Study On The Efficacy Of NSP Enzyme Evaluated With Simulated Digestion

This study was conducted to investigate the effect of extract solution and solid-liquid separation on the activity of enzymes. Then, the repeatability and additivity of reducing sugar released in feedstuff determined using computer-controlled simulated digestion system(SDS-2) to validate the method for in vitro digestion. Finally, we studied the effect of different supplemental levels of Non-starch polysaccharide Enzyme(NSPE) on the reducing sugar released in pig diets, which provides a reference for the evaluation of feed enzymes products. Specific content as follows:The objective of Exp.1 was to investigate appropriate extract solution and solidliquid separation methods for the enzyme in solid feed enzyme product. Extract solution of deionized water, acetic acid-sodium acetate buffer solution(0.1mol/L), phosphate solution(0.05mol/L) or 0.9%Nacl solution and solid-liquid separation method of no separation, 3000 rpm of centrifugation for 3 min or filtration were used in a 4 × 3 factorial arrangement. Each treatment contained 5 replicates with 2 determinations in each. The activity of enzymes in the products were determined. Then, the effect of the type of extract solution on the solubility of dry matter and protein concentration was investigated for the enzyme products(excluding α-galactosidase) dissolved and centrifuged. The results showed that the highest determined activity of xylanase products was present in the acetic acid-sodium acetate buffer solution. The similar determined activities of β-glucanase product were observed in acetic acid-sodium acetate buffer solution, phosphate solution and 0.9%Na Cl solution, and were significantly higher than that in deionized water(P < 0.05). The greatest and greater determined activity of β-mannanase product was observed in deionized water and acetic acid-sodium acetate buffer solution, respectively, and were significantly greater than these in phosphate solution or 0.9%Na Cl solution(P < 0.05). The type of extract solution had no significant effect on the determined activity of a-galactosidase product(P > 0.05). The solid-liquid separation method had no significant effect on the determined activity of xylanase product(P > 0.05). The greater determined activity of β-glucanase product was observed in centrifugation and filtration. The greatest determined activity of β-mannanase product was present in centrifugation. However, the highest determined activity of α-galactosidase product was observed in no separation. There was a significant interaction between the type of extract solution and solid-liquid separation methods in the determined activities of 4 enzyme products(P < 0.01). The highest dry matter solubility of xylanase product was observed in acetic acid-sodium acetate buffer solution. The highest dry matter solubility of-glucanase and β-mannanase was present in deionized water or 0.9%Na Cl solution. However, the lowest protein solubility of xylanase, β-glucanase and β-mannanase was observed in acetic acid-sodium acetate buffer solution.The objective of Exp.2 was conducted to investigate the linearity、repeatability and additivity of reducing sugar released in pig diets using computer-controlled simulated digestion system(SDS-2). Linearity test: The treatment with 75% corn+25% soybean meal was used in a single factorial completely randomized arrangement. Setting five test treatments: 0.2、0.4、0.6、0.8 and 1.0 g, and each treatment contained 4 replicates with 1 digestion tube per replicate. Repeatability test: The single factor completely randomized design was adopted, 3 treatments with 4 replicates per feedstuff(barley、peanut meal、rice bran、corn and soybean meal). Additivity test: Nineteen treatments consisted of 7 feedstuffs and 12 diets were used in a single factorial completely randomized arrangement. Treatments 1 to 7 were corn, barley, sorghum, soybean meal, peanut meal, cottonseed meal and rice bran, respectively. Treatments 8 to 19 were 12 diets formulated with 2 or more than 2 kinds of feedstuffs at different concentration. Each treatment contained 4 replicates with 1 digestion tube per replicate. The reducing sugar released of all treatments was determined by SDS-2. The results showed that the released total reducing sugar were increased linearly with the increase of sample volume. The linear relationship of sample volume between 0.2g to 0.8g(R2=0.9992) was better than that of sample volume between 0.2g to 1.0g(R2=0.9979), The relative released reducing sugar of sample volume between 0.2g to 0.8g varied from 559.56mg/g to 582.70mg/g, and the coefficient of variation was 1.66%. The relative released reducing sugar of sample in 1.0g decreased 5.37% than that of sample volume between 0.2g to 0.8g. With batch, between batch and total coefficient of variation of the released reducing sugar of barley, peanut meal, corn soybean meal was all less than 1.638%, The maxim absolute bias of repeated measures was controlled in 5.16, 1.17, 6.26 and 1.73 mg/g. The determined values of released reducing sugar were greater than that of calculated values of 12 diets, the coefficient of determination was 0.9996(P<0.05), and the intercept did not differ from 0(intercept=-1.99, P=0.4805), and slope did not differ from 1(slope=0.99, P=0.5141).The objective of Exp.3 was to investigate the effect of different supplemental levels of NSPE on the reducing sugar released in pig diets. Four feedstuffs(corn, wheat, barley and soybean meal) added with different levels of xylanase(0,900,3033,6066 and 10000 U/kg), β-glucanase(0,300,1567,3133 and 5000U/kg), β-mannanase(0,30,323,647 and 1000U/kg), α-galactosidase(0,300,3233,6467 and 10000U/kg) were conducted in a single factorial completely randomized arrangement, The reducing sugar released of all treatments was determined by SDS-2. The results showed as follows: the kinds and content of NSP enzyme required in feedstuffs were different. The released reducing sugar in corn, wheat and barley added with different levels of glucanase and mannanase was significantly higher than that in control group(P<0.05), while the xylanase had no significant effect on the released reducing sugar in soybean meal(P>0.05). The released reducing sugar in soybean meal was significantly higher than control group(P<0.05), when the level of glucanase, mannanase and galactosidase reached a certain level(glucanase with 3133 U/kg, mannanase with 3233 U/kg, galactosidase with 323U/kg). The released reducing sugar in wheat had an increased trend with the increase of xylanse(P=0.0743). The released reducing sugar in barley also had an increase trend with the increase of glucanase(P=0.0560), the released reducing sugar in soybean meal added with mannanase and galactosidase linearly and quadraticly increased(P<0.05). By comparing the increment of released reducing sugar in different treatment, it was shown that the increment of released reducing sugar in wheat had an increased trend with the increase of xylanse(P=0.072). The increment of released reducing sugar in soybean meal linearly and quadraticly increased with the increase of galactosidase(P<0.05).In conclusion, Acetic acid-sodium acetate buffer solution was the most efficient buffer to extract the enzyme protein from the product and display enzyme activity; The way to test the reducing sugar released in feeds with SDS-2 was feasible, and the evaluation of NSP enzyme was also verified by using this mothed.