| The physiological functions of vitamin B12 include:participating in DNA synthesis as a coenzyme;promotes the metabolism of carbohydrates,sugars and fats;to promote the maturation of red blood cells and participate in the formation of lipoprotein in nerve tissue.Vitamin B12 deficiency can lead to stunted growth,hematological and neurological symptoms.As research has progressed,vitamin B12 deficiency has been reported in many diseases in dogs and cats,including the gastrointestinal tract,liver,and pancreas.Vitamin B12 supplementation is particularly important for the treatment of companion animals with chronic gastrointestinal disease.Timely detection of vitamin B12 deficiency and targeted supplementation have a significant impact on the prognosis of many affected animals.The traditional treatment for vitamin B12 deficiency in dogs and cats is subcutaneous injection of vitamin B12.Currently,there are also studies showing that oral vitamin B12 supplementation can replace subcutaneous injection,which can reduce animal suffering and improve owner compliance.Because of its complex structure,vitamin B12 is difficult and expensive to synthesize chemically,so it is obtained mainly by fermentation of industrial strains.However,industrial fermentation usually has more adverse factors,resulting in higher production cost of vitamin B12.It is reported that some generally regarded as safe lactic acid bacteria have the ability to synthesize B vitamins,including the synthesis of vitamin B12 ability of lactic acid bacteria,However,research on vitamin B12-producing lactic acid bacteria from animal sources at home and abroad is still quite scarce.In this study,canine producing vitamin B12 lactic acid bacteria were screened to optimize their culture conditions in order to obtain high-quality vitamin B12-producing strains and simplify the production process of vitamin B12.It lays a certain theoretical foundation for the study of vitamin B12-producing lactic acid bacteria from canine sources and the prevention and treatment of canine vitamin B12 deficiency.Experiment 1 Isolation and identification of canine producing vitamin B12 lactic acid bacteriaIn this study,lactic acid bacteria in feces of healthy dogs were isolated by plate coating method,and 40 strains of suspected lactic acid bacteria that were gram-positive and catalase test-negative were preliminarily screened through morphological observation and physiological and biochemical identification.The content of vitamin B12 in the fermentation broth of the isolated strain was determined by microbiological method.The results showed that the three strains had the ability to produce vitamin B12.After 16S rDNA molecular biological identification,one strain was Lactobacillus reuteri(L-26),and the other two strains were Lactobacillus animalis(L-1 and L-8).Experiment 2 Study on biological characteristics of canine producing vitamin B12 lactic acid bacteriaIn order to screen for canine producing vitamin B12 lactic acid bacteria with excellent probiotic properties,The biological characteristics such as growth characteristics,acid production capacity,and tolerance of acid environment,bile salt,trypsin and high temperature of the strains were determined respectively.The results showed that L-26 entered the log phase after 4 h of culture,and the growth rate was relatively faster than that of L-1 and L-8.After culture for 24 h,the pH value of supernatant of each strain was between 3.80 and 4.45.After 4 h of treatment in the pH 2.0 environment,the number of viable bacteria in the L-8 and L-26 bacterial solution could reach 103 CFU/mL and 105 CFU/mL respectively,while L-1 could not tolerate the pH 2.0 acidic environment.After treatment with 0.4%bile salt for 4 h,the number of viable bacteria in the L-26 solution could reach 104 CFU/mL,while L-1 and L-8 could hardly survive.After treatment with 1,1.2 and 1.4%trypsin for 4 h,all strains were growth was normal.After the treatment at 60? for 10 min,the number of viable bacteria in the L-8 and L-26 solution could still reach 106 CFU/mL,while the number of viable bacteria in the solution decreased to 104 CFU/mL after the treatment at 60?for 8 min.The results of this experiment showed that the three lactic acid bacteria had good growth characteristics and acid production capacity,and had certain tolerance to acid,bile salt,trypsin and high temperature conditions,among which L-26 had good biological characteristics.Experiment 3 cultural condition optimization of canine producing vitamin B12 lactic acid bacteriaIn order to obtain the best yield of vitamin B12,the culture temperature,culture time,inoculation amount,medium pH and Co2+ concentration in culture medium of strain L-26 were optimized.The results showed with other culture conditions unchanged After 96 h of culture,the vitamin B12 content in the fermentation broth reached 9.54 ?g/L.After the addition of different concentrations of Co2+ to the culture medium,the vitamin B12 content in the L-26 fermentation broth increased to different degrees.Among them,when the Co2+ concentration in the culture medium was 10g/mL,the vitamin B12 content in the fermentation broth reached 5.17?g/L(48h).However,the changes of culture temperature,inoculation amount and medium pH could not significantly increase the vitamin B12 content in fermentation broth.Experiment 4 Safety evaluation of canine producing vitamin B12 lactic acid bacteriaIn this study,the safety of isolated L-26,the influence of animal intestinal flora and intestinal mucosal mechanical barrier,and the possibility of colonization in dog intestinal tract were studied by using mice and dogs as animal models.The results showed that there was no significant change in the growth status,serum biochemical indexes,and tissues and organs of the mice after continuous administration of 100 ?L conventional dose(2×108 CFU/mL)and high dose(2×1010 CFU/mL)of L-26 bacterial solution.After continuous supplementation of 5 mL of conventional dose(1×107 CFU/mL)and high dose(1×109 CFU/mL)of L-26 bacterial solution,there was no significant change in its growth status and serum biochemical indexes.The experiment was found that after conventional dose and high dose L-26 solution was administered for 14 days,the number of lactic acid bacteria in the feces of mice was significantly increased compared with the control group,the number of E.coli was significantly reduced,the length of j ejunum villi was significantly increased,and the depth of crypts was significantly reduced.V/C value increased significantly,ileum villi length and V/C value increased significantly,and crypt depth did not change significantly;After continuous supplementation with rifampicin labeled L-26 for 14 days,L-26 could be detected in dog feces,and the number of L-26 viable bacteria in dog feces could still reach 104 CFU/mL after stopping supplementation for 7 days.This experiment shows that L-26 is safe for mice and dogs.L-26 has the probiotic effect of regulating intestinal flora and promoting the development of intestinal mucosal mechanical barrier,and can survive and colonize in the digestive tract of dogs.Therefore,the isolated strain L-26 can be used as a potential probiotic for dogs,but whether it can be used in clinical application still needs further research. |
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