Application of bifidobacterium breve NX-5 in preparation of fat-reducing and weight-losing products

Abstract

The invention discloses application of bifidobacterium breve NX-5 in preparation of fat-reducing and weight-losing products, and belongs to the technical field of microorganisms. The bifidobacterium breve NX-5 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; theoretical reference and guidance basis are provided for developing probiotic preparations for treating or preventing obesity and hyperlipidemia by utilizing the bifidobacterium breve NX-5.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more specifically to the application of Bifidobacterium breve NX-5 in the preparation of lipid-lowering and weight-loss products. Background Technology

[0002] Hyperlipidemia, also known as hyperlipoproteinemia, refers to excessively high levels of lipids in the blood and is the most common form of dyslipidemia. This condition is divided into primary and secondary subtypes. Primary hyperlipidemia usually results from genetic causes (such as mutations in receptor proteins), while secondary hyperlipidemia is caused by other factors, such as diabetes. Lipid and lipoprotein abnormalities are common in the general population. Furthermore, some forms of hyperlipidemia can induce acute pancreatitis. 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.

[0003] As the application of traditional medicine becomes increasingly limited, supplementary or alternative therapies are becoming more necessary and accepted. Currently, novel lipid-lowering therapies, primarily based on probiotics, natural products, and traditional Chinese medicine, are gaining attention from medical scholars due to their natural, non-toxic, effective, mild, and safe origins. However, current domestic research on probiotics is mainly focused on basic research, with insufficient development of their lipid-lowering functions and an incomplete functional evaluation pathway. This results in a scarcity of high-quality probiotic strains with independent intellectual property rights in my country, and even fewer functional probiotic drugs used clinically as alternatives or adjunct therapy. Therefore, establishing a scientific and comprehensive functional evaluation system to screen for domestically patented drug strains with strong efficacy and high activity has significant social value and research significance.

[0004] Therefore, providing the application of Bifidobacterium breve NX-5 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

[0005] In view of this, the present invention provides the application of Bifidobacterium breve NX-5 in the preparation of lipid-lowering and weight-loss products.

[0006] 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.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] The application of Bifidobacterium breve NX-5 in the preparation of lipid-lowering and weight-loss products, wherein the preservation number of Bifidobacterium breve NX-5 is CGMCC No. 20113 (see patent number 202110372822.2).

[0009] Furthermore, the application of the aforementioned Bifidobacterium NX-5 in the preparation of products that promote lipid metabolism.

[0010] Furthermore, the application of the aforementioned Bifidobacterium NX-5 in the preparation of products that lower triglycerides and total cholesterol.

[0011] Furthermore, the Bifidobacterium NX-5 is a bacterial suspension.

[0012] Furthermore, the products mentioned are food, medicine, and health products.

[0013] At the same concentration, *Bifidobacterium breve* NX-5 showed a stronger promoting effect on fat metabolism in zebrafish than *Bifidobacterium breve* 15701, demonstrating good lipid-lowering and weight-loss effects. At the same concentration, *Bifidobacterium breve* NX-5 also showed a stronger reducing effect on triglycerides and total cholesterol in a zebrafish hyperlipidemia model than *Bifidobacterium breve* 15701, demonstrating good lipid-lowering effects.

[0014] As can be seen from the above technical solution, compared with the prior art, the present invention discloses the application of Bifidobacterium breve NX-5 in the preparation of lipid-lowering and weight-loss products. Bifidobacterium breve NX-5 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 Bifidobacterium breve NX-5. Attached Figure Description

[0015] 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.

[0016] Figure 1 The attached figure is a visual representation of the effect of the present invention, Bifidobacterium breve NX-5, on the body fat level of zebrafish;

[0017] Wherein, A: normal group; B: positive control group; C: 1×10 6CFU / mL Bifidobacterium breve 15701; D: 1×10 6 CFU / mL Bifidobacterium breve NX-5;

[0018] Figure 2 The attached figure is a statistical graph showing the effect of the present invention's Bifidobacterium NX-5 on the body fat level of zebrafish;

[0019] Figure 3 The attached figure shows the effect of Bifidobacterium breve NX-5 of the present invention on triglycerides in a zebrafish hyperlipidemia model;

[0020] Figure 4 The attached figure shows the effect of the present invention's Bifidobacterium breve NX-5 on total cholesterol in a zebrafish hyperlipidemia model. Detailed Implementation

[0021] 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.

[0022] 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.; Bifidobacterium breve 15701 (ATCC 15701) was purchased from Beijing BioBio Biotechnology Co., Ltd.

[0023] Example 1: Preparation of Bifidobacterium breve NX-5 bacterial suspension (cells)

[0024] After activation and culture, *Bifidobacterium breve* NX-5 was inoculated into BS 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 cells / mL. 6 A bacterial suspension (bacterial cells) was obtained at CFU / mL.

[0025] Example 2: Preparation of Bifidobacterium breve 15701 bacterial suspension (cells)

[0026] After activation and culture, *Bifidobacterium breve* 15701 was inoculated into BS 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.

[0027] Example 3: Effect of Bifidobacterium breve NX-5 on body fat levels in zebrafish

[0028] 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 *Bifidobacterium breve* 15701 intervention group, and a *Bifidobacterium breve* NX-5 intervention group. The normal control group received PBS, the positive control group received resveratrol solution (50 μmol / L), and the *Bifidobacterium breve* 15701 intervention group received 1 × 10⁻⁶ saturated ... 6 Add 1×10 (CFU / mL) 6 CFU / mL Bifidobacterium breve 15701, Bifidobacterium breve NX-5 intervention group (1×10 6 Add 1×10 (CFU / mL) 6 CFU / mL Bifidobacterium breve NX-5 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. The fluorescence intensity (F) in the zebrafish was quantitatively analyzed using ImageJ software. The fat level in the zebrafish was calculated as follows:

[0029]

[0030] 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.

[0031] Depend on Figure 1 and Figure 2It 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±8.23%), the fat level in the positive control group zebrafish (46.51±2.71%) was significantly reduced (p<0.005), indicating that this lipid-lowering experiment in zebrafish was effective.

[0032] Depend on Figure 1 and Figure 2 It can be seen that the intervention group of Bifidobacterium breve 15701 (1×10 6 The intensity of red fluorescence in zebrafish (CFU / mL) was similar to that in the normal group; meanwhile, the intervention group with Bifidobacterium brevis 15701 (1×10) was similar to that in the normal group. 6 The body fat level in zebrafish (CFU / mL) was 95.89±5.11%, which was not significantly different from the normal group (100.00±8.23%) (P>0.05). Furthermore, compared with the normal group, the *Bifidobacterium breve* NX-5 intervention group (1×10⁻⁶ CFU / mL) showed a significantly lower body fat level (95.89±5.11%). 6 The intensity of red fluorescence in zebrafish was reduced (CFU / mL), similar to the positive control group; meanwhile, the Bifidobacterium breve NX-5 intervention group (1×10⁻⁶ CFU / mL) showed a decrease. 6 The fat level in zebrafish (CFU / mL) was 48.50±2.83%, which was significantly different from that in the normal group (100.00±8.23%) (P<0.005). Therefore, the above results indicate that at the same concentration, *Bifidobacterium breve* NX-5 has a stronger promoting effect on fat metabolism in zebrafish than *Bifidobacterium breve* 15701, demonstrating a good lipid-lowering and weight-loss effect.

[0033] Example 4: Effects of Bifidobacterium breve NX-5 on triglycerides and total cholesterol in a zebrafish hyperlipidemia model.

[0034] 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 Bifidobacterium breve 15701 intervention group (1×10⁻⁶). 6 CFU / mL), Bifidobacterium breve NX-5 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.

[0035] 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; 1×10⁻⁵ Bifidobacterium breve 15701 intervention group was added. 6 Add 1×10 (CFU / mL) 6 CFU / mL Bifidobacterium breve 15701; Bifidobacterium breve NX-5 intervention group (1×10 6 Add 1×10 (CFU / mL) 6 CFU / mL Bifidobacterium breve NX-5, 5 mL per well; 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 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. The homogenate was 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.

[0036] 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<0.05; compared with the model group: ** P<0.01, *** P<0.005.

[0037] Depend on Figure 3 and Figure 4 It can be seen that, compared with the normal group (triglycerides: 0.10±0.01mmol / gprot, total cholesterol: 0.08±0.01mmol / gprot), the levels of triglycerides and total cholesterol (triglycerides: 0.29±0.03mmol / gprot, total cholesterol: 0.26±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.

[0038] Depend on Figure 3 and Figure 4The results showed that the triglyceride and total cholesterol levels in the zebrafish of the positive control group were 0.13±0.01 mmol / gprot and 0.12±0.01 mmol / gprot, respectively, which were significantly different from those in the model group (triglycerides: 0.29±0.03 mmol / gprot, total cholesterol: 0.26±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.

[0039] Bifidobacterium breve 15701 intervention group (1×10 6 The levels of triglycerides and total cholesterol in zebrafish (CFU / mL) were 0.27±0.02 mmol / gprot and 0.23±0.01 mmol / gprot, respectively, which showed no significant difference compared with the model group (triglycerides: 0.29±0.03 mmol / gprot, total cholesterol: 0.26±0.02 mmol / gprot) (P>0.05). Additionally, the *Bifidobacterium breve* NX-5 intervention group (1×10⁻⁶) showed no significant difference (P>0.05). 6 The levels of triglycerides and total cholesterol in zebrafish (CFU / mL) were 0.16±0.01 mmol / gprot and 0.15±0.01 mmol / gprot, respectively, which were significantly different from those in the model group (triglycerides: 0.29±0.03 mmol / gprot, total cholesterol: 0.26±0.02 mmol / gprot) (P<0.01). Therefore, these results indicate that at the same concentration, *Bifidobacterium breve* NX-5 has a stronger effect on reducing triglycerides and total cholesterol in the zebrafish hyperlipidemia model than *Bifidobacterium breve* 15701, demonstrating a good lipid-lowering effect.

[0040] 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 Bifidobacterium breve NX-5 in the preparation of lipid-lowering and weight-loss products, characterized in that, The preservation number of the Bifidobacterium breve NX-5 is CGMCC No. 20113.

2. The application of Bifidobacterium breve NX-5 according to claim 1 in the preparation of lipid-lowering and weight-loss products, characterized in that, The Bifidobacterium NX-5 was a bacterial suspension.

3. The application of Bifidobacterium breve NX-5 according to claim 1 in the preparation of lipid-lowering and weight-loss products, characterized in that, The products mentioned are food, medicine, and health products.

4. The use of the Bifidobacterium NX-5 as described in claim 1 in the preparation of products that promote lipid metabolism.

5. The application of Bifidobacterium breve NX-5 according to claim 4 in the preparation of products that promote lipid metabolism, characterized in that, The Bifidobacterium NX-5 was a bacterial suspension.

6. The application of Bifidobacterium breve NX-5 according to claim 4 in the preparation of products that promote lipid metabolism, characterized in that, The products mentioned are food, medicine, and health products.

7. The use of the Bifidobacterium breve NX-5 as described in claim 1 in the preparation of products that lower triglycerides and total cholesterol.

8. The application of Bifidobacterium breve NX-5 according to claim 7 in the preparation of products that lower triglycerides and total cholesterol, characterized in that, The Bifidobacterium NX-5 was a bacterial suspension.

9. The application of Bifidobacterium breve NX-5 according to claim 7 in the preparation of products that lower triglycerides and total cholesterol, characterized in that, The products mentioned are food, medicine, and health products.

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