CN114437968B - Lactobacillus acidophilus NX2-6 with obesity relieving function and application thereof - Google Patents
Lactobacillus acidophilus NX2-6 with obesity relieving function and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
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- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/04—Anorexiants; Antiobesity agents
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
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- A—HUMAN NECESSITIES
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Abstract
The invention provides lactobacillus acidophilus NX2-6 with an obesity relieving function and application thereof, and belongs to the field of food microorganisms. The strain NX2-6 is named after classification: lactobacillus acidophilus (Lactobacillus acidophilus) is preserved in China general microbiological culture collection center CGMCC with a preservation number of CGMCC NO.17311. The strain can effectively reduce the weight, liver and fat of a high-fat diet mouse, improve lipid metabolism in adipose tissues, inhibit accumulation of triglyceride, and enhance the function of mitochondria in adipose tissues; and effectively improves inflammatory response in mice. In addition, the strain also has the capability of optimizing the intestinal flora structure and has good probiotic function. Can be used for relieving obesity caused by high fat diet, or improving lipid metabolism and energy metabolism in adipose tissue. Or in inhibiting metabolic inflammation and regulating intestinal flora structure.
Description
1. Technical field
The invention relates to lactobacillus acidophilus NX2-6 with an obesity relieving function and application thereof, belonging to the field of food microorganisms.
2. Background art
Over the past few decades, there has been a proliferation of obese patients due to unreasonable dietary structure and lifestyle changes, and a serious public health problem worldwide. In addition, the occurrence of obesity induces other diseases such as non-alcoholic fatty liver, type II diabetes, hypertension, cardiovascular diseases, etc., and thus, obesity is seriously harmful to human health. Numerous studies have shown that the occurrence of obesity is associated with a disturbance of the structure of the intestinal flora in the body, manifested by a decrease in the diversity of the flora and the proportion of symbiotic bacteria, and an increase in the number of conditionally pathogenic bacteria.
At present, dietary intervention for obesity mainly comprises ingestion of prebiotics and probiotics, wherein the prebiotics comprise dietary fibers, polyphenols, oligosaccharides and the like, and the prebiotics and the probiotics can improve the intestinal flora structure, so that the disturbance of a micro-ecological system is effectively inhibited. However, dietary fiber and oligosaccharide taste and polyphenol absorption and processing techniques limit their development. Along with the growth of the fermentation industry in China, the variety and the quantity of products containing probiotics are increased, and the probiotics can colonise the intestinal tracts of human bodies, have the excellent characteristics of synthesizing essential vitamins, regulating an immune system, improving the intestinal flora structure, relieving the occurrence of metabolic diseases and the like, and are also gradually valued and utilized by vast consumers. Therefore, screening probiotics having a function of inhibiting lipid metabolism disorders is of great importance for the development of functional foods.
3. Summary of the invention
Technical problem
The invention aims to provide lactobacillus acidophilus NX2-6 with the function of relieving obesity caused by high-fat diet.
Technical proposal
The lactobacillus acidophilus NX2-6 with the function of relieving obesity provided by the invention has the classification and naming of the strain NX 2-6: lactobacillus acidophilus (Lactobacillus acidophilus), which has been deposited in China general microbiological culture collection center (CGMCC) 17311 in 2019, 03 and 07.
The application of the lactobacillus acidophilus NX2-6 refers to the application of the lactobacillus acidophilus NX2-6 in relieving obesity caused by high-fat diet. In particular to application of lactobacillus acidophilus NX2-6 in improving lipid metabolism and energy metabolism in adipose tissues. Or lactobacillus acidophilus NX2-6 in inhibiting metabolic inflammation. Can also refer to the application of lactobacillus acidophilus NX2-6 in the aspect of regulating the intestinal flora structure.
Advantageous effects
(1) The lactobacillus acidophilus NX2-6 provided by the invention can obviously improve lipid metabolism, effectively relieve obesity, and can be applied to improving lipid metabolism in adipose tissues.
Animal experiments prove that the lactobacillus acidophilus NX2-6 can effectively reduce the weight of obese mice, and simultaneously reduce the weight of epididymis adipose tissues and livers. Lactobacillus acidophilus NX2-6 reduced the weight of mice, epididymal adipose tissue and liver by approximately 14.58%, 16.73% and 46.00%, respectively. Apparent, lactobacillus acidophilus NX2-6 is capable of inhibiting abnormal accumulation of fat in mice.
Animal experiments prove that lactobacillus acidophilus NX2-6 can improve lipid metabolism disorder in serum and liver. After the mice ingest lactobacillus acidophilus NX2-6, the content of triglyceride, ALT activity and leptin in serum are respectively reduced by 30.55%, 34.18% and 45.66%; the triglyceride in the liver is reduced by 20.77%, so that lactobacillus acidophilus NX2-6 can reduce the blood fat of mice and inhibit the accumulation of fat in the livers of the mice.
Animal experiments prove that lactobacillus acidophilus NX2-6 promotes triglyceride decomposition and fatty acid oxidation in adipose tissues, and simultaneously inhibits synthesis of fatty acid from the head, synthesis of triglyceride and differentiation of fat cells. Rt-PCR analysis results show that after lactobacillus acidophilus NX2-6 is ingested, the expression level of a gene HSL for regulating triglyceride decomposition in the epididymal adipose tissue of the mice is increased by 1.79 times; the expression level of the genes PPAR-alpha and CPT2 for regulating and controlling the fatty acid oxidation reaction is respectively improved by 1.95 and 0.80 times; the expression level of genes FAS and Srebp-1c for regulating and controlling the fatty acid synthesis reaction is respectively reduced by 1.41 and 2.56 times; the expression level of the gene DGAT1 for regulating and controlling the triglyceride synthesis reaction is reduced by 1.08 times; the expression level of the gene C/EBP alpha for regulating the differentiation reaction of the fat cells is reduced by 0.73 times, and the results fully demonstrate that the lactobacillus acidophilus NX2-6 can directly regulate the genes related to lipid metabolism in fat tissues, thereby inhibiting fat accumulation.
(2) The lactobacillus acidophilus NX2-6 provided by the invention can effectively enhance the function of mitochondria in epididymal adipose tissue, and can be applied to improving energy metabolism in adipose tissue.
Animal experiments prove that lactobacillus acidophilus NX2-6 promotes mitochondrial synthesis in adipose tissue. The Rt-PCR analysis result shows that after the lactobacillus acidophilus NX2-6 is ingested, the expression quantity of mitochondrial biosynthesis related genes in the adipose tissues of mice, such as PGC-1 beta and SIRT1, are respectively up-regulated by 0.41 times and 0.43 times, the gene expression quantity of mitochondrial biosynthesis related regulatory factors NRF1 and TFAM is increased by 0.99 times and 1.36 times, the expression quantity of mitochondrial fusion related gene MFN2 is increased by 0.43 times, and the expression quantity of mitochondrial division related genes Fis1 and Drp1 is respectively increased by 1.03 times and 0.85 times, which fully prove that the lactobacillus acidophilus NX2-6 can directly regulate mitochondrial biosynthesis related genes in the adipose tissues, thereby improving energy metabolism.
(3) The lactobacillus acidophilus NX2-6 provided by the invention can effectively inhibit the generation of endotoxin, inhibit inflammatory reactions in serum, liver, adipose tissues and colon, and can be applied to inhibiting metabolic inflammation.
Animal experiments prove that lactobacillus acidophilus NX2-6 can inhibit metabolic inflammatory reaction. After ingestion of lactobacillus acidophilus NX2-6, the concentrations of endotoxin, TNF- α and IL-1β in the serum of mice decreased by 29.41%, 28.29% and 33.60%, respectively; meanwhile, lactobacillus acidophilus NX2-6 can also inhibit the gene expression quantity of TNF-alpha and IL-1 beta in colon and liver, and obviously up-regulate the gene expression quantity of the close-coupled proteins ZO-1, occludin and Claudin-1 in colon, and the results indicate that lactobacillus acidophilus NX2-6 can inhibit metabolic inflammatory reaction and enhance intestinal barrier.
(4) The lactobacillus acidophilus NX2-6 provided by the invention obviously optimizes the intestinal flora structure, thereby improving the intestinal microecological system and being applied to the aspect of adjusting the intestinal flora structure.
Animal experiments prove that lactobacillus acidophilus NX2-6 can effectively increase the diversity of intestinal flora and the richness of symbiotic bacteria, and simultaneously reduce the proportion of conditional pathogenic bacteria. Intestinal tract microbial sequencing results show that after ingestion by lactobacillus acidophilus NX2-6, the relative abundance of proteus, vibrio, alistipes, vibrio, helicobacter and mucispirilum in the colon of the mice is reduced by 61.71%, 350.80%, 524.99%, 487.47%, 100.61% and 193.32%, respectively; the abundance of symbiotic bacteria of the phylum bacteroides, the phylum verrucomicrobia, the phylum S24-7, the family of the lactobacillus, the genus lactobacillus and the genus parabacteroides is respectively improved by 43.99%, 435.09%, 190.47%, 395.45%, 146.21% and 164.55%, which shows that lactobacillus acidophilus NX2-6 can regulate the structure of the flora in the colon.
4. Description of the drawings
FIG. 1 shows the feed intake, water intake, body weight, liver index and epididymal adipose tissue weight of each group of mice;
FIG. 2 shows triglyceride, leptin, adiponectin, glutamic pyruvic transaminase, glutamic oxaloacetic transaminase, high density lipoprotein cholesterol, and low density lipoprotein cholesterol levels in serum from various groups of mice;
FIG. 3 shows the levels of triglyceride, high density lipoprotein cholesterol, low density lipoprotein cholesterol, superoxide dismutase, catalase, and lactate dehydrogenase in the liver of each group of mice;
FIG. 4 is a slice view of epididymal adipose tissue, colon and liver of each group of mice;
FIG. 5 shows the expression level of lipid metabolism-related genes in epididymal adipose tissues of each group of mice;
FIG. 6 shows the expression level of mitochondrial function related genes in epididymal adipose tissues of each group of mice;
FIG. 7 shows the in vivo immune related parameters of each group of mice;
FIG. 8 shows the analysis of the intestinal flora Alpha diversity of mice in each group;
FIG. 9 is an analysis of intestinal flora at the portal level for each group of mice;
FIG. 10 is an analysis of intestinal microbiota at the scientific level for each group of mice;
FIG. 11 is an analysis of intestinal flora at the genus level for each group of mice.
Preservation of organisms
Strains NX2-6, classified and named: lactobacillus acidophilus (Lactobacillus acidophilus), deposited with the chinese common microbiological bacterial deposit management center, address: the collection number of the microbiological institute of China academy of sciences is CGMCC NO.17311.
5. Detailed description of the preferred embodiments
The lactobacillus acidophilus NX2-6 is derived from the self-made mare's milk of the inner Mongolian herder and then stored in the glycerol pipe.
1. Activation and culture of Lactobacillus acidophilus NX2-6
Activating lactobacillus acidophilus NX2-6 stored in an glycerol pipe with an MRS liquid culture medium for 3 generations, coating bacterial liquid on the MRS solid culture medium, culturing for 24 hours, and then picking single bacterial colony to the liquid culture medium for culturing.
2. Preparation and test design of lactobacillus acidophilus NX2-6 bacterial suspension
Centrifuging cultured Lactobacillus acidophilus NX2-6 (8000 rpm, 10 min), discarding supernatant, washing with 0.1mol/L sterilized PBS buffer (pH=7.2) three times, and adjusting the concentration of Lactobacillus acidophilus NX2-6 to 10 with the buffer 9 cfu/mL。
Animals were tested using 4 week old mice without specific pathogen levels, and after one week adaptation, normal mice were fed with normal feed and model and sample mice were fed with high fat feed. Mice in the sample group were perfused daily with 10 9 Lactobacillus acidophilus NX2-6 of cfu, and simultaneously, the normal group and model group mice were perfused with an equal volume of sterile physiological saline, and were co-perfused with the stomach for 8 weeks, during which time the food intake and water intake of the mice were recorded, allThe test was repeated three times, 10 in parallel per group.
3. Influence of Lactobacillus acidophilus NX2-6 on mouse body weight and organs
The body weight of each mouse was recorded, and the weight of liver and epididymal fat was weighed, as shown in FIG. 1, and after eating Lactobacillus acidophilus NX2-6, the body weight of each mouse was reduced from 43.30 + -2.66 g to 36.99 + -2.11 g, the liver index was reduced from 46.13 + -7.88 mg/g to 38.41 + -2.17 mg/g, and the epididymal fat tissue weight was reduced from 1.58+ -0.58 g to 0.96+ -0.37 g, indicating that Lactobacillus acidophilus NX2-6 has the effect of inhibiting fat accumulation.
4. Influence of Lactobacillus acidophilus NX2-6 on parameters related to lipid metabolism in mouse serum
Whole blood from mice was collected using a sterile centrifuge tube, placed on ice, and centrifuged at 4℃at 5000 rpm for 10 minutes, the pale yellow liquid was serum, which was then drawn into another sterile centrifuge tube and stored at 4℃for further use, using the method of Zhu et al (Zhu et al, food function, 2018,9,3509-3522). The content of triglyceride, leptin, adiponectin, glutamic pyruvic transaminase (ALT), glutamic oxaloacetic transaminase (AST), high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C) in serum was measured with the kit, and as a result, as shown in FIG. 2, the concentration of triglyceride in serum of mice was reduced from 2.28.+ -. 0.34mmol/L to 1.58.+ -. 0.58mmol/L, the concentration of leptin was reduced from 24.93.+ -. 9.72ng/mL to 16.41.+ -. 4.29ng/mL, the activity of ALT was reduced from 14.59.+ -. 2.93U/L to 7.93.+ -. 1.86U/L, and the concentration of HDL-C was reduced from 4.57.+ -. 0.71mmol/L to 3.51.+ -. 0.84mmol/L, without significant change in the levels of adiponectin, AST and LDL-C, as compared with the mice of the high-fat model group. Thus, lactobacillus acidophilus NX2-6 effectively improves lipid metabolism in serum of high-fat diet mice.
5. Influence of Lactobacillus acidophilus NX2-6 on parameters related to lipid metabolism and oxidative stress in mouse liver
According to the method of Gao et al (Gao et al, food function, 2021,12,373-386), livers in mice were removed, rinsed with sterile physiological saline, 100 mg of liver samples were accurately weighed into sterile centrifuge tubes, 1 ml of sterile PBS buffer (0.1 mol/L, ph=7.2) was added, homogenized with an autohomogenizer until no distinct lumps were formed, and centrifuged at 4 degrees at 2000 rpm for 10 minutes, and the supernatant was aspirated into another sterile centrifuge tube and stored at 4 degrees for later use. The levels of triglyceride, HDL-C, LDL-C, superoxide dismutase (SOD), peroxidase (CAT) and Lactate Dehydrogenase (LDH) in the liver homogenate supernatant were determined using the kit, and the protein content in the liver homogenate supernatant was determined using the BCA method for calibrating the parameters.
As a result, as shown in FIG. 3, the concentration of triglyceride in liver was decreased from 18.78.+ -. 4.49mmol/mgprot to 14.88.+ -. 4.63mmol/mgprot, the concentration of HDL-C was increased from 0.38.+ -. 0.16mmol/mg prot to 0.67.+ -. 0.17mmol/mgprot, and the activity of SOD was increased from 91.16.+ -. 25.24U/gprot to 145.93.+ -. 39.25U/g prot, without significant change in the levels of LDL-C, CAT and LDH, as compared with the mice of the high-fat model group. Thus, lactobacillus acidophilus NX2-6 effectively inhibits fat accumulation in the liver and increases liver antioxidant capacity.
6. Influence of Lactobacillus acidophilus NX2-6 on tissue Structure in mice
Referring to the method of Wang et al (Wang et al, j.funct.foods,2020,68,103923), 400 mg of fresh epididymal adipose tissue, colon tissue and liver were accurately weighed, washed with sterile physiological saline, placed in a sterile centrifuge tube, 1 ml of 4% paraformaldehyde was added for fixing the tissues, and stored at 4 degrees for later use. Then, all tissues were dehydrated, embedded in paraffin, sectioned by a microtome, stained with hematoxylin and eosin, and the microstructure of the tissues was observed with a microscope.
As a result, as shown in FIG. 4, the adipocytes of the mice of the high-fat model group were significantly enlarged, the colon villi were loosely arranged and destroyed, and a large number of lipid-droplet cells were present in the liver; in contrast, mice administered Lactobacillus acidophilus NX2-6 exhibited significantly smaller adipocytes, a more orderly arrangement of colon villi, and no apparent lipid-droplet cells in the liver.
7. Effect of Lactobacillus acidophilus NX2-6 on lipid metabolism in mouse epididymal adipose tissue
In order to explore the lipid metabolism regulation mechanism of lactobacillus acidophilus NX2-6, the expression quantity of some genes related to fat metabolism is measured.
TABLE 1 primer sequences for genes related to fat metabolism
Referring to the method of Xia et al (Xia et al, biomed. Pharmacother.,2019,118,109287), 100 mg of epididymal adipose tissue was accurately weighed, placed in a sterile RNase-free centrifuge tube, placed on ice, 1 ml of Trizol lysate was added to extract total RNA from the tissue, and after the extraction of RNA, the purity of RNA, such as OD, was measured using a Nanodrop apparatus 260 /OD 280 The ratio is between 1.7 and 2.0, which means that the RNA purity is higher. Reverse transcription of higher purity RNA into cDNA, reverse transcription procedure: 15 minutes at 50 ℃; the reverse transcribed product was placed at-20℃for 5 seconds. The gene expression quantity is measured by adopting a real-time fluorescence quantitative PCR (Rt-PCR) method, and a sample adding system of the Rt-PCR method is as follows: 2. Mu.L of cDNA; 6.4. Mu.L of sterile deionized water; 1.6. Mu.L of forward and reverse primers (primer sequences see Table 1); 10 μ L SYBR GreenMaster Mix (High Rox); the reaction system of the Rt-PCR method is as follows: pre-denaturation at 95℃for 5min, a cycling stage (denaturation at 95℃for 15 sec, annealing at 60℃for 1min, 40 cycles total), a dissolution profile stage (denaturation at 95℃for 15 sec, annealing at 60℃for 1min, extension at 95℃for 15 sec), ct values were collected, and beta-actin was taken as an internal reference gene, and the reaction was carried out by 2 -ΔΔCt The relative expression level of the target gene is calculated by the method.
As shown in FIG. 5, compared with the mice of the high-fat model group, the gene expression level of the fatty acid uptake protein CD36 in the adipose tissue of the mice was reduced by about 1.24 times, the expression levels of the genes FAS and Srebp-1C acting in the synthesis of fatty acids from the head were reduced by 1.41 and 2.56 times, respectively, the expression level of the triglyceride synthesis-related gene DGAT1 was reduced by 1.08 times, and the expression level of the fatty differentiation-related gene C/EBP. Alpha was reduced by 0.73 times after the ingestion of Lactobacillus acidophilus NX 2-6; in addition, the expression level of the triglyceride decomposition related gene HSL was increased by 1.79 times, and the expression levels of the fatty acid oxidation related genes PPAR-alpha and CPT2 were increased by 1.95 and 0.80 times, respectively, so that Lactobacillus acidophilus NX2-6 effectively inhibited the accumulation of triglycerides in epididymal adipose tissue and improved lipid metabolism by improving the lipid metabolism related genes in adipose tissue.
The results show that the lactobacillus acidophilus NX2-6 provided by the invention can obviously improve lipid metabolism, effectively relieve obesity, and can be applied to improving lipid metabolism in adipose tissues.
8. Regulation and control of mitochondria-related genes in mouse epididymal adipose tissue by lactobacillus acidophilus NX2-6
In order to explore the mitochondrial regulation mechanism of lactobacillus acidophilus NX2-6, the invention determines the expression level of a plurality of genes related to mitochondrial regulation.
The measurement of the expression level of the related genes was carried out using the primer sequences of Table 2 under the conditions of total RNA and Rt-PCR of the extracted epididymal adipose tissues. As shown in FIG. 6, compared with mice of the high-fat model group, after ingestion of Lactobacillus acidophilus NX2-6, the expression levels of mitochondrial biosynthesis related genes in adipose tissue of mice, such as PGC-1 beta and SIRT1, are respectively up-regulated by 0.41 and 0.43 times, the gene expression levels of mitochondrial biosynthesis related regulatory factors NRF1 and TFAM are increased by 0.99 and 1.36 times, the expression level of mitochondrial fusion related gene MFN2 is increased by 0.43 times, and the expression levels of mitochondrial division related genes Fis1 and Drp1 are respectively increased by 1.03 and 0.85 times, which indicates that Lactobacillus acidophilus NX2-6 can regulate mitochondrial genes in epididymal adipose tissue, effectively enhance the functions of mitochondria in epididymal adipose tissue, and can be applied in improving energy metabolism in adipose tissue.
TABLE 2 primer sequences for mitochondrial biosynthesis, fusion and division related genes
9. Regulation of inflammatory response in mice by lactobacillus acidophilus NX2-6
In order to explore the regulation and control of inflammatory response by lactobacillus acidophilus NX2-6, the level of inflammatory factors in serum and the gene expression amounts of inflammatory factors and close-coupled proteins in liver and colon tissues are measured.
Taking the collected serum as a study object, and adopting an enzyme-linked immunosorbent assay (ELISA) to measure the concentration of endotoxin, TNF-alpha and IL-1 beta in the serum; the related genes in liver and colon tissues were determined using the primer sequences of Table 3 under the conditions of total RNA and Rt-PCR of epididymal adipose tissues extracted. As shown in FIG. 7, the concentrations of endotoxin, TNF-. Alpha.and IL-1β in the serum of mice were decreased from 103.14.+ -. 11.43ng/L, 1090.68.+ -. 321.98ng/L and 79.21.+ -. 14.76ng/L to 72.81.+ -. 8.39ng/L, 782.16.+ -. 123.62ng/L and 68.41.+ -. 22.44ng/L, respectively, after ingestion of Lactobacillus acidophilus NX2-6, as compared to the mice of the high-fat model group; meanwhile, the lactobacillus acidophilus NX2-6 can also inhibit the gene expression quantity of TNF-alpha and IL-1 beta in colon and liver, and obviously up-regulate the gene expression quantity of the close-coupled proteins ZO-1, occludin and Claudin-1 in colon, which shows that the lactobacillus acidophilus NX2-6 has the capability of inhibiting metabolic inflammation and enhancing intestinal barrier, and can be applied in inhibiting metabolic inflammation.
TABLE 3 primer sequences for immune-related genes
10. Regulation of intestinal flora structure by lactobacillus acidophilus NX2-6
In order to explore the influence of lactobacillus acidophilus NX2-6 on the structure of intestinal flora, colon contents are taken as a research object, and the structure of the intestinal flora in the colon of a mouse is determined by utilizing a metagenome sequencing technology.
Referring to the method of Li et al (Li et al., food res. Int.,2021,143,110270), 200mg of mouse feces were rapidly weighed into a sterilized 2mL centrifuge tube, crushed, homogenized, placed on ice, and the feces genomic DNA extraction kit was operated as follows: adding 1.4mL buffer GSL into the fecal sample, shaking for 1min to mix the sample, incubating for 10min at 70 ℃, swirling for 15s, centrifuging for 1min at 12000 Xg, transferring the supernatant into a new sterilized centrifuge tube, adding inhibitor adsorption sheet into the centrifuge tube, shaking until the adsorption sheet is completely opened, standing for 1min at room temperature, and standing for 12000XCentrifuging at g for 3min, sucking supernatant into a new centrifuge tube, centrifuging at 12000 Xg for 3min, sucking 200 μL of supernatant into another centrifuge tube, adding 15 μL of proteinase K, adding 200 μL of buffer GB, shaking vortex for 15s, incubating at 70 ℃ for 10min, adding 200 μL of absolute ethanol, vortex mixing, adding the mixed solution into an adsorption column, collecting with a sleeve, purifying DNA with buffer GD and rinsing PW, collecting DNA with buffer TB, and storing in a refrigerator at-20 ℃. The purity and concentration of DNA were measured by Nanodrop to obtain OD260/OD280 and concentration values, and the ratio of OD was about 1.8, which indicated that the DNA was of good purity. Then amplifying the V3-V4 region of the bacterial 16S rRNA, wherein the adopted primers are a forward primer 338F (5'-ACTCCTACGGGAGGCAGCA-3') and a reverse primer 806R (5 '-GGACTACHVGGGTWTCTAAT-3'), and a PCR reaction system is as follows: 1 mu LDNA template, 0.25 mu L Q DNA polymerase, 5 Xreaction buffer and 5 Xhigh GC buffer 5. Mu.L each, 10. Mu.M forward and reverse primer 1. Mu.L, 0.5. Mu.L 10mM dNTP, 11.25. Mu.L ddH 2 O. The PCR reaction conditions were: the pre-denaturation is carried out at 98 ℃ for 30s,25 cycles are denaturation at 98 ℃ for 15s, annealing at 56 ℃ for 30s and extension at 72 ℃ for 30s, and finally renaturation is carried out at 72 ℃ for 5min, and amplified products are purified and sequenced. Because the number of sequences obtained by high-throughput sequencing is huge, the original data needs to be filtered and screened to remove the chimeric sequences, so that the accuracy of the data can be improved and the working efficiency can be improved, in addition, in order to facilitate subsequent analysis, the sequences need to be clustered, and the classification units (Operational Taxonomic Unit, OTU) can be operated by clustering the sequences with the similarity of more than or equal to 97%. Based on OTU partitioning, alpha diversity analysis can be further performed to determine the abundance of the colonies within the sample, and the composition of the colonies within the sample can be further determined by different taxonomic levels.
The overall structure of each group of intestinal flora is shown in fig. 8, and compared with the mice in the high-fat model group, lactobacillus acidophilus NX2-6 significantly improves the Chao1 index, the Shannon index and the Simpson index, which indicates that lactobacillus acidophilus NX2-6 improves the richness and diversity of the intestinal flora of the mice.
In addition, the differences in flora at the portal, family and genus levels are also apparent. As shown in fig. 9-11, lactobacillus acidophilus NX2-6 inhibited proliferation of harmful flora, such as the relative abundance of proteus phylum, vibrio desulfonis, alistipes, vibrio desulfonis, helicobacter and mucispirilum, decreased by 61.71%, 350.80%, 524.99%, 487.47%, 100.61% and 193.32%, respectively, compared to mice of the high fat model group at the phylum, family and genus level; and the richness of the probiotics bacteroides, wart micro-bacteria, S24-7, lactobacillus and parabacteroides is obviously increased by 43.99%, 435.09%, 190.47%, 395.45%, 146.21% and 164.55%, which shows that lactobacillus acidophilus NX2-6 has the capability of optimizing the intestinal flora structure. On the one hand, since Proteus, vibrionaceae, vibrio, helicobacter and Mucispirillum are the main conditional pathogenic bacteria that trigger intestinal inflammation, they cause systemic inflammation and accumulation of lipids by triggering intestinal inflammation and thus destroying the intestinal barrier, leading to translocation of lipids and endotoxins in the intestinal tract into the circulatory system; on the other hand, S24-7, the Lactobacillus and the Paramycolatopsis can reduce the pH value in the intestinal tract by generating organic acid, improve the intestinal microecological system, effectively strengthen the intestinal barrier, further prevent the translocation of free fatty acid in the intestinal tract to the circulating system, and can be applied in the aspect of adjusting the structure of intestinal flora.
In addition, all animal experiments are repeated, and the results show that the body weight, liver index, epididymal adipose tissue weight, triglyceride in serum, leptin, adiponectin, ALT activity, AST activity, HDL-C, LDL-C and the activity trend of triglyceride in liver, HDL-C, LDL-C, SOD, CAT and LDH are consistent, so that the anti-obesity effect of lactobacillus acidophilus NX2-6 is further verified.
In conclusion, lactobacillus acidophilus NX2-6 has good probiotic effect by improving lipid metabolism and energy metabolism in adipose tissues, inhibiting metabolic inflammatory reaction and regulating intestinal flora structure, thereby relieving obesity caused by high-fat diet.
Sequence listing
<110> Nanjing Fosferi Biotechnology Co.Ltd, nanjing agriculture university
<120> Lactobacillus acidophilus NX2-6 with obesity relieving function and application thereof
<141> 2022-01-11
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<211> 22
<212> DNA
<213> Mice
<400> 68
tttggctgct cttgggtctg ta 22
<210> 69
<211> 20
<212> DNA
<213> Mice
<400> 69
<210> 70
<211> 20
<212> DNA
<213> Mice
<400> 70
Claims (6)
1. Lactobacillus acidophilus (Lactobacillus acidophilus) NX2-6 with the function of relieving obesity has a preservation number of CGMCC No.17311.
2. The use of lactobacillus acidophilus NX2-6 according to claim 1, in particular to the use of lactobacillus acidophilus NX2-6 for preparing a bacterial suspension for inhibiting obesity caused by high fat diet.
3. The use of lactobacillus acidophilus NX2-6 according to claim 1, in particular to the use of lactobacillus acidophilus NX2-6 for preparing a bacterial suspension for improving lipid metabolism in adipose tissue.
4. The use of lactobacillus acidophilus NX2-6 according to claim 1, in particular to the use of lactobacillus acidophilus NX2-6 for preparing a bacterial suspension for improving energy metabolism in adipose tissue.
5. The use of lactobacillus acidophilus NX2-6 according to claim 1, in particular to the use of lactobacillus acidophilus NX2-6 for preparing a bacterial suspension for inhibiting metabolic inflammatory reaction.
6. The use of lactobacillus acidophilus NX2-6 according to claim 1, in particular to the use of lactobacillus acidophilus NX2-6 for preparing a bacterial suspension for optimizing the structure of intestinal flora.
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| CN103610213B (en) * | 2013-11-26 | 2015-09-23 | 南京农业大学 | The preparation method of food-grade lactobacillus acidophilus fermentation natural antiseptic agent and product |
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