Probiotic composition for synergistically promoting Akkermansia proliferation and application of probiotic composition in weight loss

Abstract

The invention belongs to the technical field of probiotics, and particularly relates to a bifidobacterium animalis subsp. Lactis B420 and CECT8145 combination for synergistically promoting proliferation of intestinal Akkermansia and application of the bifidobacterium animalis subsp. Lactis B420 and CECT8145 combination in preparation of food for preventing or treating obesity by rapidly exerting a weight-losing effect. The invention discloses a probiotic composition capable of synergistically promoting the proliferation of Akkermansia in intestinal tracts. The bacterial strains in the probiotic composition consist of an animal bifidobacterium lactis subsp. Lactis bacterial strain B420 and a bifidobacterium lactis subsp. Lactis CECT8145, and the bacterial strains in the probiotic composition consist of an animal bifidobacterium lactis subsp. Lactis bacterial strain B420 and a bifidobacterium lactis subsp. Lactis CECT8145. The invention further discloses a preparation method of the probiotic composition capable of synergistically promoting the proliferation of the Akkermansia in the intestinal tracts of the Akkermansia in the intestinal tracts of the Akkermansia in the intestinal tracts of the Akkermansia in the intestinal tracts of the Akkermansia.

Description

Technical Field

[0001] This invention belongs to the field of probiotic technology, specifically relating to a combination of Bifidobacterium animalis subsp. lactis B420 and CECT8145 that synergistically promote the proliferation of Akkermansia in the intestine, and its application in the preparation of foods for the prevention or treatment of obesity, thereby rapidly exerting weight loss effects. Background Technology

[0002] The World Health Organization defines obesity as an abnormal or excessive accumulation of fat that can impair health, generally considered to be a BMI exceeding 25 kg / m². 2 Considered overweight, exceeding 30 kg / m 2 This is obesity. Obesity is a chronic disease that increases the risk of many diseases, including cardiovascular disease and diabetes, seriously endangering human health. Excessive intake of high-calorie foods rich in fat and sugar, coupled with reduced energy expenditure, has led to a rise in global obesity rates. Preventing and controlling obesity is of paramount importance for improving public health.

[0003] There is a complex relationship between obesity and gut microbiota. Gut microbiota dysbiosis may lead to obesity, while obesity can also affect the composition of gut microbiota. Gut microbiota is closely related to the occurrence and development of obesity by influencing nutrient absorption, appetite regulation, and inflammatory responses. Probiotics can help improve obesity by regulating gut microbiota. Probiotics can promote the growth of beneficial bacteria and inhibit harmful bacteria, thereby improving the balance of the gut microbiota and influencing weight and fat metabolism.

[0004] Akkermansia is an obligate anaerobic Gram-negative bacterium that primarily colonizes the intestinal mucus layer of humans and animals. Numerous studies have demonstrated that Akkermansia possesses various important physiological functions, including weight loss, lipid reduction, and anti-inflammation. Currently, Akkermansia has obtained safety certifications in the EU and the US, allowing its use in food and health supplements. However, it has not yet been approved as a raw material for ordinary food and health supplements in my country. Furthermore, directly ingesting live bacteria in the highly acidic environment of gastric juice can damage the cellular structure of Akkermansia, inactivating it and affecting its normal metabolism and survival ability, making it difficult for it to colonize the intestines and exert its healthy activity. In addition, while Akkermansia is generally considered safe, some risks and side effects exist, especially for individuals with weakened immune systems; regulating endogenous Akkermansia can mitigate these risks and side effects.

[0005] EP 3442550 B1 discloses the application of Bifidobacterium animalis subsp. lactis B420 probiotic in the treatment and prevention of obesity. This patent uses Bifidobacterium animalis subsp. lactis strain B420 as a probiotic and finds that it can significantly reduce energy and fat intake in humans by influencing the gut microbiota and energy homeostasis, showing great potential in weight management and interventions for obesity-related metabolic health. Strain B420 is commercially available from DuPont Nutrition Biosciences ApS.

[0006] CN 105849248A discloses the application of Bifidobacterium lactis subsp. CECT8145 probiotic (Bifidobacterium lactis subsp. CECT8145) in the treatment and prevention of obesity and related diseases. This patent utilizes the novel strain Bifidobacterium lactis subsp. CECT8145 and its bioactive compounds to regulate gene expression. It overcomes the problems of existing technologies failing to effectively induce aversion to fat and increase antioxidant resistance, achieving a significant reduction in body fat and cholesterol levels, and optimizing the management of overweight and obesity.

[0007] Studies such as Pedret et al., Int J Obes (2019), and Stenman et al., EBioMedicine (2016) have shown that Bifidobacterium animalis B420 can promote the growth of Akkermansia in the gut, which is an important pathway for its weight-loss effects. However, the ability of Bifidobacterium animalis B420 to promote Akkermansia growth is limited. After three months of human intervention, Akkermansia abundance is low, and the weight loss effect is not good. Moreover, the time required for it to exert its weight-loss effect is long, which is difficult to meet the needs of some obese patients. Therefore, there is a need to propose a method that can significantly increase the abundance of Akkermansia in the gut to achieve rapid weight loss. Summary of the Invention

[0008] The problem to be solved by this invention is to provide a probiotic combination that synergistically promotes the proliferation of Akkermansia and its application in weight loss.

[0009] To address the aforementioned problems, this invention provides a probiotic composition that synergistically promotes the proliferation of Akkermansia in the intestine. The probiotic composition comprises the strains of Bifidobacterium animalis subsp. lactis strain B420 and Bifidobacterium animalis subsp. lactis CECT8145.

[0010] As an improvement to the probiotic composition of the present invention: the ratio of the number of live bacteria of B420 to CECT 8145 in the probiotic composition is 1~10:1~10.

[0011] As a further improvement to the probiotic composition of the present invention: the ratio of the number of live bacteria of B420 to CECT 8145 in the probiotic composition is 1:1.

[0012] The present invention also provides a probiotic preparation using the above-mentioned probiotic composition, wherein the sum of the viable counts of B420 and CECT8145 is ≥5 × 10⁻⁶. 7 CFU / g (e.g., 5×10⁻⁶) 7 CFU / g ~ 1×10 10 CFU / g).

[0013] As an improvement to the probiotic agent of the present invention: the sum of the viable counts of B420 and CECT 8145 is 5 × 10⁻⁶. 7 CFU / g ~ 1×10 8 CFU / g.

[0014] As a further improvement to the probiotic agent of the present invention, the dosage form of the probiotic agent includes conventional lyophilized powder, tablets, granules, and capsules.

[0015] The present invention also provides the use of the above-mentioned probiotic composition or probiotic agent for relieving obesity symptoms.

[0016] An improvement to the use of this invention is to reduce the weight of obese individuals and shorten the time required for weight loss.

[0017] As a further improvement to the use of the present invention: increase the abundance of Akkermansia in the gut.

[0018] This invention provides a combination of Bifidobacterium lactis subsp. B420 and CECT8145, which synergistically promote the proliferation of Akkermansia in the intestine, greatly increasing its abundance, thereby rapidly exerting a weight loss effect.

[0019] This invention creatively discovers that combining Bifidobacterium animalis B420 and CECT 8145 can synergistically promote the proliferation of Akkermansia in the gut, resulting in a synergistic effect in alleviating obesity symptoms. Compared to using either strain alone, this combination achieves the aforementioned effects more rapidly and significantly, yielding technical results that are difficult for those skilled in the art to foresee, and providing a new strategy for the prevention or treatment of obesity. Both strains are probiotics permitted for food use, ensuring high safety and facilitating the development of commercially viable probiotic products.

[0020] It should be noted that: 1. Akk is a next-generation probiotic widely recognized in recent years for its immense potential, with its role in metabolic health attracting considerable attention. However, its exclusion from the "List of Strains that Can Be Used in Food" creates a core contradiction: despite its proven efficacy, companies cannot legally mass-produce Akk as a "probiotic" ingredient for use in mainstream foods and health supplements. This severely hinders its application in the Chinese market, creating a significant industrialization barrier. This invention combines two approved and safe traditional probiotics to synergistically proliferate Akk bacteria in the gut. This means that this invention solves the aforementioned problem, providing a new and compliant industrialization path for the use of Akk.

[0021] 2. As is common knowledge, different strains may have antagonistic or competitive relationships, or even weaken each other's functions, making the results of combination highly uncertain. Therefore, the rational selection of strains, such as how to combine them to achieve the desired effect, requires creative effort.

[0022] In terms of technical effectiveness, this invention utilizes two types of Bifidobacterium animalis to synergistically proliferate Akk, achieving a better and faster weight loss effect, rather than simply summing the effects of the two strains. Industrially, this invention further solves a pressing regulatory challenge in the field, providing a legal and efficient solution to achieve functions that previously required unapproved strains. This technical solution, proposed to address a practical industrial dilemma, is significantly innovative.

[0023] The probiotics involved in this invention have a simple preparation process, are suitable for large-scale industrial production, and have significant practical value.

[0024] The probiotic preparation of this invention is taken orally, for example, approximately (6.5~13) × 10 ml per day. 8 CFU. Attached Figure Description

[0025] Figure 1 This is a statistical graph showing the results of the weight changes of mice in each group; Figure 2 This is a statistical graph showing the final weight change of mice in each group; Figure 3 This is a statistical chart showing the time required for mice in each treatment group to achieve the same weight gain (15%). Figure 4 This is a statistical graph showing the relative abundance of Akkermansia in the intestines of mice in each group; Figure 5 This is a statistical graph showing the serum TNF-α measurement results of each group of mice; In the above figures, "*" indicates p < 0.05, and "****" indicates p < 0.0001. Detailed Implementation

[0026] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto: The Bifidobacterium animalis subsp. lactis CECT8145 and Bifidobacterium animalis subspecies lactis strain 420 (B420) used in this invention are both existing known bacteria.

[0027] B420 bacterial culture (5×10) 7 The preparation method for CFU / mL is as follows: Take one cryovial of Bifidobacterium animalis B420 stored at -80℃ and thaw it on ice. Under aseptic conditions, take 100 μL of bacterial culture and add it to MRS broth supplemented with 0.05% cysteine ​​hydrochloride. Amplify to the logarithmic growth phase under anaerobic conditions at 37℃. After amplification, measure OD600 and calculate the current concentration of bacterial culture using the CFU-OD curve.

[0028] Take an appropriate amount of bacterial suspension, centrifuge at 4000 rpm for 5 min at 4 ℃, and discard the supernatant. Thoroughly pipette the bacterial sludge with physiological saline, resuspend, and dilute to 5 × 10⁻⁶. 7 CFU / mL is sufficient.

[0029] CECT4145 bacterial culture (5×10) 7 The preparation method for CFU / mL is the same as above, except that the strain is changed to CECT4145.

[0030] B420 + CECT4145 mixed bacterial culture (total viable count 5 × 10⁻⁶) 7 The preparation method for B420 bacterial suspension (CFU / mL, with a viable count ratio of 1:1) is as follows: 5 × 10⁻⁶ CFU / mL of the prepared B420 bacterial suspension (5 × 10⁻⁶ CFU / mL, with a viable count ratio of 1:1) is as follows: 7 CFU / mL) and CECT4145 bacterial culture (5×10) 7 When CFU / mL is mixed in equal volumes under aseptic conditions, a mixed bacterial suspension of B420 + CECT4145 (total viable count 5 × 10⁻⁶ CFU / mL) is prepared. 7 CFU / mL, with a viable bacteria ratio of 1:1.

[0031] Example 1: This example studies the effects of a 1:1 ratio of B420 and CECT8145 on body weight and gut microbiota in high-fat diet-induced obese mice. (1) Grouping of animals: Fifty SPF-grade male C57BL / 6J mice (6 weeks old, 18-22g) were randomly divided into 5 groups of 10 mice each: control group (Chow), model group (HFD), B420 group, CECT8145 group, and B420+CECT8145 group. The mice were housed at a constant temperature of 25±2℃, humidity of 50±5%, noise level ≤60 dB, and illumination of 15-20 LX.

[0032] (2) Modeling and intervention methods: Mice were first acclimatized for 7 days. Starting from day 8, they were fed a high-fat diet (consisting of 60% regular feed, 20% sucrose, 17.5% lard and 2.5% cholesterol) to the model group mice and the probiotic intervention group mice (including the B420 group, CECT8145 group, and B420+CECT8145 group). The control group was fed a regular diet.

[0033] In addition, mice in the B420 group, CECT8145 group, and B420+CECT4145 group were administered 0.2 mL of B420 bacterial solution (5 × 10⁻⁶) by gavage daily starting from day 8. 7 CFU / mL), CECT4145 bacterial culture (5×10) 7 CFU / mL), B420+CECT4145 mixed bacterial culture (total viable count 5×10⁻⁶) 7 The CFU / mL count was 1:1 (the ratio of viable bacteria between the two groups was 1:1). The control and model groups were administered an equal volume of physiological saline by gavage. The gavage treatment lasted for 8 weeks.

[0034] Note: After 7 days of acclimatization feeding, there was no significant difference in body weight among the mice.

[0035] (3) During the experiment, the weight of the mice was measured weekly after the start of gavage.

[0036] like Figure 1 and 2 As shown, the body weight of mice in the model group (HFD) was significantly higher than that in the control group (Chow). Compared with the model group mice, the body weight of mice in all groups was significantly lower at week 8 after being fed the probiotics and their combinations involved in this invention; however, the body weight gain of mice in the B420+CECT8145 group was significantly lower than that of mice in the B420 and CECT4145 groups.

[0037] (4) The time required to achieve the same weight gain is used as an evaluation indicator: At the end of the experiment (56 days after gavage), the final weight gain in the Chow group was 19.1%.

[0038] During the experiment, it was found that the model group (HFD) mice gained weight significantly faster than other groups. They reached the same weight gain level as the Chow group in just 17 days after gavage.

[0039] The weight gain of mice treated with strains B420 or CECT8145 alone was delayed, taking approximately 25 to 28 days after gavage to reach the final weight gain level of the Chow group, but the effect of delaying weight gain was limited.

[0040] When the two strains were used in combination (B420 + CECT8145), the time required for mice to reach the final weight gain level of the Chow group was significantly prolonged to approximately 42 days after gavage, which was highly significant compared to the HFD group and significantly better than the single-strain group. This indicates that the combination of B420 and CECT8145 can have a synergistic effect, significantly delaying the process of weight gain and demonstrating a weight loss potential superior to that of a single strain.

[0041] (5) After the experiment (i.e., 56 days after gavage), blood was drawn for testing: like Figure 4 As shown, compared to the control group (Chow), the serum TNF-α level in the model group (HFD) mice was significantly increased. Treatment groups treated with either strain B420 or CECT8145 alone showed some degree of decrease in TNF-α levels, but the effect was limited. However, when both strains B420 and CECT8145 were used in combination, the TNF-α level was significantly reduced, approaching normal levels, and the difference was highly significant compared to the HFD group, indicating a significantly better anti-inflammatory effect than either strain. This suggests that bacterial agents containing B420, CECT8145, or B420+CECT8145 can significantly improve the inflammatory state caused by obesity, and that the combination of B420 and CECT8145 can synergistically exert an anti-inflammatory effect.

[0042] (6) After the experiment (i.e., 56 days after gavage), feces were collected for 16S rDNA sequencing to obtain the relative abundance of Akk.

[0043] like Figure 5 As shown, fecal microbiota analysis revealed that the spatial distribution of the model group samples significantly deviated from the control group, B420 group, CECT8145 group, and B420+CECT4145 group; relatively speaking, the B420+CECT4145 group was closest to the control group, suggesting that the probiotics and their combinations involved in this invention can significantly regulate the composition and structure of the intestinal microbiota, with the B420+CECT4145 group showing the best effect. Figure 5As shown, the relative abundance of Akkermansia in the control group, B420 group, CECT8145 group, and B420+CECT4145 group was significantly higher than that in the model group; the B420+CECT4145 group had the highest abundance, which was significantly higher than that in the B420 group and the CECT8145 group, suggesting that B420 and CECT4145 synergistically promote the proliferation of Akkermansia in the intestine.

[0044] The experimental results of this embodiment are shown in Table 1 below.

[0045] Table 1

[0046] Example 2: This example studies the effects of a 10:1 ratio of B420 and CECT8145 on body weight and gut microbiota in high-fat diet-induced obese mice. The difference from Example 1 is as follows: Mice in the B420 group, CECT8145 group, and B420+CECT4145 group were administered 0.2 mL of B420 bacterial solution (1×10⁻⁶) by gavage daily starting from day 8. 8 CFU / mL), CECT4145 bacterial culture (1×10) 7 CFU / mL), B420+CECT4145 mixed bacterial culture (total viable count 1×10⁻⁶) 8 CFU / mL, with a viable bacteria ratio of 10:1.

[0047] The rest is the same as in Example 1.

[0048] The results are shown in Table 2 below.

[0049] Table 2

[0050] Example 3: This example studies the effects of a 1:10 ratio of B420 and CECT8145 on body weight and gut microbiota in high-fat diet-induced obese mice. The difference from Example 1 is as follows: Mice in the B420 group, CECT8145 group, and B420+CECT4145 group were administered 0.2 mL of B420 bacterial solution (1×10⁻⁶) by gavage daily starting from day 8. 7 CFU / mL), CECT4145 bacterial culture (1×10) 8 CFU / mL), B420+CECT4145 mixed bacterial culture (total viable count 1×10⁻⁶) 8 CFU / mL, with a viable bacteria ratio of 1:10.

[0051] The rest is the same as in Example 1.

[0052] The results are shown in Table 3 below.

[0053] Table 3

[0054] Comparative Example 1 Replace “B420” or “CECT4145” with Bifidobacterium animalis ssp. lactis BB-12 (BB-12) for use in combination. The rest is the same as in Example 1. When BB-12 strain is used alone, the weight gain and Akkermansia relative abundance at week 8 are 44.9% and 2.96%, respectively.

[0055] The weight gain and Akkermansia relative abundance at week 8 of BB-12+CECT4145 were 34.2% and 12.46%, respectively.

[0056] The weight gain and Akkermansia relative abundance in week 8 of BB-12+B420 were 42.7% and 5.68%, respectively.

[0057] Finally, it should be noted that the above examples are merely some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. A probiotic composition synergistically promoting proliferation of Akkermansia in the gut, characterized in that: The strains of the probiotic combination consist of Bifidobacterium animalis subspecies lactis strain B420 and Bifidobacterium animalis subsp. lactis CECT8145.

2. The probiotic composition according to claim 1, characterized in that: In the probiotic composition, the ratio of viable counts of B420 to CECT8145 is 1~10:1~10.

3. The probiotic composition for synergistically promoting proliferation of Akkermansia in the intestine according to claim 2, characterized by: In the probiotic composition, the ratio of viable counts of B420 to CECT8145 is 1:

1.

4. A probiotic agent prepared using the probiotic composition according to any one of claims 1 to 3, characterized by: The sum of viable counts of B420 and CECT 8145 < 5 x 105CFU / g. 7 CFU / g.

5. The probiotic agent of claim 4, characterized in that: The sum of viable counts of B420 and CECT 8145 was 5 x 10 7 CFU / g ~ 1 x 10 8 CFU / g.

6. The probiotic agent of claim 4 or 5, characterized in that: The dosage form of the probiotic agent includes a freeze-dried powder, a tablet, a granule, and a capsule.

7. The probiotic composition according to any one of claims 1 to 3 or the use of a probiotic agent according to any one of claims 4 to 6, characterized in that: Relieving obesity symptoms.

8. Use according to claim 7, characterized in that: Reducing the body weight of obese people and shortening the time required for weight loss.

9. Use according to claim 7 or 8, characterized in that: Increasing the abundance of Akkermansia in the intestinal tract.

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