Probiotic-prebiotic composition and its application in preparing obesity prevention product

Abstract

This invention relates to a probiotic and prebiotic composition and its application in the preparation of products for preventing obesity. The probiotics include *Lactobacillus johnsonii*, and the prebiotics are xylooligosaccharides and isomaltooligosaccharides in a mass ratio of (0.5-2.5):(0.5-2.5). The probiotics and prebiotics in the composition have a synergistic effect, inhibiting weight gain in mice on a high-fat diet, improving blood lipid levels in mice on a high-fat diet, reducing total cholesterol, triglycerides, or low-density lipoprotein cholesterol in mouse serum, increasing high-density lipoprotein cholesterol in serum, improving ectopic fat deposition, and reducing liver and epididymal fat weight. It can be used to prepare drugs for preventing obesity or non-alcoholic fatty liver disease, and has broad application prospects, providing a new approach for the prevention or treatment of obesity.

Description

Technical Field

[0001] This invention relates to the field of medical technology, and in particular to probiotic and prebiotic compositions and their application in the preparation of products for preventing obesity. Background Technology

[0002] Obesity is a chronic and complex metabolic disease characterized by excessive accumulation of body fat. When the body consumes more calories than it burns, the excess calories are stored as fat. When the amount of fat accumulated exceeds normal physiological needs and reaches a certain level, it develops into obesity. Obesity not only affects an individual's appearance and self-confidence but is also closely related to other health problems. Since 1990, the global adult obesity rate has doubled, while the adolescent obesity rate has quadrupled, making obesity one of the most serious public health problems worldwide. Obesity not only severely impacts individual health but also has negative effects on socioeconomic and psychological well-being. Therefore, the prevention and treatment of obesity are of great significance for improving the overall health of human society and reducing the burden on healthcare.

[0003] According to the consensus statement of the International Scientific Association for Probiotics and Prebiotics (ISAPP), prebiotics are substances that can be selectively utilized by host microorganisms and can confer health benefits to the host. Among them, non-digestible oligosaccharides, composed of 2-10 sugar units linked by glycosidic bonds, are an important class of prebiotics.

[0004] Despite this, the application of non-digestible oligosaccharides (NOS) in the prevention and treatment of obesity still faces numerous challenges. While a single type of NOS may exhibit significant probiotic effects in specific areas, its effects are often limited. Long-term intake of the same oligosaccharide alone may lead to an imbalance in the gut microbiota, thus affecting its long-term benefits. Furthermore, differences in gut microbiota composition and metabolic capacity among individuals make it difficult to predict and standardize the effects of a single oligosaccharide across different populations. To overcome the limitations of single oligosaccharides and enhance their anti-obesity effects, recent studies have increasingly focused on the combined use of NOS. This combination strategy aims to achieve more comprehensive gut health regulation and more significant anti-obesity effects through the complementary effects of different types of oligosaccharides.

[0005] However, there are many types of non-digestible oligosaccharides, each with different structures, molecular weights, solubilities, and metabolic pathways in the gut, leading to specific effects on the gut microbiota. Using multiple non-digestible oligosaccharides in a compound significantly increases product formulation complexity. Therefore, these factors must be carefully considered during the compounding process to ensure overall product quality and stability. This complexity may increase production and regulatory difficulties, as well as production costs. Although the initial intention of compounding is to achieve synergistic effects, the interaction mechanisms between different oligosaccharides are not fully understood. Therefore, in practical applications, it is difficult to accurately predict and control the specific effects of compounded products on the gut microbiota, metabolic pathways, and human health. This uncertainty may lead to compounded products not achieving the expected results. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a composition of probiotics and prebiotics and its application in the preparation of products for preventing obesity. This invention investigated the anti-obesity effects of different combinations of non-digestible oligosaccharides and determined the most effective combination by adjusting the ratios, namely xylooligosaccharide and isomaltooligosaccharide. Subsequently, the intestinal probiotic *Lactobacillus johnsonii* was used to ferment the xylooligosaccharide and isomaltooligosaccharide composition, verifying that the fermentation product further enhanced the anti-obesity effect.

[0007] The first object of the present invention is to provide a composition of probiotics and prebiotics, wherein the probiotics include Lactobacillus johnsonii, and the prebiotics are xylooligosaccharides and isomaltooligosaccharides, wherein the mass ratio of xylooligosaccharides to isomaltooligosaccharides is (0.5-2.5):(0.5-2.5).

[0008] Furthermore, the mass ratio of xylooligosaccharide to isomaltooligosaccharide is (0.5-1.5):(1.5-2.5).

[0009] Preferably, the mass ratio of xylooligosaccharide to isomaltooligosaccharide is 1:2.

[0010] Furthermore, the viable count of the *Lactobacillus johnsonii* is (1-5) × 10⁻⁶. 5 CFU / mL.

[0011] Preferably, the viable count of the *Lactobacillus johnsonii* is 1 × 10⁻⁶. 5 CFU / mL.

[0012] A second objective of this invention is to provide the use of the above-described composition in the preparation of products for preventing obesity.

[0013] Furthermore, the obesity mentioned is obesity induced by a high-fat diet.

[0014] Furthermore, the obesity prevention product is a drug.

[0015] A fourth object of the present invention is to provide a medicament for preventing obesity, comprising the above-described composition.

[0016] Furthermore, the drug is ingested via the digestive tract.

[0017] Furthermore, the drug is in the form of tablets, capsules, pills, granules, powders, or oral liquids.

[0018] A fifth object of the present invention is to provide the use of the above composition in the preparation of products for the prevention of non-alcoholic fatty liver disease.

[0019] A sixth object of the present invention is to provide the use of the above composition in the preparation of products for the prevention of hyperlipidemia.

[0020] A seventh object of the present invention is to provide the use of the above composition in the preparation of products that increase high-density lipoprotein cholesterol in serum.

[0021] An eighth object of the present invention is to provide the use of the above-described composition in the preparation of products that lower serum total cholesterol, triglycerides or low-density lipoprotein cholesterol.

[0022] The beneficial effects of this invention are:

[0023] The probiotic and prebiotic composition provided by this invention can inhibit weight gain in mice on a high-fat diet, improve blood lipid levels in mice on a high-fat diet, reduce total cholesterol, triglycerides, or low-density lipoprotein cholesterol in mouse serum, and increase high-density lipoprotein cholesterol in serum. Simultaneously, this composition can improve ectopic fat deposition, reduce liver and epididymal fat weight, and inhibit the enlargement of white adipocytes and the whitening of brown adipocytes. Compared with mice on a high-fat diet, the combination of xylooligosaccharides and isomaltooligosaccharides upregulated the expression levels of stearoyl-CoA desaturase 1 (Scd1) and peroxisome activating receptor α (Pparα) mRNA in the liver, which is beneficial for the oxidative breakdown of fatty acids. It also promoted the production of acetic acid and propionic acid by the intestinal microbiota of mice on a high-fat diet, and reorganized the intestinal microbiota community structure of mice on a high-fat diet. The abundance of Lactobacillus and Lactobacillus johnsonii was significantly upregulated. Therefore, this combination has a significant synergistic effect. Prebiotics can promote the proliferation of probiotics, and the efficacy of prebiotics is further enhanced under the action of probiotics. It can be used to prepare drugs for the prevention of obesity and hyperlipidemia, and has broad application prospects. Attached Figure Description

[0024] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0025] Figure 1 This is an evaluation of the anti-obesity efficacy of the non-digestible oligosaccharide composition in mice after 12 weeks of dietary intervention in mice, where AD represents the change in mouse body weight, E represents the energy intake of mice, F represents the weight of mouse liver, GJ represents the levels of total cholesterol, triglycerides, low-density lipoprotein cholesterol and high-density lipoprotein cholesterol in mouse serum, and K represents the weight of epididymal fat in mice.

[0026] Figure 2 This describes the regulatory effect of the non-digestible oligosaccharide composition on lipid metabolism in mice after 12 weeks of dietary intervention in Example 3 of the present invention, where A represents white adipose tissue, B represents brown adipose tissue, C represents liver tissue, and D represents the expression level of lipid metabolism-related mRNAs in the liver of mice.

[0027] Figure 3 This is the regulatory effect of the non-digestible oligosaccharide composition on the intestines of mice after 12 weeks of dietary intervention in Example 4 of the present invention. In this example, A is the content of short-chain fatty acids, B is the principal component analysis diagram of mouse intestinal flora, C is the abundance of Lactobacillus, and D is the abundance of Lactobacillus johnsonii.

[0028] Figure 4 This is an evaluation of the anti-obesity efficacy of different ratios of xylooligosaccharide and isomaltooligosaccharide compositions in mice after 12 weeks of dietary intervention, where A represents the change in mouse body weight and B represents the energy intake of mice. Detailed Implementation

[0029] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0030] Example 1: Dietary intervention strategy for non-digestible oligosaccharide compositions

[0031] Fifty-four C57BL / 6J mice were housed at a temperature of 20-26℃, relative humidity of 40-70%, noise level less than 60dB, and illuminance of 15-20lx, with a 12-hour light-dark cycle. Different types and formulations of functional oligosaccharides were used for dietary intervention. To ensure consistent energy levels in all groups, 3% of non-digestible oligosaccharides replaced the dietary fiber in the high-fat (HFD) diet. All mice were randomly divided into nine groups of six. The feed formulations for each group are shown in Table 1, where " / " indicates no addition.

[0032] (1) Standard diet (Chow) group;

[0033] (2) High-fat diet (HFD) group;

[0034] (3) High-fat diet + 3% xylooligosaccharide (HFD+XOS) group;

[0035] (4) High-fat diet + 3% galactooligosaccharides (HFD+GOS) group;

[0036] (5) High-fat diet + 3% isomaltooligosaccharide (HFD+IMO) group;

[0037] (6) High-fat diet + 1.5% xylooligosaccharide + 1.5% galactooligosaccharide (HFD+XOS+GOS) group;

[0038] (7) High-fat diet + 1.5% xylooligosaccharide + 1.5% isomaltooligosaccharide (HFD+XOS+IMO) group;

[0039] (8) High-fat diet + 1.5% galactooligosaccharide + 1.5% isomaltooligosaccharide (HFD+GOS+IMO) group;

[0040] (9) High-fat diet + 1% xylooligosaccharide + 1% galactooligosaccharide + 1% isomaltooligosaccharide (HFD+XOS+GOS+IMO) group.

[0041] Table 1 Experimental feed formulation

[0042]

[0043]

[0044] Example 2: Evaluation of the anti-obesity efficacy of non-digestible oligosaccharide compositions

[0045] Different types and ratios of functional oligosaccharides were used to treat mice ingesting HFD through a 12-week dietary intervention. Figure 1 B indicates that, compared to mice fed an HFD diet, adding a combination of XOS and IMO to the HFD diet, starting from week 8 of dietary intervention, significantly inhibited weight gain in mice. After 12 weeks of feeding, the HFD+XOS+IMO group showed a 10.80% weight loss compared to the HFD group, while adding XOS or IMO alone to the diet did not achieve this effect. Furthermore, there was no significant difference in energy intake among all mice except the Chow group. Figure 1 E), indicating that the XOS and IMO combination synergistically inhibited HFD diet-induced weight gain in mice. Furthermore, after 12 weeks of dietary intervention, the liver weight in the HFD+XOS+IMO group was reduced by 18.58% compared to the HFD group. Figure 1 F), the epididymal fat weight decreased by 18.33% ( Figure 1K). The non-digestible oligosaccharide composition improved blood lipid levels in HFD mice. Compared with the HFD group, the serum low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) levels in the HFD+XOS+IMO group decreased by 24.79% and increased by 34.43%, respectively. Figure 1 I and Figure 1 J).

[0046] Example 3: Evaluation of the efficacy of non-digestible oligosaccharide compositions in improving lipid metabolism

[0047] A 12-week dietary intervention using a single type and non-digestible oligosaccharide combination in mice fed with HFD resulted in improved pathological morphology of white adipose tissue, brown adipose tissue, and liver tissue in the HFD+XOS+IMO group. Compared with mice on a high-fat diet, the HFD+XOS+IMO group inhibited the accumulation of triglycerides in white and brown adipocytes, and suppressed the enlargement of white adipocytes and the albinism of brown adipocytes. Figure 2 (A and 2B). Meanwhile, the HFD+XOS+IMO group reduced fatty infiltration in the liver of mice fed a high-fat diet, thereby alleviating the occurrence of non-alcoholic fatty liver disease. Figure 2 C). Real-time quantitative PCR was used to analyze the expression of lipid metabolism-related gene mRNAs, including Cd36 (fatty acid transporter), Pparγ (peroxisome-activated receptor γ), Acc1 (acetyl-CoA carboxylase), Fasn (fatty acid synthase), Srebp1c (sterol regulatory element-binding protein), Scd1 (stearoyl-CoA desaturase 1), Cpt1a (carnitine palmitoyltransferase 1a), and Pparα (peroxisome-activated receptor). The results are as follows: Figure 2 As shown in D. Compared with mice on a high-fat diet, the combination of xylooligosaccharides and isomaltooligosaccharides upregulated the expression levels of stearoyl-CoA desaturase 1 (Scd1) and peroxisome activating receptor α (Pparα) mRNA in the liver, which is conducive to the oxidative breakdown of fatty acids.

[0048] Example 4: Non-digestible oligosaccharide composition regulates gut microbiota

[0049] Mice ingesting HFD were subjected to a 12-week dietary intervention using a single type and a non-digestible oligosaccharide composition. The content of short-chain fatty acids in the cecal contents of the mice was then determined. Figure 3A shows that the concentrations of acetic acid and propionic acid in the cecal contents of mice in the HFD group were 29.94 μmol / g and 9.70 μmol / g, respectively, while the concentrations of acetic acid and propionic acid in the HFD+XOS+IMO group increased to 37.95 μmol / g and 18.32 μmol / g, respectively. Adding XOS or IMO alone to the diet did not achieve this effect. Acetic acid produced by the gut microbiota can serve as an adjunct therapy for managing metabolic syndrome by altering the metabolism of taurine-bound bile acids, while propionic acid affects fatty acid metabolism in the liver by regulating transporter activity. Principal component analysis of the mouse gut microbiota yielded the following results: Figure 3 As shown in Figure B, the HFD+XOS+IMO group altered the gut microbiota structure of mice on a high-fat diet. Further analysis of the gut microbiota revealed a significant upregulation of *Lactobacillus* spp. abundance in the HFD+XOS+IMO group, with the relative abundance increasing from 4.26% to 13.48%. Figure 3 C), among which the abundance of Lactobacillus johnsonii was most significantly increased, from 2.33% to 10.87%. Figure 3 D).

[0050] Example 5: Dietary intervention strategies for different ratios of xylooligosaccharide and isomaltooligosaccharide compositions

[0051] Twenty-four C57BL / 6J mice were housed at a temperature of 20-26℃, relative humidity of 40-70%, noise level less than 60dB, and illuminance of 15-20lx, with a 12-hour light-dark cycle. Dietary interventions were conducted using different ratios of xylooligosaccharides and isomaltooligosaccharides. To ensure consistent energy levels in the diets across all groups, 3% xylooligosaccharides and isomaltooligosaccharides were used to replace the dietary fiber in the HFD diet. All mice were randomly divided into four groups of six each. The feed formulations for each group are shown in Table 1.

[0052] (1) High-fat diet + 1% xylooligosaccharide + 2% isomaltooligosaccharide (HFD + 1% XOS + 2% IMO) group;

[0053] (2) High-fat diet + 2% xylooligosaccharide + 1% isomaltooligosaccharide (HFD + 2% XOS + 1% IMO) group;

[0054] (3) High-fat diet + 0.5% xylooligosaccharide + 2.5% isomaltooligosaccharide (HFD + 0.5% XOS + 2.5% IMO) group;

[0055] (4) High-fat diet + 2.5% xylooligosaccharide + 0.5% isomaltooligosaccharide (HFD + 2.5% XOS + 0.5% IMO) group.

[0056] Table 2 Experimental feed formulation

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[0058]

[0059] Example 6: Evaluation of the anti-obesity efficacy of different ratios of xylooligosaccharide and isomaltooligosaccharide compositions

[0060] Mice ingesting HFD were subjected to a 12-week dietary intervention using different ratios of xylooligosaccharides and isomaltooligosaccharides. Figure 4 As shown in Figure B, the energy intake of each group of mice was comparable during the dietary intervention period. Compared to the HFD+1.5%XOS+1.5%IMO group (data from Example 2), the HFD+1%XOS+2%IMO group and the HFD+0.5%XOS+2.5%IMO group had a more positive effect on combating obesity, with the HFD+1%XOS+2%IMO group showing the most significant effect. After 12 weeks of dietary intervention, the body weight of the HFD+1%XOS+2%IMO group decreased by 6.41% compared to the HFD+1.5%XOS+1.5%IMO group. Figure 4 A) Serum levels of total cholesterol, triglycerides, and LDL-C decreased by 8.24%, 4.51%, and 26.90%, respectively, while HDL-C levels increased by 8.04%, and liver and epididymal fat weight decreased by 8.57% and 16.24%, respectively (Table 3).

[0061] Table 3. Anti-obesity effects of different ratios of xylooligosaccharide and isomaltooligosaccharide compositions.

[0062]

[0063] Example 7: Probiotic Fermented Functional Oligosaccharide Composition

[0064] MRS medium without added glucose was used as the basal medium. The medium formula was as follows: 10.0 g peptone, 10.0 g beef extract, 4.0 g yeast extract, 2.0 g anhydrous sodium acetate, 2.0 g diammonium citrate, 2.6 g dipotassium hydrogen phosphate trihydrate, 0.1 g magnesium sulfate heptahydrate, 0.05 g manganese sulfate monohydrate, 1.0 g Tween, and 0.5 g L-cysteine ​​hydrochloride. The above ingredients were added to distilled water and the volume was adjusted to 900 mL. The pH was adjusted to 6.2-6.4 and autoclaved at 121 °C for 20 min.

[0065] 100 mL of a 0.2 g / mL xylooligosaccharide and isomaltooligosaccharide combination solution filtered through a 0.22 μm aqueous membrane was added to MRS medium as a carbon source, with a final concentration of 20 g / L in the fermentation broth. Lactobacillus johnsonii in the logarithmic growth phase was added to the culture medium for fermentation of the xylooligosaccharide and isomaltooligosaccharide combination, adjusting the initial concentration of Lactobacillus johnsonii in the fermentation broth to 10 g / L. 5 CFU / mL, 37℃, anaerobic culture for 24 h. After the fermentation period, the fermentation broth was centrifuged at 5000 rpm for 15 min, the supernatant of the culture broth was collected, and the supernatant was freeze-dried under vacuum for 48 h to obtain the fermentation product. The product was then administered to C57BL / 6J mice on a high-fat diet by gavage at a dose of 0.4 mg / kg of mouse body weight every 2 days for 12 weeks.

[0066] Different ratios of xylooligosaccharide and isomaltooligosaccharide combinations were fermented using *Lactobacillus johnsonii*. C57BL / 6J mice were fed a high-fat diet along with the fermentation products for 12 weeks. Table 4 shows that the fermentation products of the xylooligosaccharide and isomaltooligosaccharide combinations further enhanced the anti-obesity effect, with the HFD+1%XOS+2%IMO group showing the most significant effect. Furthermore, the serum lipid levels in the HFD+1%XOS+2%IMO group were closer to normal values, with total cholesterol, triglycerides, LDL cholesterol, and HDL cholesterol levels of 6.63, 1.21, 1.25, and 4.19 mmol / L, respectively.

[0067] Table 4. Anti-obesity effects of probiotic fermentation products with different ratios of xylooligosaccharide and isomaltooligosaccharide.

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[0069]

[0070] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A composition of probiotics and prebiotics, characterized in that: The probiotics include Lactobacillus johnsonii, and the prebiotics are xylooligosaccharides and isomaltooligosaccharides, with a mass ratio of xylooligosaccharides to isomaltooligosaccharides of (0.5-2.5):(0.5-2.5).

2. The composition according to claim 1, characterized in that: The mass ratio of xylooligosaccharide to isomaltooligosaccharide is (0.5-1.5):(1.5-2.5).

3. The composition according to claim 1, characterized in that: The viable count of the Lactobacillus johnsonii was (1-5) × 10⁻⁶. 5 CFU / mL.

4. The use of the composition according to any one of claims 1-3 in the preparation of an obesity prevention product.

5. The application according to claim 4, characterized in that: The obesity mentioned refers to obesity induced by a high-fat diet.

6. The application according to claim 4, characterized in that: The obesity prevention product mentioned is a medicine.

7. A drug for preventing obesity, characterized in that: Includes the composition according to any one of claims 1-3.

8. The drug according to claim 7, characterized in that: The drug is ingested via the digestive tract.

9. Use of the composition according to any one of claims 1-3 in the preparation of a product for the prevention of non-alcoholic fatty liver disease.

10. The use of the composition according to any one of claims 1-3 in the preparation of a product for preventing hyperlipidemia.

11. Use of the composition according to any one of claims 1-3 in the preparation of a product that increases high-density lipoprotein cholesterol in serum.

12. Use of the composition according to any one of claims 1-3 in the preparation of products that lower total cholesterol, triglycerides or low-density lipoprotein cholesterol in serum.

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