercholesterolemic mice
Strain culture and animal experiments are shown in examples 1 and 2.
The collected mouse blood is centrifuged to obtain upper serum, and the upper serum is detected by using TC and LDL-C, HDL-C detection kit.
The main symptom of hypercholesterolemia is an increase in serum cholesterol levels, leading to an increased risk of cardiovascular and cerebrovascular diseases. Multiple studies have shown that LDL-C is more likely to deposit on the inner wall of blood vessels, causing atherosclerosis. HDL-C can transport cholesterol in blood vessel to liver catabolism, can reduce cholesterol deposition on inner wall of blood vessel, and has anti-atherosclerosis effect.
FIG. 3 shows that the TC concentration in the serum of the model group was significantly increased to 7.13mmol/L (p < 0.05) compared to the blank group, and that Lactobacillus johnsonii CCFM1376 was able to significantly reduce the TC concentration in the serum to 4.93 (p < 0.05), whereas Lactobacillus johnsonii QJSWX M2 had no effect.
FIG. 4 shows that the LDL-C concentration of the model group serum was significantly increased to 3.13mmol/L (p < 0.05) compared to the blank group, and that Lactobacillus johnsonii CCFM1376 was able to significantly reduce the LDL-C concentration in the serum to 2.02mmol/L (p < 0.05), whereas Lactobacillus johnsonii QJSWX M2 did not.
FIG. 5 shows that Lactobacillus johnsonii CCFM1376 significantly increases HDL-C concentration in serum to 2.01mmol/L (p < 0.05) compared to the blank, with a significant decrease in HDL-C concentration in serum to 1.47mmol/L (p < 0.05) for the model group.
Example 4: lactobacillus johnsonii CCFM1376 improves liver cholesterol in cholesterolemia mice
Strain culture and animal experiments are shown in examples 1 and 2.
The collected livers of mice were homogenized and tested with TC and LDL-C test kits.
The liver is one of the important organs for regulating cholesterol level in the body, and long-term intake of high cholesterol foods can cause the liver to fail to normally metabolize, resulting in a higher concentration of cholesterol in the liver.
FIG. 6 shows that the model group liver TC concentration was significantly increased to 0.17mmol/L (p < 0.05), the Lactobacillus johnsonii CCFM1376 significantly reduced the serum TC concentration to 0.12mmol/L (p < 0.05), and the Lactobacillus johnsonii QJSWX M2 did not.
FIG. 7 shows that Lactobacillus johnsonii CCFM1376 can significantly reduce the LDL-C concentration in serum to 0.10mmol/L (p < 0.05) compared to the blank, with a significant increase in the LDL-C concentration in the model liver to 0.14mmol/L (p < 0.05).
Example 5: lactobacillus johnsonii CCFM1376 improves liver pathology in hypercholesterolemic mice
Strain culture and animal experiments are shown in examples 1 and 2.
During dissection, a portion of the liver was fixed using 4% neutral paraformaldehyde solution, the fixed liver was dehydrated, embedded, sectioned, and finally stained with hematoxylin and eosin stain (hematoxylin and eosin, H & E). Liver tissue was scored according to the SAF scoring system from three aspects, the scoring rules being as follows: liver cell adiposity: <5% >: 0min, 5% -33%:1 min, 34-66%:2 min, >66%:3 minutes; intralobular inflammation (20-fold mirror count necrotic lesions): the method is free of: 0 minutes, < 2:1 minute, 2-4: 2 minutes, > 4:3 minutes; hepatocyte balloon-like changes: the method is free of: 0 minutes, rare: 1 min, see: 2 minutes.
And observing whether the liver cell shape is complete, whether the nucleus is enlarged, whether the liver lobular shape is complete and the like through H & E sections of the liver. Integrated scoring assessment of liver injury and liver fibrosis can be performed simultaneously by SAF integration.
FIG. 8 shows that the degree of pathology in the liver of the model group was significantly increased compared to the blank group, with a pathology score of 6.5 points (p < 0.05), and that Lactobacillus johnsonii CCFM1376 reduced the degree of pathology in the liver, with a pathology score of 4.5 points, whereas Lactobacillus johnsonii QJSWX M2 did not.
Example 6: lactobacillus johnsonii CCFM1376 improves fecal flora diversity in hypercholesterolemic mice
Strain culture and animal experiments are shown in examples 1 and 2.
Mouse faeces were collected before sacrifice and stored in a-80 ℃ refrigerator for later experiments. After fecal DNA was extracted using a rapid DNA extraction kit, 16s rDNA sequencing was performed for analysis of changes in the flora.
Stabilization of intestinal micro-ecology is very important, and hypercholesterolemic patients are often accompanied by dysbacteriosis in the intestinal tract. Mainly manifested by a reduced diversity of intestinal flora. Restoring the homeostasis of intestinal flora can effectively relieve hypercholesterolemia. The ratio of the phylum firmicutes to the phylum bacteroides is considered as an important index for evaluating the risks of diabetes and obesity, and the increase of the ratio of the phylum firmicutes to the phylum bacteroides often has the risks of diseases such as obesity, metabolic disorder and the like.
FIG. 9 shows that the reduced flora diversity of the model group compared to the blank group, the presence of Lactobacillus johnsonii CCFM1376 significantly increased the flora diversity (p < 0.05), whereas the presence of Lactobacillus johnsonii QJSWX M160M 2 did not. Compared to the blank, the ratio of firmicutes/bacteroides in the model mice was significantly increased (p < 0.05), while the CCFM1376 significantly reduced the ratio of firmicutes/bacteroides in the mice (p < 0.05).
Example 7: lactobacillus johnsonii CCFM1376 for regulating composition of mouse fecal bile acid
Sample pretreatment method: the freeze-dried feces are precisely weighed into a centrifuge tube with a concentration of 20mg to 1.5mL, and 1mL chromatographic grade methanol is added for homogenization. Standing at room temperature for 1h, centrifuging (4deg.C, 12000 Xg, 15 min), collecting supernatant, adding 1mL of methanol to the precipitate, mixing, standing for 15min, centrifuging (4deg.C, 12000 Xg, 15 min), collecting supernatant, mixing with supernatant, and repeating the process. The supernatant was freeze-dried until no liquid was present, 1mL of chromatographic grade methanol was added, and after sufficient dissolution, the supernatant was centrifuged (4 ℃,12000 Xg, 15 min) and filtered using a 0.22 μm filter membrane, and then an appropriate amount of sample was taken and transferred into a sample bottle.
The bile acid content was determined using LC-MS. The detection conditions and program configuration are Thermo U3000, using ACQUITYHSS T3.8 μm (2.1X100 mM) column, automatic injector temperature 15 ℃, flow rate 0.30mL/min, column temperature 35 ℃, sample volume 2. Mu.L mode gradient elution, mobile phase 1mM ammonium acetate aqueous solution (A), 1mM ammonium acetate methanol solution (B). Gradient elution procedure 0min,20% b; 0-6 min, 20-60% B; 6-26 min,100% B; 26-28 min, 100-50% B; 28-30 min, 50-20% B; 30-32 min,20% B.
When the BSH activity in the intestinal tract is increased, more conjugated bile acid is converted into free bile acid, the proportion of the free bile acid is increased, and the free bile acid has stronger hydrophobicity and is not easy to be absorbed by the intestinal tract to be deposited along with excrement and discharged out of the body. Thus, the content of bile acid is lower, and the liver is promoted to synthesize cholesterol into bile acid so as to maintain the balance of the bile acid in the liver and intestine.
FIG. 10 shows that Lactobacillus johnsonii CCFM1376 increases the proportion of free bile acids in mouse fecal bile acids compared to the modeling module. Wherein the concentration of free bile acid beta-MCA, DCA, LCA, UDCA in the faeces of the mice of the group CCFM1376 of lactobacillus johnsonii is 0.76 mug/mg, 14.91 mug/mg, 0.0123 mug/mg and 0.0472 mug/mg respectively, which are significantly higher than 0.59 mug/mg, 10.47 mug/mg, 0.0077 mug/mg and 0.0130 mug/mg of the model building group.
Example 8: lactobacillus johnsonii CCFM1376 modulates FXR-FGF15 signalling pathway
Strain culture and animal experiments are shown in examples 1 and 2.
A portion of the liver was extracted with Trizol to obtain total RNA, which was reverse transcribed into cDNA and detected by quantitative PCR (qPCR).
FXR plays a key role in negative feedback regulation in the bile acid metabolic pathway, and its gene expression inhibits the conversion of liver cholesterol to bile acids. Bile acid synthase CYP7A1 is the rate-limiting enzyme for bile acid synthesis. Activation of FXR can negatively feedback inhibit transcription of bile acid synthase gene CYP7A1, affecting the rate of cholesterol synthesis of bile acid, so CYP7A1 is an important research target for FXR regulation pathway in cholesterol lowering research. Fibroblast growth factor (FGF 15) is a key signaling molecule for the ileal FXR regulatory pathway.
FIG. 11 shows that Lactobacillus johnsonii CCFM1376 significantly down-regulates the relative expression of FXR gene to 0.61 (p < 0.05) compared to the modeling group, whereas Lactobacillus johnsonii QJSWX160M2 does not.
FIG. 12 shows that Lactobacillus johnsonii CCFM1376 significantly down-regulates the relative expression of FGF15 gene to 0.76 (p < 0.05) compared to the modeling group, whereas Lactobacillus johnsonii QJSWX160M2 does not.
FIG. 13 shows that Lactobacillus johnsonii CCFM1376 significantly up-regulates the relative expression of the CYP7A1 gene to 1.34 (p < 0.05) compared to the modeling group, whereas Lactobacillus johnsonii QJSWX160M2 does not.
The experimental results show that the lactobacillus johnsonii CCFM1376 with higher BSH activity can relieve the hypercholesterolemia of mice, particularly reduce the concentration of serum TC and LDL-C, increase the concentration of serum HDL-C and reduce the concentration of liver TC, and has an improvement effect on the pathological conditions of the liver. In addition, the effect of alleviation is due to the regulation of intestinal flora diversity, the alteration of bile acid composition, and the regulation of cholesterol metabolism key gene expression.
In summary, lactobacillus johnsonii CCFM1376 can significantly improve hypercholesterolemia, and can be used for preparing medicines or health care products for preventing and treating high single ply, or producing foods beneficial to hypercholesterolemia, such as food additives for probiotic beverages, sour soybean milk, fermented jelly, fermented tea beverages or dairy products (such as yogurt, cheese products, lactic acid bacteria, milk powder) and the like.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. A strain of lactobacillus johnsonii (Lactobacillus johnsonii) CCFM1376, wherein the lactobacillus johnsonii (Lactobacillus johnsonii) has been deposited with the collection of microorganisms and cell cultures, cantonese province, at 2 nd month 2 of 2024 under accession number GDMCC No:64367, the preservation address is building 5 of road 100 university, no. 59 in Guangzhou city martyr.
2. A microbial agent comprising lactobacillus johnsonii CCFM1376 of claim 1.
3. Use of lactobacillus johnsonii CCFM1376 according to claim 1 for the manufacture of a medicament for the prevention and/or treatment of hypercholesterolemia.
4. The use according to claim 3, wherein the viable count of lactobacillus johnsonii CCFM1376 in the pharmaceutical product is not less than 10 9 CFU/g.
5. The use according to claim 3 or 4, wherein the medicament further comprises a pharmaceutical carrier and/or a pharmaceutical excipient.
6. A product comprising lactobacillus johnsonii CCFM1376 as claimed in claim 1.
7. The product of claim 6, wherein the viable count of lactobacillus johnsonii CCFM1376 of claim 1 is not less than 10 9 CFU/g.
8. The product of claim 6 or 7, wherein the product comprises a food or pharmaceutical product.
9. The product of claim 8, wherein the food product comprises a health food product; or the food comprises dairy products, bean products, meat products or fruit and vegetable products; the medicine also comprises a medicine carrier and/or a pharmaceutic adjuvant.
10. A method for degrading conjugated bile acid, wherein lactobacillus johnsonii CCFM1376 according to claim 1 or a microbial agent according to claim 2 is added to a system containing conjugated bile acid for fermentation.
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| CN202410489363.XA CN118291334A (en) | 2024-04-23 | 2024-04-23 | Lactobacillus johnsonii for relieving hypercholesterolemia and application thereof |
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