Application of lactobacillus rhamnosus E2 in preparation of fat-reducing and weight-losing products

Abstract

The invention discloses application of lactobacillus rhamnosus E2 in preparation of fat-reducing and weight-losing products, and belongs to the technical field of microorganisms. The lactobacillus rhamnosus E2 disclosed by the invention can remarkably promote fat metabolism in a zebra fish body in vivo, can remarkably reduce the content of triglyceride and total cholesterol in a hyperlipidemia zebra fish body, and has the potential of being applied to losing weight and regulating the blood fat level; the lactobacillus rhamnosus E2 provides theoretical reference and guidance basis for developing probiotic preparations for treating or preventing obesity and hyperlipidemia by utilizing the lactobacillus rhamnosus E2.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more specifically to the application of Lactobacillus rhamnosus E2 in the preparation of lipid-lowering and weight-loss products. Background Technology

[0002] Hyperlipidemia refers to excessively high blood lipid levels, which can directly cause several serious health problems. Numerous studies have shown that hyperlipidemia is a risk factor for stroke, coronary heart disease, myocardial infarction, and sudden death. Furthermore, hyperlipidemia is also a significant risk factor for hypertension, impaired glucose tolerance, and diabetes. Hyperlipidemia can also lead to fatty liver, cirrhosis, gallstones, pancreatitis, retinal hemorrhage, blindness, peripheral vascular disease, claudication, and hyperuricemia. Therefore, the dangers of hyperlipidemia must be taken very seriously, and active prevention and treatment are essential. Currently, to achieve a more comprehensive lipid-lowering effect, it is often necessary to take two lipid-lowering drugs with different mechanisms of action. For example, statins lower triglycerides, but drugs that raise high-density lipoprotein cholesterol are not ideal, increasing treatment costs and side effects. Based on this, screening for functional probiotics with cholesterol-lowering effects in the human body has become a research hotspot. Probiotics, as a class of microorganisms beneficial to human health, can exert beneficial health effects by altering the intestinal microecology when a certain number of live probiotics colonize the host's intestines.

[0003] Therefore, providing the application of Lactobacillus rhamnosus E2 in the preparation of lipid-lowering and weight-loss products is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0004] In view of this, the present invention provides the application of Lactobacillus rhamnosus E2 in the preparation of lipid-lowering and weight-loss products.

[0005] Because zebrafish possess a digestive system similar to humans, including a liver and intestines, their digestion, nutrient absorption, and transport are highly similar to those of humans. The yolk sac of a zebrafish is approximately 70% neutral fat. Promoting the absorption of the yolk sac is equivalent to promoting the breakdown and metabolism of fat, similar to burning fat within the body. After fat-specific fluorescent staining (which produces red), the zebrafish yolk sac is stained red and can be clearly observed.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The application of Lactobacillus rhamnosus E2 in the preparation of lipid-lowering and weight-loss products, wherein the Lactobacillus rhamnosus E2 has the accession number CGMCC No.21770 (see patent number 202210394834.X).

[0008] Furthermore, the application of the aforementioned Lactobacillus rhamnosus E2 in the preparation of products that promote lipid metabolism.

[0009] Furthermore, the application of the aforementioned Lactobacillus rhamnosus E2 in the preparation of products that lower triglycerides and total cholesterol.

[0010] Furthermore, the Lactobacillus rhamnosus E2 is a bacterial suspension.

[0011] At the same concentration, *Lactobacillus rhamnosus* E2 had a stronger promoting effect on fat metabolism in zebrafish than *Lactobacillus rhamnosus* 7469, demonstrating a good lipid-lowering and weight-loss effect. At the same concentration, *Lactobacillus rhamnosus* E2 had a stronger reducing effect on triglycerides and total cholesterol in a zebrafish hyperlipidemia model than *Lactobacillus rhamnosus* 7469, demonstrating a good lipid-lowering effect.

[0012] As can be seen from the above technical solution, compared with the prior art, the present invention discloses the application of Lactobacillus rhamnosus E2 in the preparation of lipid-lowering and weight-loss products. Lactobacillus rhamnosus E2 can significantly promote fat metabolism in zebrafish in vivo, and can significantly reduce the content of triglycerides (TG) and total cholesterol (TC) in zebrafish with high blood lipids. It has the potential to be used for weight loss and regulating blood lipid levels. This provides a theoretical reference and guiding basis for developing probiotic preparations for the treatment or prevention of obesity and hyperlipidemia using Lactobacillus rhamnosus E2. Attached Figure Description

[0013] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0014] Figure 1 The attached figure is a visual representation of the effect of Lactobacillus rhamnosus E2 of the present invention on the body fat level of zebrafish;

[0015] Wherein, A: normal group; B: positive control group; C: 1×10 6 CFU / mL Lactobacillus rhamnosus 7469; D: 1×10 6 CFU / mL Lactobacillus rhamnosus E2;

[0016] Figure 2 The attached figure is a statistical graph showing the effect of Lactobacillus rhamnosus E2 of the present invention on the body fat level of zebrafish;

[0017] Figure 3 The attached figure shows the effect of Lactobacillus rhamnosus E2 of the present invention on triglycerides in a zebrafish hyperlipidemia model;

[0018] Figure 4 The attached figure shows the effect of Lactobacillus rhamnosus E2 of the present invention on total cholesterol in a zebrafish hyperlipidemia model. Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] Resveratrol was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; Nile Red was purchased from Sigma-Aldrich, Inc.; Atorvastatin Calcium was purchased from Shanghai Luyuan Biotechnology Co., Ltd.; Both regular and high-cholesterol feeds were purchased from Nantong Trofi Feed Technology Co., Ltd.; Triglyceride assay kits and total cholesterol assay kits were purchased from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.; Lactobacillus rhamnosus 7469 (ATCC 7469) was purchased from Beijing Bio-Bio Biotechnology Co., Ltd.

[0021] Example 1: Preparation of Lactobacillus rhamnosus E2 bacterial suspension (cells)

[0022] Lactobacillus rhamnosus E2 was activated and cultured, then inoculated into MRS liquid medium and cultured at 37°C for 24 h. The culture was then centrifuged at 4°C and 6000 rpm for 10 min to obtain a bacterial pellet. The bacterial pellet was washed twice with PBS, and the cells were resuspended in PBS to adjust the cell concentration to 1 × 10⁻⁶. 6 A bacterial suspension (bacterial cells) was obtained at CFU / mL.

[0023] Example 2: Preparation of Lactobacillus rhamnosus 7469 bacterial suspension (cells)

[0024] Lactobacillus rhamnosus 7469 was activated and cultured, then inoculated into MRS liquid medium and cultured at 37°C for 24 h. The culture was then centrifuged at 4°C and 6000 rpm for 10 min to obtain a bacterial pellet. The bacterial pellet was washed twice with PBS, and the cells were resuspended in PBS to adjust the cell concentration to 1 × 10⁻⁶. 6 A bacterial suspension (bacterial cells) was obtained at CFU / mL.

[0025] Example 3: Effect of Lactobacillus rhamnosus E2 on body fat levels in zebrafish

[0026] Healthy wild-type AB strain zebrafish that had reached 3 days post-fertilization (dpf) were selected and placed in 6-well cell culture plates, 15 zebrafish per well. The experiment included a normal control group, a positive control group (resveratrol), a *Lactobacillus rhamnosus* 7469 intervention group, and a *Lactobacillus rhamnosus* E2 intervention group. The normal control group received PBS, the positive control group received resveratrol solution (50 μmol / L), and the *Lactobacillus rhamnosus* 7469 intervention group received 1 × 10⁻⁶ saturated ... 6 Add 1×10 (CFU / mL) 6 CFU / mL Lactobacillus rhamnosus 7469, Lactobacillus rhamnosus E2 intervention group (1×10 6 Add 1×10 (CFU / mL) 6 CFU / mL Lactobacillus rhamnosus E2 was added to each well (5 mL) and incubated at 28°C for 24 hours. The solution was replaced with fresh solution after 48 hours. After incubation, the solution was discarded, and 10 ng / mL Nile Red solution was added to each well (5 mL). The mixture was incubated at 28°C in the dark for 24 hours. The zebrafish were then washed three times with PBS, and the fluorescence intensity was observed and photographed under a fluorescence microscope. ImageJ software was used for quantitative statistical analysis of the fluorescence intensity (F) in the zebrafish. The body fat level in the zebrafish was calculated as follows:

[0027]

[0028] Data were statistically processed using SPSS 19.0 software. Experimental data are expressed as mean ± SEM values. T-tests were used for analysis, comparing the results with the normal group: *** P<0.005.

[0029] Depend on Figure 1 and Figure 2 It can be seen that the intensity of red fluorescence in zebrafish reflects the level of fat in zebrafish. Compared with the normal group, the intensity of red fluorescence in the positive control group (resveratrol) zebrafish was weakened, indicating that the fat level in the positive control group zebrafish was reduced. At the same time, compared with the normal group (100.00±6.16%), the fat level in the positive control group zebrafish (45.94±2.99%) was significantly reduced (p<0.005), indicating that this lipid-lowering experiment in zebrafish was effective.

[0030] Depend on Figure 1 and Figure 2 It can be seen that the Lactobacillus rhamnosus 7469 intervention group (1×10) 6 The intensity of red fluorescence in zebrafish (CFU / mL) was similar to that in the normal group; meanwhile, the Lactobacillus rhamnosus 7469 intervention group (1×10) 6The body fat level in zebrafish (CFU / mL) was 89.35±4.79%, which was not significantly different from the normal group (100.00±6.16%) (P>0.05). Furthermore, compared with the normal group, the *Lactobacillus rhamnosus* E2 intervention group (1×10⁻⁶ CFU / mL) showed a significantly lower body fat level (89.35±4.79%). 6 The intensity of red fluorescence in zebrafish was reduced (CFU / mL), similar to the positive control group; meanwhile, the *Lactobacillus rhamnosus* E2 intervention group (1×10⁻⁶ CFU / mL) showed a decrease. 6 The fat level in zebrafish (CFU / mL) was 65.68±5.66%, which was significantly different from that in the normal group (100.00±6.16%) (P<0.005). Therefore, the above results indicate that at the same concentration, Lactobacillus rhamnosus E2 has a stronger promoting effect on fat metabolism in zebrafish than Lactobacillus rhamnosus 7469, demonstrating a good lipid-lowering and weight-loss effect.

[0031] Example 4: Effects of Lactobacillus rhamnosus E2 on triglycerides and total cholesterol in a zebrafish hyperlipidemia model.

[0032] Model construction: Normally developed wild-type AB zebrafish (5 dpf) were selected under a microscope and divided into a normal group, a model group (high cholesterol), a positive control group (atorvastatin calcium), and a Lactobacillus rhamnosus 7469 intervention group (1×10⁻⁶). 6 CFU / mL), Lactobacillus rhamnosus E2 intervention group (1×10 6 (CFU / mL). Each group had 6 replicates, with 30 fish per well and 5 mL PBS per well. The zebrafish in the normal group were fed a normal diet (20 mg each time); the zebrafish in the model group, positive control group, and strain intervention group were fed a high-cholesterol diet (20 mg each time), 3 times a day for 7 days, with the PBS changed daily.

[0033] Intervention: PBS solution was added to both the normal group and the model group; 0.25 μg / mL atorvastatin calcium solution was added to the positive control group; Lactobacillus rhamnosus 7469 intervention group (1×10 6 Add 1×10 (CFU / mL) 6 CFU / mL Lactobacillus rhamnosus 7469; Lactobacillus rhamnosus E2 intervention group (1×10 6 Add 1×10 (CFU / mL) 6CFU / mL Lactobacillus rhamnosus E2, 5 mL per well; simultaneously, zebrafish in the normal group were fed a normal diet (20 mg each time); zebrafish in the model group, positive control group, and strain intervention group were all fed a high-cholesterol diet (20 mg each time), three times a day for 5 days, with the solution changed daily. After the intervention, zebrafish were collected into 1.5 mL centrifuge tubes, 6 tubes from each experimental group; after removing the water from the centrifuge tubes, PBS buffer was added at a mass (mg): volume (μL) ratio of 1:9, and the zebrafish were homogenized using an S-18KS handheld micro-electric tissue homogenizer until no obvious tissue fragments remained. Centrifuged at 15000×g, 4℃ for 15 min, and the supernatant was collected. The triglyceride and total cholesterol concentrations of each group were measured using a triglyceride assay kit and a total cholesterol assay kit.

[0034] Data were statistically processed using SPSS 19.0 software. Experimental data are expressed as mean ± SEM values. T-tests were used for analysis, comparing the results with the normal group: ### P<0.005; compared with the model group: ** P<0.01, P<0.005.

[0035] Depend on Figure 3 and Figure 4 It can be seen that, compared with the normal group (triglycerides: 0.11±0.01mmol / gprot, total cholesterol: 0.09±0.01mmol / gprot), the levels of triglycerides and total cholesterol (triglycerides: 0.31±0.03mmol / gprot, total cholesterol: 0.29±0.02mmol / gprot) in the zebrafish of the model group were significantly increased (P<0.005), indicating that the zebrafish hyperlipidemia model was successfully established.

[0036] Depend on Figure 3 and Figure 4 The results showed that the triglyceride and total cholesterol levels in the zebrafish of the positive control group were 0.16±0.01 mmol / gprot and 0.14±0.01 mmol / gprot, respectively, which were significantly different from those in the model group (triglycerides: 0.31±0.03 mmol / gprot, total cholesterol: 0.29±0.02 mmol / gprot) (P<0.005). Therefore, atorvastatin calcium has a lipid-lowering effect, consistent with clinical results, indicating that this lipid-lowering efficacy evaluation trial was effective.

[0037] Lactobacillus rhamnosus 7469 intervention group (1×10 6The levels of triglycerides and total cholesterol in zebrafish (CFU / mL) were 0.28±0.02 mmol / gprot and 0.26±0.02 mmol / gprot, respectively, which showed no significant difference compared with the model group (triglycerides: 0.31±0.03 mmol / gprot, total cholesterol: 0.29±0.02 mmol / gprot) (P>0.05). Additionally, the *Lactobacillus rhamnosus* E2 intervention group (1×10⁻⁶) showed no significant difference. 6 The levels of triglycerides and total cholesterol in zebrafish (CFU / mL) were 0.19±0.02 mmol / gprot and 0.18±0.02 mmol / gprot, respectively, which were significantly different from those in the model group (triglycerides: 0.31±0.03 mmol / gprot, total cholesterol: 0.29±0.02 mmol / gprot) (P<0.01). Therefore, these results indicate that at the same concentration, *Lactobacillus rhamnosus* E2 has a stronger effect on reducing triglycerides and total cholesterol in the zebrafish hyperlipidemia model than *Lactobacillus rhamnosus* 7469, demonstrating a good lipid-lowering effect.

[0038] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The application of Lactobacillus rhamnosus E2 in the preparation of lipid-lowering and weight-loss products, characterized in that, The preservation number of Lactobacillus rhamnosus E2 is CGMCC No. 21770.

2. The application of Lactobacillus rhamnosus E2 according to claim 1 in the preparation of lipid-lowering and weight-loss products, characterized in that, The Lactobacillus rhamnosus E2 is a bacterial suspension.

3. The use of Lactobacillus rhamnosus E2 as described in claim 1 in the preparation of products that promote lipid metabolism.

4. The application of Lactobacillus rhamnosus E2 according to claim 3 in the preparation of products that promote lipid metabolism, characterized in that, The Lactobacillus rhamnosus E2 is a bacterial suspension.

5. The use of Lactobacillus rhamnosus E2 as described in claim 1 in the preparation of products that lower triglycerides and total cholesterol.

6. The application of Lactobacillus rhamnosus E2 according to claim 5 in the preparation of products that lower triglycerides and total cholesterol, characterized in that, The Lactobacillus rhamnosus E2 is a bacterial suspension.

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