The Preparation Of Compound Milk-clotting Enzyme Capsules And Its Effect On Functional Dyspepsia

Our laboratory had found a B-.subtilis(Bacillus subtilis)YB-3 strain which can secrete the MCE(milk-clotting enzyme)with a high yield in the preliminary study.In addition to having a stronger proteolytic activity to a-casein,the MCE YS-1 also can hydrolyze ?-casein and?-casein.However the animal rennet has almost no hydrolysis to ?-casein.So the MCE exhibits broad-spectrum proteolytic capacities and has the potential to replacing the animal rennet in the compound rennet capsules.For these reasons,the thesis aims to make a deep research on the pharmaceutical properties of MCE YS-1 with following four aspects:the fermentation production process,the extraction and purification process of MCE YS-1,the pharmaceutics studies of MCE YS-1 and the treatment effect of composite MCE capsule on functional dyspepsia.The main contents and conclusions on this paper are as follows:1.The effect of fermentation parameter on the yield of MCE in the shake-flask culture and the pilot fermentation were respectively studied.The enzyme activity could reach the maximum of 85.41SU/ml with a inoculation quantity 1.67%(v/v)in the shake-flask culture stages.As the B-.subtitle-3 is a aerobic bacteria,enough oxygen can play an important role in increasing the amount of MCE in the shake flask culture stages.So the culture broth quantity in 500ml flask is 30ml.In the shake flask culture stages,6-14h were the logarithmic phase of the bacteria and the seed age in the pilot scale fermentation should be 12h.The stirring speed must be set to 600 rpm for ensuring the normal growth of bacteria and enzyme secretion in the pilot fermentation stage.Feeding the beef extract solution after 10 h in the fermentation could relieve the lack of nutrients and significantly delay the decrease in the enzyme activity.The fermentation process should be stopped after 10 h because the enzyme activity reached maximum at this point.2.This paper removed the bacteria in the fermented liquid by centrifuge and enriched the sterile fermentation liquid by flowing through a 10 kDa hollow fiber membrane.The concentration rate was 2.7 times and the enzyme yield was 73%.The MCE can be purified to 2.05 times and the milk-clotting activity and protein content ratio can reach 884.12 SU/mg by fractional precipitation with 20-60%ethanol.Then,we compared the MCE yield of the ethanol precipitation dissolving solution which were respectively dried by ambient pressure drying and vacuum drying.The result is that the MCE obtained by these two kinds of drying methods had a similar yield(86.60%:89.97%).However,the ambient pressure drying is easy operation and lower cost when compared with the vacuum drying.So we chose the 40? ambient pressure drying to dry the ethanol precipitation dissolving solution and then ground into powder.3.The article first studied the characters of MCE powder and found that it was pale yellow or gray,slight odor powder,could extraordinarily lead to wet and slightly dissolve in water.The milk-clotting activity was no less than 80000SU/g.Then we used the MCE and pepsin as the main drugs and screened the dosage of starch,mannitol and other excipients.The result is that the best prescription is pepsin 20mg,MCE 56mg,starch 67.1 mg,lactose 17.1mg,mannitol 18mg,magnesium stearate 1.8mg per 180mg prescription powder.We chose the capsule(5)for filling the prescription powder.In the end,this paper examined the dry weightlessness,filling quantity differences and disintegration time of the capsule according to the provisions of capsule on Ch.p 2010(2)and found that dry weightlessness,filling quantity differences and disintegration time were within the scope of the requirements.4.The paper studied the treatment effect of the composite MCE capsule on functional dyspepsia.First of all,we judged the FD rats model success by the significantly decreased of the diet amount and the rats weight growth rate of modeling group compared with the blank group in the modeling time and the none obvious organic disease in the modeling rats after the end of building model in the anatomical observation.Then we found that compound MCE,composite MCE and domperidone can improve the appetite and gastrointestinal dynamics of FD rats according to the results of the rats weight growth rate and gastric emptying rate in each rats groups after the final administration.Compound MCE capsule and composite MCE capsule which had been enrolled have the same treatment effect on the FD rats and were little lower than the effect of domperidone.