The Development Of Microcapsule Of Akkermansia Muciniphila For Preventing Obesity In Beagle Dog

In this study,we isolated Akkermansia muciniphila from the feces of IFI204^-/-（Interferon-activated gene 204 deletion）mice.This study aims to investigate the roles of A.muciniphila in the prevention of obesity in mice and beagle dogs and to develop microcapsule of A.muciniphila and lay a foundation for its application.In prior works,we found that the abundance of A.muciniphila in the intestine of IFI204^-/-mice was significantly higher than wild type mice.In this study,we isolated two A.muciniphila（named AKK1 and AKK2）from the feces of IFI204^-/-mice by PCR identification.A phylogenetic tree was constructed based on the16S rRNA sequences,and the alignment results revealed that AKK1 and AKK2 isolates were in the same class as Akkermansia muciniphila,which has been submitted into the database,the characteristics of A.muciniphila isolates were determined,including growth curve,acid resistance,and bile salt resistance.The results suggested that the log period of A.muciniphila isolates was about 72 hours.The bacterial suspension of AKK2 was higher than AKK1（1.6×10⁸ CFU/mL）.Tolerance assays showed that the acid resistance of AKK2 was stronger than AKK1.Collectively,we selected that isolate AKK2 for further study due to its excellent characteristics.Recent studies have shown that the pasteurized A.muciniphila（70℃for 30min）could effectively inhibit the obesity in murine diabetes model compared with live probiotics.In this study,the murine obesity model was established through high-fat diets and pasteurized AKK2 was applied for intervention to detect the parameters of serum.Consistent with previous studies,we found that the weight gain of obese mice induced by high-fat diets and the regulation of blood sugar were improved.Our data further confirmed that AKK2 could improve obesity induced by high-fat diets in mice through inhibit fat accumulation and decrease the blood lipids.Histopathological analysis of mice liver showed that AKK2 could reduce liver weight,inhibited hepatic lipid accumulation,and protected the liver induced by high-fat diets in mice.In summary,AKK2 could inhibit obesity in mice by inhibiting the fat synthesis and enhancing energy metabolism responses,such as non-alcoholic fatty liver disease.The successful establishment of the murine obesity model lays the foundation for investigating the functions of AKK2 in canine obesity.Moreover,the beagle dog obesity model induced by a high diet was established.The result revealed that the Body Condition Scoring（BCS）and weight of dogs in HFD group significantly increased in 9 weeks and high blood lipid level was also increased,which indicated that the dog obesity model was successfully established.The OGTT test showed that the blood sugar of the‘HFD+AKK’group treated with AKK2 was decreased.We also found that AKK2 might play a vital role in the improvement of serum parameters,including TG,TC,LDL-C,and HDL-C.In addition,the detection of liver function suggested that AKK2 could prevent damage induced by a high-fat diet,which was consistent with the results obtained in the murine model.Importantly,no adverse effects were observed in dogs treated with A.muciniphila.In mammals,it has been found that A.muciniphila survived in various hosts.However,the presence of A.muciniphila in the gastrointestinal tract of dogs has not been reported.Furthermore,we detected A.muciniphila survived in feces samples of the‘HFD+AKK’group by RT-PCR method,which indicating that AKK2 could be recognized and through the intestine of dogs.Intestinal flora plays an important role in the regulation of physiological and pathological development,and it is also an important factor in obesity.Therefore,the 16S rRNA sequencing was performed to analyze the effect of the dog intestinal microbiota in ND,HFD,and‘HFD+AKK’groups.The results showed that microbiota species in‘Phylum Firmicutes’were decreased in the HFD group.In contrast,the levels of‘Bacteroides’,‘Proteobacteria’,and‘Actinomycetes’was increased in the HFD group.Thus,we speculated that A.muciniphila could regulate the balance of intestinal microbiota in the dog obesity model induced by high-fat diets through promoting the growth of probiotics.Our data would contribute to regulate the intestinal microbiota using probiotics,which indicates that alternative therapies for dog obesity or metabolic diseases.However,the presence of A.muciniphila in dog intestine was not determined prior to the experiment.It is difficult to maintain long-term colonization after oral administration with live bacteria.To improve the application of A.muciniphila,the microcapsule of A.muciniphila was prepared,which would contribute to enhance the resistance to stomach and bile acids and maintain the number of living bacteria.The sodium alginate,pectin,and chitosan were used as materials,and A.muciniphila was entrapped using microcapsulation with an entrapment rate of 81.8%.The result of gastrointestinal stability by stimulation in vitro revealed that the survival rate of bacteria coated by microcapsule was significantly increased compared with the control group.The number of living bacteria in microcapsule was 1.57×107 CFU/g stored at 4℃,which consistent with the standard of International Dairy Federation（107 CFU/g）.Therefore,this preparation of microcapsule could be contributed to improve the efficiency of A.muciniphila.