Use of akkermansia muciniphila in combination with human milk oligosaccharides in the preparation of a product for alleviating depression

Abstract

The present application relates to probiotics and the field of biological medicine, and more particularly to the application of mucinophilic Akkermansia and human milk oligosaccharides in the preparation of products for relieving depression. The present application provides the application of mucinophilic Akkermansia and human milk oligosaccharides in the preparation of products for relieving depression, wherein the mucinophilic Akkermansia is named as mucinophilic Akkermansia EAZY1, which is preserved in the China General Microbiological Culture Collection Center with the preservation number of CGMCC NO. 46444. The present application proves through the experiment of combined medication that compared with single use of mucinophilic Akkermansia or human milk oligosaccharides, the present application can play a synergistic effect in the treatment of depression by using mucinophilic Akkermansia and human milk oligosaccharides in combination, and effectively enhances the treatment effect of depression. Therefore, the present application is beneficial to improve the drug efficacy, so as to bring new product research and development ideas and research and development direction for the clinical treatment of depression.

Description

Technical Field

[0001] This invention relates to the fields of probiotics and biomedicine, and in particular to the application of Akkermansia myxophilus combined with human milk oligosaccharides in the preparation of products for relieving depression. Background Technology

[0002] Depression, also known as depressive disorder, is a common mental health problem worldwide. According to incomplete statistics, approximately 200 to 300 million people globally suffer from depression. The causes of depression are complex, usually influenced by both exogenous and endogenous factors. Exogenous factors mainly include environmental stress and lifestyle habits, while endogenous factors are mainly related to neurotransmitter imbalances, inflammatory responses, and gut microbiota dysbiosis.

[0003] In recent years, a growing body of research has shown that the gut microbiota plays a crucial role in mood regulation through the gut-brain axis. Specific probiotics can influence brain function by modulating gut microbiota structure, reducing inflammation levels, and improving neurotransmitter balance.

[0004] Akkermansia myxophilus ( Akkermansia muciniphila Akkella (Bacillus subtilis), as a novel next-generation probiotic, plays an important role in host metabolic health, inflammation regulation, and gut homeostasis maintenance. Previous studies have also found its potential benefits in mood regulation. However, the effect of Akkella alone on mood regulation and depression relief is relatively limited. Further enhancing the role and efficacy of Akkella in alleviating depression could provide new ideas and directions for product development and clinical treatment of depression. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, one objective of this invention is to provide the application of the combined use of *Akermansia myxophilus* and human milk oligosaccharides in the preparation of products for alleviating depression. This invention, through the combined use of *Akermansia myxophilus* and human milk oligosaccharides, has discovered that their combined action exhibits a significant synergistic effect in alleviating depression, thus demonstrating potential for development in products for the prevention and treatment of depression.

[0006] The second objective of this invention is to provide a product that alleviates depression.

[0007] To achieve the above objectives, the first aspect of the present invention is implemented using the following technical solution:

[0008] The application of Akkermansia myxophilus in combination with human milk oligosaccharides in the preparation of products for relieving depression, wherein the Akkermansia myxophilus is named Akkermansia myxophilus ( Akkermansia muciniphila) EAZY1, which is preserved in China General Microbiological Culture Collection Center, and the preservation number is CGMCC NO. 46444; the product for relieving depression is a functional food or a drug.

[0009] Preferably, the use amount ratio of the Akkermansia muciniphila to the human milk oligosaccharide is (1x10 9 -5x10 9 ) CFU: (100-500) mg.

[0010] Further preferably, the use amount ratio of the Akkermansia muciniphila to the human milk oligosaccharide is 2.5x10 9 CFU: 250 mg.

[0011] Further preferably, the human milk oligosaccharide is a mixture of 2'-fucosyllactose and 6'-sialyllactose.

[0012] In a second aspect of the present application, the following technical solution is adopted:

[0013] A product for relieving depression, which comprises the Akkermansia muciniphila and the human milk oligosaccharide as described above.

[0014] Further preferably, the product for relieving depression further comprises an excipient. The skilled person can select a suitable type of excipient according to the specific requirements of the product, and the present application does not particularly limit it. For example, the excipient that can be used includes malt dextrin, fruit juice powder, corn starch, vitamins and other common excipients that can be used in functional foods and drugs.

[0015] The beneficial effects of the above technical solution of the present application are as follows:

[0016] The Akkermansia muciniphila screened by the present application has been verified to have a good anti-depression effect. Further, through experiments of drug monotherapy and drug combination, it is proved that, compared with the scheme of using the Akkermansia muciniphila or the human milk oligosaccharide alone, the Akkermansia muciniphila and the human milk oligosaccharide used in combination in the present application can exert a synergistic effect in the treatment of depression, thereby effectively enhancing the treatment effect of depression. Therefore, the present application proves that the Akkermansia muciniphila and the human milk oligosaccharide used in combination have a synergistic effect in relieving depression, which is conducive to improving the drug efficacy, thereby bringing new research ideas and research directions for the clinical treatment and product development of depression. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 The weight change results of mice in different groups in the experimental examples of the present application;

[0018] Figure 2The results of the sucrose preference test, forced swimming test and tail suspension test of different groups of mice in the experimental examples of this invention;

[0019] Figure 3 These are the test results of the serum levels of 5-HT, IL-1β, IL-10, and TNF-α in different groups of mice in the experimental examples of this invention;

[0020] Figure 4 These are the test results of 5-HT and BDNF levels in the brain tissue of mice in different groups in the experimental examples of this invention;

[0021] Figure 5 The results show the detection of corticosterone in the serum of mice in different groups in the experimental examples of this invention. Detailed Implementation

[0022] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments. Specific conditions not specified in the embodiments are performed according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, all materials used are conventional products obtained through commercial channels.

[0023] The biological preservation information involved in this invention is as follows:

[0024] Akkermansia myxophilus EAZY1, classified as Akkermansia muciniphila It is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.46444, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on May 26, 2025.

[0025] In the following embodiments of the present invention, the human milk oligosaccharides used are 2'-fucosylated lactose (2'-FL) and 6'-sialylated lactose (6'-SL), provided by Chr. Hansen. In the following embodiments of the present invention, the comparative strain involved is *Akermansia myxophilus* (…). Akkermansia muciniphila DSM 22959, which comes from Beijing Beina Innovation Biotechnology Research Institute.

[0026] In the following embodiments of the present invention, the isolation and identification process of *Ackermania myxophilus* EAZY1 is as follows: *Ackermania myxophilus* EAZY1 was isolated from a fecal sample of a healthy adult in Harbin. During isolation, a mucin-selective medium was used, and the strain was enriched and screened under anaerobic conditions to obtain purified strains. The purified colonies were round and convex, 0.5-1 mm in diameter, smooth, and with regular edges. Subsequently, amplification and sequencing were performed using universal 16S rRNA primers for gene identification. After gene sequencing identification and comparison with NCBI, it was confirmed as *Ackermania myxophilus*, and named *Ackermania myxophilus* EAZY1. The mucin-selective medium was a modified BHI medium, prepared by taking 38.5 g of brain heart extract broth (BHI), 4.0 g of mucin, 0.5 g of L-cysteine, 0.005 g of vancomycin, and bringing the volume to 1 L with sterile water.

[0027] Akkermansia myxophilus ( Akkermansia muciniphila The 16S rRNA sequence of EAZY1 is as shown in SEQ ID NO.1:

[0028] Example 1

[0029] This embodiment provides a product for relieving depression, comprising *Ackermania myxotroph* and human milk oligosaccharides. The *Ackermania myxotroph* is named *Ackermania myxotroph* (…). Akkermansia muciniphila EAZY1, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.46444, contains *Ackermania mucilage* at a ratio of 2.5 × 10⁻⁶ to human milk oligosaccharides. 9 CFU: 250 mg. Human milk oligosaccharides are a mixture of 2'-fucosylated lactose and 6'-sialylated lactose in a mass ratio of 1:1. Comparative Example 1

[0030] This comparative example provides a product for alleviating depression, comprising *Akkermansia myxophilus* and human milk oligosaccharides. The ratio of *Akkermansia myxophilus* to human milk oligosaccharides is 2.5 × 10⁻⁶. 9 CFU: 250 mg. Human milk oligosaccharides are a mixture of 2'-fucosylated lactose and 6'-sialylated lactose in a 1:1 mass ratio. *Ackermania myxophilus* is a commercially available strain, specifically *Ackermania myxophilus* (…). Akkermansia muciniphila DSM 22959.

[0031] Experimental Example

[0032] This experiment established a mouse chronic unpredictable stress model (CUMS), which involves inducing experimental animals to exhibit depressive-like behavioral and physiological changes by applying a series of unpredictable mild stressors. Different drugs were then used for intervention to evaluate the effect of drug intervention on improving depression in mice.

[0033] (I) Experimental Methods

[0034] The experimental animals were male BALB / c mice, weighing 20-22g. CUMS modeling: Mice were randomly subjected to different mild stresses each day (fasting and water deprivation, tilted cage, swimming in ice water, reversed day and night, etc.) to construct a mouse model of mood depression.

[0035] Specifically, after one week of adaptive feeding, the mice in each group were randomly divided into 6 groups, 20 mice in each group. Except for the control group, the remaining groups were modeled by CUMS. Different types of mild stress were randomly applied every day, including inverted day-night cycle (24 h), fasting (24 h), water deprivation (12 h), wet bedding (12 h), inclined cage (45°, 12 h), cold stimulation (4°C, 10 min), strong light irradiation (24 h), group or single feeding (12 h), etc. 1-2 types per day, for 3 consecutive weeks. When determining the stress type, in order to control the variables, the mice in each group received the same type of stress stimulation on the same day to ensure consistency within the group and enhance the comparability of the experiment.

[0036] From the 5th week, the drug intervention groups used single Akkermansia muciniphila EAZY1 (Example 1), Akk EAZY1 group), single human milk oligosaccharides (HMOs group), the Akkermansia muciniphila DSM22959 + human milk oligosaccharides composition (Example 1), Akk DSM 22959 + HMOs group), the Akkermansia muciniphila EAZY1 + human milk oligosaccharides composition (Example 1), Akk EAZY1 + HMOs group) were dissolved in PBS in the corresponding amount, and the mice were gavaged in an amount of 0.2 mL per mouse per day. The control group and the model group were gavaged with an equal amount of normal saline. The dosage of AKK bacteria and HMOs in the combination group was halved compared to the single use group. The experimental grouping and dosing measures are shown in Table 1.

[0037] Table 1, Experimental grouping and dosing measures (n = 20)

[0038]

[0039] (B) Experimental results

[0040] The experimental results were analyzed using GraphPad Prism 9.0 software, p <0.05,** p <0.01,*** p <0.001,**** p <0.0001, indicating a significant difference.

[0041] 2.1, Analysis of mouse daily behavior and general state

[0042] During the experiment, the body weight and behavior of the mice were recorded within 1-6 weeks, and their food intake, water intake, hair condition, etc. were observed. The body weight changes of the mice recorded during the experiment and the body weight determination results on the last day of intervention are shown in Table 2. Figure 1 Figure 1 ​In this study, A represents the results of changes in body weight of mice, and B represents the results of the last day of body weight test of mice.

[0043] From Figure 1 It can be seen that the body weight of mice in all groups showed a growth trend. On the last day of the end of the experimental treatment, the body weight of mice in the model group was significantly higher than that in the control group (P < 0.05). p <0.05). There was no significant difference between the HMOs group and the model group, but the Akk EAZY1 group, the Akk DSM 22959+HMOs group and the Akk EAZY1+HMOs group were significantly higher than the model group (P < 0.05). p <0.05). Among them, the body weight of mice in the Akk EAZY1+HMOs group was significantly higher than that in the HMOs group (P < 0.05), and there was no significant difference between the Akk EAZY1 group and the Akk DSM 22959+HMOs group (P > 0.05), but the body weight of mice in the control group was the closest to the model group, indicating that Akk EAZY1 and HMOs have interaction, and can obtain a more excellent effect of improving the body weight of mice than single component. p p <0.05), but the body weight of mice in the control group was the closest to the model group, indicating that Akk EAZY1 and HMOs have interaction, and can obtain a more excellent effect of improving the body weight of mice than single component.

[0044] At the same time, the daily life state of mice was observed, and it was found that the activity of depressed mice in the model group decreased, and they were still or curled up in the corner for a long time, and their reaction to external stimuli was slow; at the same time, the food and water intake decreased, the hair was messy, and the self-cleaning behavior was lacking; there was also social withdrawal, and they were reluctant to contact their peers. The drug intervention group significantly improved the above-mentioned conditions.

[0045] 2.2, Behavioral analysis of mice

[0046] Sucrose Preference Test (SPT) is used to evaluate the pleasure of mice, mainly to detect the degree of preference for sugar water, and a decrease in preference is considered to be a manifestation of Anhedonia. Forced Swim Test (FST) assesses despair behavior by measuring immobility time, which can be used to judge the depressive-like state. Tail Suspension Test (TST) also measures the degree of behavioral despair by immobility time, which is complementary to FST. These three behavioral experiments can effectively evaluate the degree of depression of mice from different dimensions.

[0047] ​The sucrose preference test (SPT) procedure is as follows: Before the formal experiment, mice are trained to adapt to sucrose. Two days before the end of the experiment, two bottles of 1% sucrose solution are placed. On the last day, one bottle of 1% sucrose solution is placed, and one bottle of purified water is used to replace one of the sucrose solution bottles. The positions of the two bottles are randomized, and mice have free access to water during this period. After a 12-hour fast and water restriction, all mice are given one bottle of purified water and one bottle of 1% sucrose solution placed randomly for the sucrose consumption experiment. The sucrose solution and purified water are weighed before the experiment. After 1.5 hours, the positions of the water bottles are exchanged. After 3 hours, the masses of the sucrose solution and purified water are weighed again, and the sucrose preference rate is calculated based on the difference between the two weighings. Sucrose preference rate = Sucrose solution consumption / (Sucrose solution consumption + Purified water consumption).

[0048] The forced swimming test (FST) is conducted as follows: After the experiment, 10 mice from each group are randomly selected and placed in a water tank at a temperature of 25°C and a depth of 15cm. The time they remain still is observed to reflect despair behavior.

[0049] The procedure for the suspended tail test (TST) is as follows: After the experiment, 10 mice from each group are randomly selected, their tails are fixed and they are suspended upside down. The time they remain still within 6 minutes is recorded.

[0050] The results of the sugar water preference test (SPT), forced swimming test (FST), and tail suspension test (TST) are as follows: Figure 2 As shown. Figure 2 In the table, A through C represent the results of the sugar water preference experiment, the forced swimming experiment, and the tail suspension experiment, respectively.

[0051] Depend on Figure 2 It can be seen that, compared with the control group, the model group showed a significantly lower liking for sugary drinks. p <0.05), in the FST and TST experiments, the immobility time and cessation time of the model group mice were significantly prolonged ( p <0.05 indicates that the mice exhibited significant depressive-like behavior. In the intervention group, the sucrose preference rate in the Akk EAZY1 + HMOs combined treatment group was significantly higher than that in the model group ( p <0.05). Among them, the sucrose preference rate of mice in the Akk EAZY1+HMOs group was significantly higher than that in the Akk EAZY1 group, HMOs group, and Akk DSM 22959+HMOs group, indicating that it was more effective in restoring pleasure. Meanwhile, in the FST and TST experiments, the immobility time of mice in the Akk EAZY1+HMOs combined treatment group was also significantly lower than that in the model group (…). p <0.05, and significantly lower than the two single intervention groups in the FST experiment; significantly lower than the Akk EAZY1 group, HMOs group and AkkDSM 22959+HMOs group in the TST experiment ( pThe value was <0.05, indicating stronger proactive coping behaviors and more significant improvement in mood. This suggests that the combined intervention of Akk EAZY1 and HMOs is more effective in alleviating CUMS-induced depressive-like behaviors, demonstrating a synergistic effect.

[0052] 2.3 Detection of mouse biomarkers

[0053] This experiment measured the levels of 5-HT, IL-1β, IL-10, and TNF-α in mouse serum, the levels of 5-HT and BDNF in mouse brain tissue, and the level of corticosterone (CORT) in mouse serum.

[0054] 1) Serum levels of 5-HT, IL-1β, IL-10, and TNF-α

[0055] 5-HT is an important neurotransmitter regulating mood, and its decrease is associated with depression. IL-1β and TNF-α are pro-inflammatory factors, and their elevation reflects increased inflammation, while IL-10 is an anti-inflammatory factor, and its decrease indicates a decline in anti-inflammatory capacity. The method for testing the levels of 5-HT, IL-1β, IL-10, and TNF-α was as follows: After the intervention, blood was collected from the decapitation of mice in each group. After centrifugation, serum was collected, and the levels of 5-HT, IL-1β, IL-10, and TNF-α in the serum of mice in each group were analyzed using an ELISA kit to assess the content of each neurotransmitter in the mouse serum. Figure 3 The results show the levels of 5-HT, IL-1β, IL-10, and TNF-α in the serum of mice from different groups. Figure 3 In the table, A through D represent the experimental results for 5-HT, IL-1β, IL-10, and TNF-α, respectively.

[0056] Depend on Figure 3 It can be seen that, compared with the control group, the model group mice showed significantly decreased levels of 5-HT and IL-10, and significantly increased levels of IL-1β and TNF-α. p <0.05 indicates increased inflammation and neurotransmitter imbalance in the model group. Akk EAZY1 intervention significantly restored 5-HT levels ( p <0.05), and significantly reduced inflammatory factors IL-1β and TNF-α ( p <0.05. There was no significant difference between the HMOs group and the model group ( p>0.05). Both the Akk DSM 22959+HMOs group and the Akk EAZY1+HMOs group effectively restored 5-HT and IL-10 levels and reduced inflammatory factors IL-1β and TNF-α. Among them, the Akk EAZY1+HMOs combination therapy group showed significantly higher efficacy in regulating serum 5-HT, IL-10, and TNF-α levels than the Akk EAZY1 group, the HMOs group, and the Akk DSM 22959+HMOs group. p <0.05), which can significantly improve inflammatory state and alleviate depression, demonstrating a synergistic effect.

[0057] 2) Brain tissue 5-HT and BDNF levels

[0058] 5-HT, also known as serotonin, is an important neurotransmitter that plays a crucial role in mood regulation, cognitive ability, psychology, and sleep, and is closely related to the occurrence and development of depression. Brain-derived neurotrophic factor (BDNF) is one of the most important neurotrophic factors, playing a vital role in neurogenesis, neural structural growth and remodeling, and nerve impulse transmission. Abnormal BDNF function can easily lead to depression and is often considered a peripheral biomarker for depression. The method for testing 5-HT and BDNF levels in mouse brain tissue is as follows: After intervention, mice are anesthetized, quickly euthanized by decapitation, and brain tissue is collected, weighed, and frozen. An appropriate amount of PBS or lysis buffer is added for homogenization, and the mixture is centrifuged at 12,000 rpm for 10 minutes at 4°C. The supernatant is used for ELISA to detect 5-HT and BDNF levels in mouse brain tissue. Figure 4 The results show the levels of 5-HT and BDNF in the brain tissue of mice from different groups. Figure 4 In the figure, A and B are the experimental results of 5-HT and BDNF, respectively.

[0059] Depend on Figure 4 It can be seen that the levels of 5-HT and BDNF in the brain tissue of the model group mice were significantly reduced. p <0.05, leading to neurotransmitter imbalance. Post-intervention results showed that, compared to the model group, both the Akk EAZY1 group and the HMOs group significantly increased 5-HT and BDNF levels ( p <0.05 indicates that both interventions have neuroprotective effects. The combined use of Akk EAZY1 and HMOs showed the most significant improvement in 5-HT and BDNF levels, with significant differences compared to the model group ( p <0.05), and higher than Akk The results of combining the EAZY1 group and the HMOs group indicate a synergistic enhancing effect. Furthermore, the Akk EAZY1 + HMOs group was significantly higher than the Akk DSM22959 + HMOs group. p<0.05), indicating that Akk EAZY1 combined with HMOs has a stronger effect than Akk DSM 22959+HMOs in regulating neurotransmitters, with strain specificity. Akk EAZY1 combined with HMOs can promote the content of monoamine neurotransmitters (5-HT), increase the expression of BDNF, enhance the emotional regulation ability, and thus play an antidepressant role. In summary, the combination of Akk EAZY1 and HMOs has a synergistic effect in regulating neurotransmitters, and its antidepressant effect is significantly better than that of either component alone, and has strain specificity.

[0060] 3) Serum Corticosterone (CORT) levels

[0061] Corticosterone is a glucocorticoid that plays a key role in stress response and HPA axis regulation, and its elevated levels are often associated with anxiety and depressive-like behaviors. The test method for corticosterone levels is as follows: after the intervention, the mice in each group were decapitated and blood was collected, and after centrifugation, the serum was collected and the serum CORT level was measured using an ELISA kit to assess HPA axis activity. The results of the detection of serum corticosterone in mice in different groups are shown in Figure 5 .

[0062] As shown in Figure 5 , the serum corticosterone levels of the model group mice were significantly elevated ( p <0.05), indicating that the HPA axis was overactivated, which may lead to abnormal emotional regulation. In contrast, both the Akk EAZY1 group and the HMOs group significantly reduced the level of cortisol ( p <0.05), indicating that they have a positive effect on regulating HPA axis function and relieving stress response. It is worth noting that the combination of Akk EAZY1 bacteria and human milk oligosaccharides (Akk EAZY1+HMOs combination group) showed the best intervention effect, with a higher inhibitory effect on cortisol than the Akk EAZY1 group, the HMOs group, and the Akk DSM 22959+HMOs group, further verifying the synergistic effect of the two in regulating HPA axis homeostasis and relieving stress-related depressive symptoms, as well as the strain specificity of Akk EAZY1 in antidepressant.

[0063] From the above experimental results, it is proved by the combination drug experiment that, compared with single use of mucinophilic Akkermansia or human milk oligosaccharides, the use of mucinophilic Akkermansia and human milk oligosaccharides in the treatment of depression can play a synergistic effect, and effectively enhance the treatment effect of anti-depression. In addition, it is also proved that the combination of Akk EAZY1 and human milk oligosaccharides has strain specificity and potential advantages in regulating neurotransmitters and inhibiting inflammatory response. Therefore, it is proved that there is a mutual promotion synergistic effect between mucinophilic Akkermansia and human milk oligosaccharides, which is beneficial to improve the drug efficacy, so as to bring new research ideas and research directions for the product development and clinical treatment of depression.

[0064] The above embodiments are only preferred embodiments of the present application, and cannot be used to limit the scope of protection of the present application. Any non-essential changes and substitutions made by those skilled in the art on the basis of the present application shall fall within the scope of protection of the present application.

Claims

1. The application of Akkermansia myxophilus combined with human milk oligosaccharides in the preparation of products for relieving depression, characterized in that, The name of the amycophilic Akkermansia is Amycophilus. Akkermansiamuciniphila EAZY1, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO. 46444; the product for relieving depression is a pharmaceutical product; the ratio of *Akermansia myxophilus* to human milk oligosaccharides is 2.5 × 10⁻⁶. 9 CFU: 250 mg; the human milk oligosaccharide is a mixture of 2'-fucosylated lactose and 6'-sialylated lactose.

2. A product for relieving depression, characterized in that, The product for relieving depression includes Akkermansia mycotoxin as described in claim 1 and human milk oligosaccharides; the product for relieving depression is a pharmaceutical product.

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