Protease Evaluation By The In Vitro Method And Application In Broiler Diet

The shortage of protein-feed resource is the key factor that restrict the development of livestock industry in our country. Feed-protease can improve the utilization rate of feed protein, to some extent, relieve the shortage of protein resource. As the increasing of protease products as well as the time-consuming of traditional evaluation method "animals trial", it is increasingly important and meaningful to build a method which could screen and evaluate the protease fast and accurate. The in vitro method can be used for rapid evaluation of feedstuff which has been accepted, and the attention on the use of in vitro method to evaluate the enzyme has been gradually increased. The protease is different from other feedenzyme for the interaction effect with the endogenous digestive enzymes. Therefore, the in vitro method is more sensitive to the parameters of animals, especially the digestive enzymes activity. Thus, an in vitro method for evaluating the protease performance is indeed needed. According to the research on influencing factors of protease effect, it is meaningful to provide a method for the scientific application and accurate evaluation of protease. This trial built an in vitro methed which is used to evaluate the effect of protease on chicken by measuring the actual gastrointestinal parameters of 31d broiler. To selecte the optimum level of four proteases (acid protease, neutral protease, alkaline protease and keratinase), in four addition levels (0.2,0.4,0.8, 1.6 U/g substrate), on two feedstuff’s (corn protein powder and soybean meal), the in vitro method was used. In addition, the possible factors that influence the effect of protease was analysed, and in order to prove the veracity of the in vitro method, an in vivo trial was also adopted. The result is as follows:1. The trial build an in vitro method to evaluate the performance of protease on broiler chicks:in a centrifuge tube,1.0000 g material was suspended in 3.5 mL of HC1 ensuring the pH is 4.9, in order to keep from the microbe,0.5 mL chloramphenicol solution was added,40 ℃ for 50 min. The solution was added 0.5 mL HCI to adjust the pH to 3.3, before 0.5 mL pepsin-HCl was added the solution was blended,40 ℃ for 61 min. Then the pH of the solution was adjusted to 6.2 by adding 1 mL NaCO3,40 ℃ for 163 min.The protein was precipitated by 5 mL sulphosalicylic acid (20%), after 30 min standing, the solids were separated by centrifuging.2. Study on the optimum level and the effects of four proteases on feedstuff. The addition of proteases on soybean meal significantly increased the in vitro crude protein digestibility (IVCPD) (P<0.05). For corn protein powder, the IVCPD has also been improved more or less. The optimal level of acid protease, neutral protease, alkaline protease and keratinase for soybean meal and corn protein powder were respectively: 1.6、0.8、0.8、1.6 U/g substrate and 0.8、1.6、0.4、0.2 U/g substrate. The acid protease at the optimum level alone for corn and soybean meal (7:3) was the best, significantly increase IVCPD than control (P<0.05), and the effects of combining with neutral protease and alkaline protease has no difference with the acid protease alone.The single use of neutral protease, alkaline protease and keratinase has no significant effect on IVCPD of corn and soybean meal (7:3). These indicate the influence of protease on feed ingredients deponds on the tipe of substract as well as the kind and the level of protease, in addition, the combination of acid protease with other protease had no synergistic effect (P>0.05).3. The study on main factors that influence protease effect. The activity of neutral protease, alkaline protease and keratinase in pH 3.30 is low to 10% of the optimal level, and these three proteases all showed renaturation when faced a hostile pH 3.30. The activity of keratinase could recover to 37.68% which shows it could act well than other enzymes in small intestine after the digestion of stomach. The IVCPD of corn and maize protein powder have been significant increased by the use of neutral protease, alkaline protease and keratinase by the addition from crop and small intestine respectively (P<0.05), and it was better to add protease from crop than from small intestine (P<0.05), which shows the digestion time may a critical factor on the performance of protease and the protease could still action after gastric. The addition of four proteases in soybean meal and corn protein significantly improved the IVCPD during the crop phase, the neutral protease showed the best performance than others. Which suggested the crop may play a catalytic role in providing a moderate environment for protease, and the function of acid protease may influenced by other factors besides pH. Acid protease could improve the activity of trypsin, it reached significant level when adding 0.2 and 0.8 U (P<0.05); Neutral protease had no significant influence on activity of trypsin (P>0.05); while the alkaline protease and keratinase can significantly reduce the activity of trypsin (P<0.05). The use of four proteases had no significant effect on chymotrypsin activity (P>0.05). The acid protease increase protein digestibility may though the way of improving the activity of trypsin, while alkaline protease and keratinase may have inhibitory effect on protein digestibility.4. The effects on growth performance of broilers (31 d) and ICPD (in vivo crude protein digestion) of corn-soybean meal by the addition of four proteases at the optimal level. Acid protease alone could significant increase ADFI and ADG of 31d broiler chicken (P<0.05), had no significant difference with the combine of acid protease and neutral protease, acid protease and alkaline protease (P>0.05). While the single use of other three proteases had no significant effect (P>0.05). On the contrary, protease showed a negative influence on ICPD of broilers (31 d) (P<0.05), except the Tyr and Pro, the digestibility of other amino acid were decreased as the use of proteases.In conclusion, the performance of protease is influenced by the enzymatic characteristics, and the addition level and the interaction with endogenous enzymes also have influence on the protease performance. The in vitro method in this trial could be used to evaluate the effects of protease on the performance of broilers, while is not available to evaluate the CP digestibility.