Study On The Preparation Of Mimic Intestinal Fluid And Simulated Digestion Method For Determination Of Digestible Nutrient Content Of Feeds For Pigs

This study was conducted to investigate the relationship between digestive enzyme of jejunal fluid and nutrient digestion, develop a method for purification of digestive enzyme and preparation of mimic intestinal fluid(MIF), then evaluate the feasibility of simulated digestion method for determination of digestible energy(DE) and digestible amino acid(DAA) using the simulative digestion system(SDS-Ⅱ) and MIF for pig feed. Five parts were included:The objective of Exp. 1 was to investigate the digestive enzyme activity of jejunal fluid in pigs fed different diets at different growing phases to establish a standard of enzyme activities for preparation of MIF. Two diets(corn-soybean meal or corn-DDGS-rice bran meal) and two growing phases(growing period(60-100 d) or fattening period(100-140 d)) were used in a 2×2 factorial arrangement in a completely randomized design. The amylase, trypsin, chymotrypsin and lipase activity, concentration of total protein and p H of jejunal fluid were determined. The results showed that growing phases and diets had no significant effects on the enzyme activity and p H of jejunal fluid(P>0.0.5). The activities of amylase, trypsin, chymotrypsin, lipase and p H were 189.79 U/ml, 62.10 U/ml, 7.36 U/ml, 4.91U/ml and p H 6.44, respectively.The objective of Exp. 2 was to investigate the relationship between digestive enzyme activity of jejunal fluid and dietary nutrient digestibility to establish a standard of error control in digestive enzyme activity for preparation of MIF. Sixteen replicates with 1 pig in each were used in Corn-Soybean meal and Corn-DDGS-rice bran meal, respectively. The contents of DM, AIA, CP and GE in the diets and feces, and digestive enzyme activity of jejunal fluid were determined. The relationship between digestive enzyme activities of jejunal fluid and dietary nutrient digestibility were calculated with simple correlation analysis and canonical correlation analysis. The results showed as follows:1) The CV of 4 digestive enzymes were over 20 % and CV of digestibility of DM, CP and GE were no more than 1.5 % in pigs fed both diets; 2) No significant correlation were observed between the nutrient digestibility of diets and activity of amylase, trypsin or chymotrypsin(P >0.05). There was low-middle correlation between nutrient digestibility and lipase activity, however, the contribution of lipase activity to the variation of nutrient digestibility was little.Two parts were involved in Exp. 3, the objective of part 1was to develop a purification method for the digestive enzyme of jejunal fluid from growing pigs, and part 2 was to compare the digestive ability of MIF prepared by reagent enzyme and PPIF. Two diets and two growing phases were used in a 2×2 factorial arrangement in a completely randomized design. The digestive enzyme of jejunal fluid were purified using ice ethanol-precipitated followed freeze-dried method(IAPFD). The specific activities of enzyme in powder purified from intestinal fluid(PPIF) were determined, then the loss of enzyme activity in the process of purification was calculated. The enzymatic hydrolysate gross energy(EHGE) of diets were determined using simulated intestinal fluid prepared by reagent enzyme(RE), PPIF from intra-pigs with RE, and PPIF from total pooled pigs with RE in accordance with each of the enzyme activity of 4 treatments. Then EHGE values were compared with the in vivo DE values. The results showed as follows: 1) Diet and growing phase didn’t significantly influence the enzyme activity of PPIF. The recovery of enzyme activity were more than 60 %. 2) The loss of enzyme activity in the precipitate process were significantly higher than that in the freeze-drying process(P<0.01). 3) EHGE determined with MIF prepared by PPIF were higher than reagent enzyme. The correlation coefficient between EHGE and DE was also higher in MIF prepared by PPIF than reagent enzyme. In conclusion, PPIF is better in preparation of MIF.The objective of Exp. 4 was to predict the DE from EHGE determined with the simulated digestion method based on pepsin-MIF-Viscozyme compound enzyme in cottonseed meal diets for pigs. The EHGE values of 11 dephenolization cottonseed proteins and 19 cottonseed meal diets were determined with MIF prepared by PPIF and reagent enzyme, respectively. Then the EHGE were compared with the DE. The results showed as follows:1) High correlation between EHGE determined with PPIF and reagent enzyme was observed in dephenolization cottonseed protein(P < 0.05, r=0.96). 2) Higher correlation was observed between DE and EHGE determined with MIF prepared by PPIF than reagent enzyme(r=0.97 VS 0.87). Therefore, the EHGE determined with the MIF prepared by PPIF is better than that determined with reagent enzyme in cottonseed meal.The objective of Exp.5 was to predict the DAA content by the two-stage of simulated digestion method based on pepsin-MIF in cottonseed meals for pigs. The DAA contents determined with SDS-Ⅱ and in vivo method were compared in 10 cottonseed meals. The results showed that significant correlation was observed between in vitro content of DAA exclued methionine and in vivo value(P<0.05), which indicated that MIF prepared by PPIF can be used to determine the DAA content of cottonseed meal.