Akkermansia muciniphila strain, preparation method and application thereof

Abstract

The present application relates to a strain of Akkermansia muciniphila and its application, and belongs to the technical field of microbial strains. Specifically, the strain is Akkermansia muciniphila AUSA024, and its preservation number in Guangdong Microbial Culture Collection Center is GDMCC No. 65717. The Akkermansia muciniphila AUSA024 has important application value in preparing preparations for reducing homocysteine and preventing and treating cardiovascular diseases.

Description

Technical Field

[0001] This invention relates to a strain of Akkermansia myxophilus AUSA024 and its use in preparing products that lower homocysteine ​​and prevent stroke, belonging to the field of microbial technology. Background Technology

[0002] Homocysteine ​​(Hcy) is a sulfur-containing amino acid and an intermediate metabolite of methionine (an essential amino acid). Under normal circumstances, homocysteine ​​is metabolized in the human body primarily through two pathways: methylation and transsulfurization. Abnormalities in either of these pathways can lead to hyperhomocysteinemia. Studies have confirmed that hyperhomocysteinemia is a significant risk factor for many diseases, including cardiovascular disease, neurodegenerative diseases, diabetes, osteoporosis, and autoimmune diseases. Currently, cardiovascular disease remains one of the most serious diseases affecting human health and quality of life. Therefore, lowering homocysteine ​​levels to reduce the risk of cardiovascular disease is particularly important.

[0003] Currently, the main methods for lowering homocysteine ​​levels include lifestyle interventions and nutritional supplements, but these methods have certain limitations. Lifestyle interventions, such as dietary adjustments and increased exercise, help lower homocysteine ​​levels, but patient adherence is poor, making long-term maintenance difficult. Nutritional supplements and drug therapy, while effective, have less than ideal results in some patients due to genetic polymorphisms affecting absorption and metabolism. Although these supplements can provide essential nutrients, their dosage and form are difficult to standardize, and they may interact with other medications.

[0004] Probiotics can improve the balance of gut microbiota and enhance intestinal barrier function. With in-depth research, the application areas of probiotics are becoming increasingly widespread. However, the application of probiotics also faces some challenges. For example, the selection of effective strains and the determination of dosage still require further research. Furthermore, different strains have significantly different mechanisms of action and effects, necessitating the screening of the most suitable strains and the determination of effective dosages for specific therapeutic purposes. In addition, technical issues such as the stability, survival rate, and production processes of probiotic preparations urgently need to be addressed to ensure their activity and efficacy during production and storage.

[0005] Akkermansia muciniphila, or AKK for short, is a beneficial gut bacterium. As enteritis progresses, the number of AKK bacteria gradually decreases. Supplementation with AKK bacteria or its outer membrane protein Amuc_1100 can alleviate enteritis symptoms, regulate immune responses in the spleen, intestines, and mesenteric lymph nodes, reduce intestinal inflammation levels, and prevent colorectal cancer caused by enteritis. Furthermore, AKK bacteria are negatively correlated with obesity, diabetes, and cardiovascular metabolic diseases. In a mouse model of ALS (Amyotrophic Lateral Sclerosis), increasing the concentration of Akkermansia muciniphila can improve symptoms and prolong survival. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing homocysteine-lowering products and to provide a strain of Akkermansia myxophilus and its agent for preparing a product for lowering homocysteine ​​levels and preventing stroke.

[0007] The technical solution adopted in this invention is:

[0008] The first objective of this invention is to provide a strain of Akkermansia muciniphila, AUSA024, which was deposited on January 3, 2025, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC) with accession number GDMCC No. 65717, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Taxonomically, it is named Akkermansia muciniphila.

[0009] In this invention, the myxotrophic Akkermansia AUSA024 was isolated from infant feces.

[0010] In this invention, the Akkermansia muciniphila AUSA024 includes one or more of live bacteria, dead bacteria, cell fragments, fermentation supernatant, modified bacteria, mutant bacteria and / or induced mutant bacteria.

[0011] The second objective of this invention is to provide a culture medium for *Akermansia myxophilus*, comprising: 5-20 g / L yeast extract, 5-20 g / L tryptone, 5-30 g / L glucose, 2-10 g / L threonine, 1-10 g / L N-acetylglucosamine, and vitamin B1. 12 50-200 μg / L, Vitamin B9 50-200 μg / L.

[0012] In the culture medium provided by the present invention, as a preferred embodiment, the culture medium composition is: yeast extract 10 g / L, tryptone 10 g / L, glucose 15 g / L, threonine 5 g / L, N-acetylglucosamine 3 g / L, and vitamin B1. 12100 μg / L, Vitamin B9 100 μg / L.

[0013] The present invention also provides the use of the culture medium in culturing Akkermansia myxophilus.

[0014] A third objective of this invention is to provide a microbial inoculant containing Akkermansia myxophilus AUSA024 or Akkermansia myxophilus AUSA024 cultured in the above-mentioned culture medium.

[0015] As a preferred embodiment, the microbial agent provided by the present invention contains prebiotics, which are selected from one or more of the following: mannose oligosaccharides, fructooligosaccharides, maltodextrin, isomaltitol, isomaltooligosaccharides, galactooligosaccharides, xylooligosaccharides, inulin, etc. As a more preferred embodiment, the prebiotics are a mixture comprising fructooligosaccharides, isomaltitol, and isomaltooligosaccharides.

[0016] In this invention, the microbial agent can be prepared into tablets, capsules, powders, granules, semi-solid preparations, liquid preparations, etc. The semi-solid preparations include gels, and the liquid preparations include oil drops, aqueous solutions, etc.

[0017] In the microbial agent provided by this invention, the viable count of *Ackermania pseudomallei* AUSA024 is 1 × 10⁻⁶. 8 ~1×10 11 CFU / g; preferably, the viable count of the myxotrophic Akkermansia AUSA024 is 1×10⁻⁶. 9 ~1×10 10 CFU / g.

[0018] A fourth object of the present invention is to provide the use of Akkermansia myxophilus AUSA024 or an agent containing Akkermansia myxophilus AUSA024 for the preparation of products that lower homocysteine.

[0019] A fifth object of the present invention is to provide the use of Akkermansia myxophilus AUSA024 or an agent containing Akkermansia myxophilus AUSA024 in the preparation of products for the prevention of stroke. The stroke includes ischemic stroke and hemorrhagic stroke.

[0020] The *Ackermannii myxophilus* AUSA024 strain provided by this invention was isolated from infant feces. This strain has the advantage of utilizing mucin in the intestine for growth, exhibiting strong colonization ability and being difficult to eradicate. The *Ackermannii myxophilus* AUSA024 culture medium provided by this invention is prepared by adding vitamin B to the culture medium. 12In conjunction with vitamin B9, this method achieves a surprisingly high-density growth and enrichment of Akkermansia myxophilus AUSA024. This culture medium is also suitable for other Akkermansia myxophilus strains. The Akkermansia myxophilus AUSA024 or a bacterial agent containing Akkermansia myxophilus AUSA024 provided by this invention exhibits a non-obvious effect in reducing homocysteine ​​levels and preventing stroke, with significant efficacy. Attached Figure Description

[0021] Figure 1 Image: This is a fluorescence quantitative PCR detection image of Akkermansia AUSA024, a myxotrophic strain, provided by the present invention.

[0022] 1. *Akermansia myxophilus* AUSA024; 2. Water

[0023] Figure 2 Gram staining image of Akkermansia AUSA024, a myxotrophic strain provided by this invention.

[0024] Figure 3 : An phylogenetic tree diagram of Ackermania AUSA024 and other Ackermania species provided by this invention. Detailed Implementation

[0025] The present invention will be further described below with reference to specific embodiments, but this is not intended to limit the present invention. Any equivalent substitutions made in the art in accordance with the content of the present invention shall fall within the protection scope of the present invention.

[0026] Example 1: Isolation and identification of Akkermansia AUSA024 provided by the present invention

[0027] 1. Strain isolation:

[0028] The separation steps are as follows:

[0029] (1) Sample collection: Feces were collected from a healthy infant volunteer in Shenzhen.

[0030] (2) High-throughput screening of strains: 1g of collected infant fecal samples were added to 9mL of sterile physiological saline and mixed thoroughly to prepare a sample suspension. 1mL of the sample suspension was added to 9mL of sterile physiological saline and diluted a series of 10-fold values. The resulting 10-fold concentrations were then collected. -5 10 -6 and 10 -70.1 mL of the diluted solution was spread onto solid plates of AKK strain-specific selection medium (Table 1), with three replicates for each dilution. The plates were anaerobically incubated at 37°C for 72 h. Round, transparent, small (<1 mm) single colonies were then picked up with a sterile toothpick and inoculated into 96-well plates for further culture. 200 μL of AKK strain-specific selection liquid medium was added to each well of the 96-well plate. The 96-well plates were then anaerobically incubated at 37°C for 48 h.

[0031] Table 1: Formulation of Specific Screening Culture Medium for Akkermansia myxophilus (AKK)

[0032]

[0033] (3) Initial screening of strains: 1 μL of bacterial culture from a 96-well plate was used as a DNA template. qPCR (quantitative real-time polymerase chain reaction) was performed using specific primers for AKK strains (F: CAATCATGCGGAAGATATC, R: GAAGGTTTTGGATGCAAA) (Table 2). Wells showing fluorescent signals were identified as potential AKK strains. Figure 1 These bacteria were then subjected to further streak purification culture.

[0034] Table 2: qPCR system for screening Akkermansia myxophilus AUSA024

[0035]

[0036] (4) Observation of strain morphology: The selected strains were Gram-stained and their morphology was observed under a microscope. Figure 2 ).

[0037] 2. Strain identification:

[0038] (1) 16S rDNA sequencing analysis:

[0039] Strain DNA was extracted using a bacterial genomic DNA extraction kit, and the full-length 16S rDNA was amplified by PCR technology. Sequencing analysis was then performed to identify the species of the strains isolated in the above steps.

[0040] The 16S rDNA gene sequence of the Akkermanisa muciniphila AUSA024 is as follows:

[0041]

[0042] The obtained full-length 16S rDNA sequence was searched and compared for similarity using the BLAST webpage of the National Center for Biotechnology Information (NCBI) (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). The results showed greater than 99% identity with the 16S rDNA sequences of the first 100 reported Akkermanisa muciniphila strains, thus identifying it as Akkermanisa muciniphila. The identity with the 16S rDNA of the first 100 reported Akkermanisa muciniphila strains is not entirely identical, indicating that the Akkermanisa muciniphila AUSA024 strain provided in this invention is original.

[0043] The bacterial culture medium was mixed with 50% glycerol at a ratio of 2:1 and stored at -80℃ for later use.

[0044] (2) Whole genome sequence analysis

[0045] DNA was extracted from the bacterial strain using a bacterial genomic DNA extraction kit, and whole-genome sequencing analysis was performed to further identify the genomic characteristics of the strain. The whole genome sequence of the *Akkermanisa muciniphila* AUSA024 has been uploaded to the National Center for Biotechnology Information (NCBI) in the United States, with Accession No. CP186328.

[0046] Example 2: High-density culture method of a strain of Akkermansia myxophilus AUSA024

[0047] Traditional Akkermansia myxophilus cultivation requires the addition of a mixture of animal-derived mucin and vitamins (as shown in Table 1). By analyzing the metabolic pathways of the Akkermansia myxophilus genome, carbon, nitrogen, and vitamin sources were prepared according to Table 3 to replace the mucin and vitamin mixture in the control medium. After cultivation, the OD600 values ​​were measured and compared with those of the medium formulated in Table 1 as a control medium to determine whether each candidate medium component could replace the mucin and vitamin mixture and achieve high-density growth of Akkermansia myxophilus.

[0048] The specific steps are as follows: Akkermansia muciniphila AUSA024, preserved at -80℃, was activated for 48 hours in the control medium (Table 1) mentioned above. Then, it was inoculated at a 1% inoculum and cultured anaerobicly (10% H2, 10% CO2, 80% N2) for 48 hours. The OD600 (200 μL / well) was then measured using a microplate reader, and the results are shown in Table 3. The results showed that without the addition of mucin, ATCC vitamin Mix, and ATCC trace minerals mix, increasing the levels of yeast extract (10 g / L), tryptone (10 g / L), glucose (15 g / L), threonine (5 g / L), N-acetylglucosamine (3 g / L), and vitamin B1 significantly improved the results. 12 100 μg / L, Vitamin B9 100 μg / L, Akkermansia myxophilus AUSA024 OD 600 It can reach 1.6, and the viable count can reach 1×10⁶. 9 CFU / ml.

[0049] Table 3: Optimization of high-density culture of Akkermansia myxophilus AUSA024

[0050]

[0051] Example 3: Preparation of freeze-dried bacterial powder containing Akkermansia myxophilus AUSA024 as described in this invention 1. Preparation process of Akkermansia myxophilus AUSA024 powder:

[0052] (1) The *Akermansia myxophilus* strain AUSA024 was activated by streaking onto solid culture medium prepared according to the optimized formula in Table 3 of Example 3, and incubated at 37°C for 72 h. After activation, single colonies were picked and cultured anaerobically in 10 ml of liquid culture medium at 37°C for 48 h, with an inoculum size of 5%.

[0053] (2) The culture was expanded again to 100 ml under the same culture medium and conditions to serve as the seed culture for fermentation. 5% of the seed culture was inoculated into 4 L of fermentation medium. The fermentation conditions were 37°C, pH 6.5, nitrogen flow rate of 0.5 L / min, and fermentation time of 44–48 h. After fermentation, the mycelial sludge was collected by centrifugation at 6000 rpm for 10 min.

[0054] (3) Freeze-drying. The mycelial sludge and a preservative (preservative preparation method: mix 12g skim milk powder, 5g sucrose, 3g lactose, 3g trehalose, and 5g fructooligosaccharides in 100ml water) were mixed at a weight ratio of 1:2, and freeze-dried for 30-48 hours. The prepared freeze-dried mycelial powder had a viable count greater than 1.0 × 10⁻⁶. 9 CFU / g.

[0055] Example 4: Effects of the myxotrophic Akkermansia strain AUSA024 provided by the present invention on rats with hyperhomocysteinemia

[0056] SPF-grade SD rats, half male and half female, 8 weeks old, purchased from Beijing Vital River Co., Ltd., were included in the experiment after passing inspection. Ten rats were selected as the control group and fed ordinary purified feed (AIN93M). The remaining rats were fed a low-folate purified feed for 4 weeks. Blood was collected from the orbital vein to measure homocysteine ​​(Hcy) concentration. Rats with Hcy concentration greater than 10 μmol / L were selected as the experimental group. Ten rats from the experimental group with Hcy concentration greater than 10 μmol / L were selected as the model group. The remaining rats were randomly divided into groups of 10 each and administered via gavage according to Table 4. The control group and the model group were given physiological saline. Akkermansia myxophilus ATCCBAA-835 was purchased commercially. The low, medium, and high dose groups of AUSA024 were diluted with physiological saline to the corresponding total bacterial content in the low, medium, and high dose groups of this invention. The dosage design for each group was based on references and guidelines. The recommended dose of probiotics for rats was 1 × 10⁻⁶. 8- 1×10 10 CFU / day, therefore three doses were designed: low, medium, and high (Table 4). The AUSA024+prebiotic group was administered by gavage with Akkermansia muciniphila AUSA024 and a prebiotic solution (the prebiotic solution contained a mixture of 0.25 mg / ml fructooligosaccharides, 6.2 mg / ml maltodextrin, and 1 mg / ml isomaltooligosaccharides).

[0057] Eight weeks after gavage, blood was collected from the abdominal aorta of all rats, and Hcy levels were measured using an automated biochemical analyzer.

[0058] The results were statistically processed using GraphPad Prism 5 statistical analysis software; all data are expressed as mean ± standard deviation. t-tests were performed between groups, with P < 0.05 considered statistically significant and P > 0.05 considered not statistically significant.

[0059] Table 4: Effects of AUSAO24 on Hcy levels in rats ( n=10)

[0060]

[0061] Note: Compared with the control group, ## P<0.01; compared with the model group, * P<0.05, ** P<0.01; compared with the ATCC BAA-835 group, ▲ P<0.05. ▲ P<0.01.

[0062] Table 4 shows the experimental results:

[0063] Compared with the control group, the homocysteine ​​level of rats fed a low-folate diet was significantly increased (P<0.01) and exceeded 10 μmol / L, proving that a rat model of hyperhomocysteinemia had been successfully established.

[0064] Compared with the model group, the Hcy level in the ATCC BAA-835 group only showed a decreasing trend, but there was no significant difference, indicating that this Akkermansia myxophilus had no significant effect on improving homocysteine ​​levels.

[0065] Compared with the model group, the homocysteine ​​levels in rats in the low-dose, medium-dose, and high-dose AUSA024 groups of Akkermansia myxophilus were significantly decreased (P<0.05 or P<0.01), indicating that AUSA024 has a significant effect in reducing homocysteine ​​(Hcy). The decrease in homocysteine ​​levels in the low-dose, medium-dose, and high-dose AUSA024 groups gradually increased with increasing AUSA024 dosage, showing a clear dose-response relationship.

[0066] Compared with the ATCC BAA-835 group, the low, medium and high dose groups of AUSA024 showed significant differences in the reduction of Hcy levels (P<0.05 or P<0.01), indicating that although they belong to the same genus, AUSA024 has the effect of reducing homocysteine.

[0067] The Hcy level in the AUSA024 + prebiotic group not only showed a highly significant statistical difference compared with the model group, but also decreased further compared with the high-dose AUSA024 group at the same dose, indicating that the combined use of AUSA024 and prebiotics has a better effect on reducing homocysteine.

[0068] Example 5: Effect of Akkermansia myxophilus AUSA024 on the incidence of stroke in rats.

[0069] I. Methods

[0070] Male stroke-susceptible spontaneously hypertensive rats (SHR-SP) aged 10-11 weeks were housed in a laboratory environment with a room temperature of 22±2℃ and a relative humidity of 50%, and were given purified water and fed ordinary feed.

[0071] The animals were randomly divided into 6 groups (n=12 per group) according to Table 5 below and administered the medication by gavage. The administration was once daily for 16 consecutive weeks. *Akkermansia myxophilus* ATCC BAA-835 lyophilized bacterial powder was purchased commercially. The low, medium, and high dose groups of AUSA024 were prepared by diluting the lyophilized powder (prepared in Example 1 of this invention) with physiological saline to the corresponding total bacterial count. The AUSA024 + prebiotic group was simultaneously administered AUSA023 and a prebiotic solution (the prebiotic solution contained a mixture of 0.5 mg / ml fructooligosaccharides, 15.6 mg / ml isomaltitol, and 2.5 mg / ml isomaltooligosaccharides) by gavage. The model group received physiological saline. A WKY control group of 10 animals was also included, administered the same volume of physiological saline by gavage.

[0072] The dosage settings are the same as in Example 4.

[0073] Observation indicators:

[0074] (1) The incidence of cerebral infarction in rats was observed and statistically analyzed using a neurological function grading evaluation method. The specific method was as follows:

[0075] During the gavage period, the animals' diet, survival, and behavioral activity were observed daily, and the neurological function of each group of rats was graded and evaluated.

[0076] Based on the Bederson neurological functional classification, the following 5 levels were established, and observations were conducted once daily.

[0077] Grade 0 (Normal): No neurological deficit symptoms.

[0078] Grade 1 (mild): Forelimbs are curled or flexed, mainly with persistent wrist flexion or shoulder adduction accompanied by elbow extension.

[0079] Grade 2 (moderate): Persistent flexion or curling of the forelimbs, with complete flexion of the wrist and elbow, and adduction or internal rotation of the shoulder.

[0080] Grade 3 (Severe): Persistent limb curling or flexion, persistent weakening of unilateral limb pushing resistance, and no limb circling behavior during voluntary activities.

[0081] Level 4 (Very Severe): Persistent curling or flexion of both limbs, persistent weakening of pushing force and resistance of one limb, and circling behavior of limbs during voluntary activities.

[0082] According to the above neurological function grading, a neurological deficit grade of 1 or above can be defined as a stroke.

[0083] II. Results

[0084] The results are shown in Table 5 below:

[0085] Table 5: Effects of continuous gavage for 16 weeks on stroke in SHR-SP rats ( n=12)

[0086]

[0087] Note: χ² test, compared with the WKY group, ## P<0.01; compared with the model group, * P<0.05, ** P<0.01; compared with the ATCCBAA-835 group, ▲ P<0.05, ▲▲ P<0.01.

[0088] Rats in the model group began to show stroke symptoms from approximately 13 to 14 weeks of age, and all 12 rats exhibited signs of stroke throughout the study period. Following the stroke, the SHR-SP rats in the model group displayed significant lethargy and drowsiness, with ruffled, dry, or lusterless fur, weight loss, significantly reduced voluntary activity, bradykinesia, and varying degrees of limb paralysis. Some rats also experienced convulsions, diarrhea, urinary incontinence, and ocular hemorrhage at the onset of the stroke; severely affected rats died within hours. Between 12 and 16 weeks after grouping, 10 rats in the model group died, while the WKY group rats did not exhibit the aforementioned abnormalities.

[0089] There was no statistically significant difference between the ATCC BAA-835 group and the model group.

[0090] Compared with the model group, rats in the low-dose, medium-dose, and high-dose AUSA024 groups showed milder neurological deficits, significantly better mental state, increased spontaneous activity, active foraging and drinking, minimal weight loss, and smoother, shinier fur than the model group. The incidence of stroke and mortality were significantly reduced in all AUSA024 groups, exhibiting a dose-dependent effect.

[0091] Compared with the ATCC BAA-835 group, the low, medium and high dose groups of AUSA024 showed significant differences in the reduction of stroke incidence and mortality (P<0.05 or P<0.01).

[0092] Compared with the high-dose AUSA024 group, the AUSA024 + prebiotic group showed a further reduction in the incidence of stroke and mortality.

[0093] The results of this experiment show that *Ackermania myxophilus* AUSA024 can significantly reduce the incidence and mortality of stroke in SHR-SP rats, and improve neurological functional classification and pathological changes. This indicates that *Ackermania myxophilus* AUSA024 has a significant effect in preventing stroke. Furthermore, adding prebiotics to AUSA024 can further reduce the incidence and mortality of stroke, indicating that AUSA024 combined with prebiotics has a better effect in preventing stroke.

Claims

1. A strain of Akkermansia muciniphila AUSA023 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on January 3, 2025, with accession number GDMCC No. 65716.

2. The *Ackermania* AUSA023 according to claim 1, characterized in that, The strain was isolated from infant feces.

3. A culture medium for *Akermansia myxophilus*, comprising: yeast extract 5–20 g / L, tryptone 5–20 g / L, glucose 5–30 g / L, threonine 2–10 g / L, N-acetylglucosamine 1–10 g / L, and vitamin B1. 12 50-200 μg / L, Vitamin B9 50-200 μg / L; as a preferred embodiment, the culture medium composition is: yeast extract 10 g / L, tryptone 10 g / L, glucose 15 g / L, threonine 5 g / L, N-acetylglucosamine 3 g / L, Vitamin B9 50-200 μg / L; 12 100 μg / L, Vitamin B9 100 μg / L.

4. Use of the culture medium according to claim 3 in culturing Akkermansia myxophilus.

5. A microbial agent containing Akkermansia myxophilus AUSA023 as described in claim 1 or Akkermansia myxophilus AUSA023 cultured in accordance with claim 3.

6. The microbial agent according to claim 5, characterized in that, The microbial agent contains prebiotics, which are selected from one or more of the following: mannose oligosaccharides, fructooligosaccharides, maltodextrin, isomaltitol, isomaltooligosaccharides, galactooligosaccharides, xylooligosaccharides, inulin, etc.

7. The microbial agent according to claim 5, characterized in that, The microbial agent can be prepared into tablets, capsules, powders, granules, semi-solid preparations, liquid preparations, and other formulations.

8. The microbial agent according to claim 5, characterized in that, The viable count of the *Ackermania* strain was 1 × 10⁻⁶. 8 ~1×10 11 CFU / g; preferably, the viable count of the myxotrophic Akkermansia is 1×10⁻⁶. 9 ~1×10 10 CFU / g.

9. Use of the Akkermansia myxophilus AUSA023 of claim 1 or the microbial agent of claim 5 in the preparation of a product for reducing serum trimethylamine oxide or treating diseases related to elevated blood trimethylamine oxide levels.

10. Use of the Akkermansia myxophilus AUSA023 of claim 1 or the bacterial agent of claim 5 in the preparation of a product for reducing trimethylamine or treating trimethylamineuria.

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