Application of lactobacillus acidophilus LA-03 in preparation of fat-reducing and weight-losing products

Abstract

The invention discloses application of lactobacillus acidophilus LA-03 in preparation of fat-reducing and weight-losing products, and belongs to the technical field of microorganisms. The lactobacillus acidophilus LA-03 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, has the potential of being applied to losing weight and regulating the blood fat level, and can be used for preventing and treating hyperlipidemia. Theoretical reference and guidance basis are provided for developing probiotic preparations for treating or preventing obesity and hyperlipidemia by using the lactobacillus acidophilus LA-03.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more specifically to the application of Lactobacillus acidophilus LA-03 in the preparation of lipid-lowering and weight-loss products. Background Technology

[0002] Hyperlipidemia is a disease characterized by excessive lipid metabolism, primarily manifested as dizziness, headache, chest tightness and pain, abdominal distension, and obesity. With improved living standards, the number of patients with this condition is increasing. While there are many medications available to treat hyperlipidemia, their biggest drawbacks are low cure rates, high prices, predominance of Western medicine, and significant 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 Lactobacillus acidophilus LA-03 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 Lactobacillus acidophilus LA-03 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 Lactobacillus acidophilus LA-03 in the preparation of lipid-lowering and weight-loss products, wherein the preservation number of Lactobacillus acidophilus LA-03 is CGMCC No.22239 (see patent number 202111249043.X).

[0009] Furthermore, the application of the aforementioned Lactobacillus acidophilus LA-03 in the preparation of products that promote fat metabolism.

[0010] Furthermore, the application of the aforementioned Lactobacillus acidophilus LA-03 in the preparation of products that lower triglycerides and total cholesterol.

[0011] Furthermore, the Lactobacillus acidophilus LA-03 is a bacterial suspension.

[0012] At the same concentration, *Lactobacillus acidophilus* LA-03 promoted fat metabolism in zebrafish more strongly than *Lactobacillus acidophilus* 4356, demonstrating a good lipid-lowering and weight-loss effect. At the same concentration, *Lactobacillus acidophilus* LA-03 also reduced triglycerides and total cholesterol in a zebrafish hyperlipidemia model more strongly than *Lactobacillus acidophilus* 4356, demonstrating a good lipid-lowering effect.

[0013] As can be seen from the above technical solution, compared with the prior art, the present invention discloses the application of Lactobacillus acidophilus LA-03 in the preparation of lipid-lowering and weight-loss products. Lactobacillus acidophilus LA-03 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 acidophilus LA-03. Attached Figure Description

[0014] 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.

[0015] Figure 1 The attached figure is a visual representation of the effect of Lactobacillus acidophilus LA-03 of the present invention on the body fat level of zebrafish;

[0016] Wherein, A: normal group; B: positive control group; C: 1×10 6 CFU / mL Lactobacillus acidophilus 4356; D: 1×10 6 CFU / mL Lactobacillus acidophilus LA-03;

[0017] Figure 2 The attached figure is a statistical graph showing the effect of Lactobacillus acidophilus LA-03 of the present invention on the body fat level of zebrafish;

[0018] Figure 3 The attached figure shows the effect of Lactobacillus acidophilus LA-03 of the present invention on triglycerides in a zebrafish hyperlipidemia model;

[0019] Figure 4 The attached figure shows the effect of Lactobacillus acidophilus LA-03 of the present invention on total cholesterol in a zebrafish hyperlipidemia model. Detailed Implementation

[0020] 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.

[0021] 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 acidophilus 4356 (ATCC 4356) was purchased from Beijing Bio-Bio Biotechnology Co., Ltd.

[0022] Example 1: Preparation of Lactobacillus acidophilus LA-03 bacterial suspension (cells)

[0023] Lactobacillus acidophilus LA-03 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.

[0024] Example 2: Preparation of Lactobacillus acidophilus 4356 bacterial suspension (cells)

[0025] Lactobacillus acidophilus 4356 was activated and cultured, then inoculated into MRS liquid medium and cultured at 37°C for 24 h. After centrifugation at 4°C and 6000 r / min for 10 min, bacterial cells were obtained. The bacterial cells were washed twice with PBS, and then resuspended in PBS. The cell concentration was adjusted to 1×106 CFU / mL to obtain a bacterial suspension (bacterial cells).

[0026] Example 3: Effect of Lactobacillus acidophilus LA-03 on body fat levels in zebrafish

[0027] Healthy wild-type AB zebrafish that had reached 3 days post-fertilization (dpf) were selected and placed in 6-well cell culture plates, 15 fish / well. The experiment included a normal control group, a positive control group (resveratrol), a *Lactobacillus acidophilus* 4356 intervention group, and a *Lactobacillus acidophilus* LA-03 intervention group. The normal control group received PBS, the positive control group received resveratrol solution (50 μmol / L), and the *Lactobacillus acidophilus* 4356 intervention group received 1 × 10⁻⁶ saturated ... 6 Add 1×10 (CFU / mL) 6 CFU / mL Lactobacillus acidophilus 4356, Lactobacillus acidophilus LA-03 intervention group (1×10 6 Add 1×10 (CFU / mL) 6 CFU / mL Lactobacillus acidophilus LA-03 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:

[0028]

[0029] 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.

[0030] 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±5.26%), the fat level in the positive control group zebrafish (47.72±3.64%) was significantly reduced (p<0.005), indicating that this lipid-lowering experiment in zebrafish was effective.

[0031] Depend on Figure 1 and Figure 2 It can be seen that the Lactobacillus acidophilus 4356 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 acidophilus 4356 intervention group (1×10) 6The body fat level in zebrafish (CFU / mL) was 93.77±2.86%, which was not significantly different from that in the normal group (100.00±5.26%) (P>0.05). Furthermore, compared with the normal group, the Lactobacillus acidophilus LA-03 intervention group (1×10⁻⁶ CFU / mL) showed a significantly lower body fat level (93.77±2.86%). 6 The intensity of red fluorescence in zebrafish was reduced (CFU / mL), similar to the positive control group; meanwhile, the Lactobacillus acidophilus LA-03 intervention group (1×10⁻⁶ CFU / mL) showed a decrease. 6 The fat level in zebrafish (CFU / mL) was 57.18±4.14%, which was significantly different from that in the normal group (100.00±5.26%) (P<0.005). Therefore, the above results indicate that at the same concentration, Lactobacillus acidophilus LA-03 has a stronger promoting effect on fat metabolism in zebrafish than Lactobacillus acidophilus 4356, demonstrating a good lipid-lowering and weight-loss effect.

[0032] Example 4: Effects of Lactobacillus acidophilus LA-03 on triglycerides and total cholesterol in a zebrafish hyperlipidemia model.

[0033] 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 acidophilus 4356 intervention group (1×10⁻⁶). 6 CFU / mL), Lactobacillus acidophilus LA-03 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.

[0034] 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 acidophilus 4356 intervention group (1×10 6 Add 1×10 (CFU / mL) 6 CFU / mL Lactobacillus acidophilus 4356; Lactobacillus acidophilus LA-03 intervention group (1×10 6 Add 1×10 (CFU / mL) 6CFU / mL Lactobacillus acidophilus LA-03, 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.

[0035] Data were statistically processed using SPSS 19.0 software. All experimental data were analyzed using... Data are presented, and analyzed using a t-test, compared to the normal group: ### P<0.05; compared with the model group: ** P<0.01, *** P<0.005.

[0036] 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±0.02mmol / gprot, total cholesterol: 0.28±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.

[0037] 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 0.14±0.01 mmol / gprot and 0.17±0.01 mmol / gprot, respectively, which were significantly different from those in the model group (triglycerides: 0.31±0.02 mmol / gprot, total cholesterol: 0.28±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.

[0038] Lactobacillus acidophilus 4356 intervention group (1×10 6The levels of triglycerides and total cholesterol in zebrafish (CFU / mL) were 0.28±0.02 mmol / gprot and 0.25±0.02 mmol / gprot, respectively, which showed no significant difference compared with the model group (triglycerides: 0.31±0.02 mmol / gprot, total cholesterol: 0.28±0.02 mmol / gprot) (P>0.05). Additionally, the *Lactobacillus acidophilus* LA-03 intervention group (1×10⁻⁶) showed no significant difference compared with the model group (P>0.05). 6 The levels of triglycerides and total cholesterol in zebrafish (CFU / mL) were 0.21±0.01 mmol / gprot and 0.20±0.02 mmol / gprot, respectively, which were significantly different from those in the model group (triglycerides: 0.31±0.02 mmol / gprot, total cholesterol: 0.28±0.02 mmol / gprot) (P<0.01). Therefore, these results indicate that at the same concentration, *Lactobacillus acidophilus* LA-03 had a stronger effect on reducing triglycerides and total cholesterol in the zebrafish hyperlipidemia model than *Lactobacillus acidophilus* 4356, demonstrating a good lipid-lowering effect.

[0039] 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 acidophilus LA-03 in the preparation of lipid-lowering and weight-loss products, characterized in that, The Lactobacillus acidophilus LA-03 has the accession number CGMCC No. 22239.

2. The application of Lactobacillus acidophilus LA-03 according to claim 1 in the preparation of lipid-lowering and weight-loss products, characterized in that, The Lactobacillus acidophilus LA-03 is a bacterial suspension.

3. The use of Lactobacillus acidophilus LA-03 as described in claim 1 in the preparation of products that promote fat metabolism.

4. The application of Lactobacillus acidophilus LA-03 according to claim 3 in the preparation of products that promote lipid metabolism, characterized in that, The Lactobacillus acidophilus LA-03 is a bacterial suspension.

5. The use of Lactobacillus acidophilus LA-03 as described in claim 1 in the preparation of products that lower triglycerides and total cholesterol.

6. The application of Lactobacillus acidophilus LA-03 according to claim 5 in the preparation of products that lower triglycerides and total cholesterol, characterized in that, The Lactobacillus acidophilus LA-03 is a bacterial suspension.

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