Research On Biological Evaluation Technique Of Feed Enzyme

With the widely application of feed enzyme in feed industry, the evaluation of feed enzyme effect is becoming the focus of enterprises and farmers. At present, there are two ways to evaluate the effect of enzyme, One is enzyme activities determination, the other is to determine digestibility in vivo.The method of activities determination have done under strict experimental condition with sepicial enzymes,but the digested conditions in animals are very complex system which have effected the evaluation of enzyme activities by variety of factors. Animal experiment is a good approach to evaluate the biological value of feed enzyme .But the experiment is easily to be affected by environment, animal species, physiological stage,and the feeds the animals intake ,etc.So it's necessary to establish a rapid, accurate and reliable method for enzyme evaluation. In this experiment, we choose the piglets as experimental animals because of sensibility to enzyme.We determine the actual digestive environment of piglet's stomach and small intestine, combined with relative references , on the basis of determination, we imitate the digestion environment in vitro to find out the conditions that affect the evaluation of this in vitro method,and then, we choose the best conditions of this in vitro method, and we use phytase, xylanase as a model in vivo method to verificate the above established methods and use the method to evaluate the biological value of mannanase. All of the experiments were as follows:Experiment 1.Determine the main digestive parameters of piglet.Six healthy piglet its bodyweight is 15kg were provide with NRC corn-soybean meal. During the trial period, all animals were fed with three times a day and drunk water ad libltum.The management keep accordence with normal piglets. After 15 days, the piglets were slaughtered when they were feeded in 1h and rapidly splited the abdominal cavity, ligated pyloric valve and ileocecal valve. The sample took from the stomach and small intestine were immediately determined some parameters including pH and tempreture. The samples were pretreatment in liquid nitrogen and stored at -70℃,digestive enzyme activities of chyme in duodenum, jejunum and ileum will be measured after treatment.The results as follows:the tempreture of the stomach: 37.5～38.4℃,pH value:1.85～3.48,pepsin activity:192.53～255.67 U/ml ; the enzymes activities of small intestine(U/g chyme): amylase:714.08～893.75;lipase:720.09～1156.69;trypsin:522.96～653.08;chymotrypsin:432.10～595.81.pH:6.77-7.91.Experiment 2. Establishing a digestion method in vitro to imitate digestion in vivo and its optimization parameters.Two orthogonal design were conducted to imitate the important factors of the stomach and small intestine.Tempreture of the stomach: 37℃,38℃,39℃;pH:1.5,2.5,3.5;pepsin level:150 U/ml,220 U/ml,290 U/ml;time:2h,3h,4h.The screening indexes include digestibility of dry matter and crude protein. After stomach digestion, the best parameters of the stomach is 39℃,pH:1.5, pepsin concentration: 220 U/ml, time:4h. Base on the optimization condition, DM and CP have the highest digestibility. After stomach digestion, imitate the optimization parameters of the small intestine, pH value:6.0,7.0,8.0; the retention time of chyme: 3h,6h,12h; buffer :300ml 400ml 500ml.enzyme activity 25mg/g,35 mg/g,45mg/g chyme.Considered DM and CP digestibility as determined items.The results suggest:the best digestion parameters of the intestine is pH6.0,enzyme :45mg/g chime,retention time: 12h, buffer: 300ml.Therefore, the DM and CP have the higher digestibility than the others in the small intestine.Experiment 3: Veirfy the in vitro method by studying the effect of additive phytase on piglet productivity and digestibilities of nutrient in vitro and in vivo. The result shows:the digestibility of DM, CP, DE, Ca ,P are high correlation coefficient between in vitro and in vivo methods(P<0.05). the R2 were 0.88,0.59,0.65,0.90,0.95. It was concluded that the methods we found was efficient and reliable. It can be used to evaluate feed enzyme biological value. Additive phytase to low- phosphorus diet has significant effect on the digestibility of Ca and P and has high correlation coefficient between in vitro and in vivo methods(P<0.05).So the digestibility of Ca and P in vitro could be used to predict the biological evaluation of phytase.Experiment 4: In order to verify the methods efficiency and reliability in diferent enzymes, the diet applied with xylanase got the further research.The result shows: The digestibility of DM CP DE CF EE are high correlations between in vitro and in vivo methods(P<0.01),the correlation coefficient is respectively of 0.87,0.83,0.94,0.95,0.89.It showed that the methods that had been established in experiments were efficiency and reliability and can be used to evaluate biological value of different enzymes . Additive xylanase to low-energy diet has significant effect on the digestibility of DE and CF and has high correlation coefficient between in vitro and in vivo methods(P<0.01).So the digestibility of DE and CF in vitro could be used to predict the biological evaluation of xylanase.Experiment 5:Evaluation the biological value of mannanase by using the in vitro method . The results show: mannanase had increased the digestibility of DM, CP and DE of corn-soybean diet in vitro .The group which was applied with 600U mannanase had the highest digestibility in DM,CP and DE in experiment.The results are agree with many other references and verify the method are practical.