| Phytate,as an anti-nutritional factor in feed,can inhibit the absorption of protein,mineral elements,glucose and amino acids in broilers,thus affecting the growth and development of broilers.Phytase,as a feed additive,can degrade phytate and release inorganic phosphorus in feed,improve the digestibility of phosphorus in broilers,and help the normal growth and development of broilers.However,phytase,as a kind of protein,is vulnerable to the interference of adverse factors in the external environment and loses its activity,which limits the use range and conditions of phytase.It is an effective way to improve the stability of phytase to physically embed phytase with chitosan nanoparticles which are non-toxic,cheap,biodegradable and biocompatible.In this paper,chitosan as the substrate,sodium tripolyphosphate as the cross-linking agent,phytase as the active ingredient,using ionic gel method to prepare chitosan-embedded acidase nanoparticles(CSNPs-Phy),through single factor experiment and orthogonal experiment to study the factors affecting the preparation of CSNPs-Phy and optimize the preparation process.In addition,the related properties of the coated phytase material in vitro,including high temperature resistance,strong acid,strong alkali,trypsin degradation resistance,in vitro protein release,enzyme preparation storage and so on,were further studied and discussed.Finally,the effects of free phytase and coated phytase on the release of phosphorus and the digestibility of dry matter in the feed of broilers were investigated by a two-step in vitro digestion model of broiler feed.The main conclusions are as follows:1.CSNPs-Phy was prepared by ionic gel method,and it was found that the chitosan nanoparticles prepared by chitosan with low molecular weight had smaller particle size.Meanwhile,the effects of chitosan solution concentration,reaction system p H,initial enzyme concentration and crosslinking time on the encapsulation of phytase by chitosan nanoparticles were analyzed.In addition,by optimizing the preparation process conditions,the optimal conditions for preparing CSNPs-Phy were determined as follows: the concentration of chitosan solution was 4.0 mg/m L,the p H of the reaction system was 5.00,the cross-linking time was 45 min,and the concentration of initial phytase was 5.0 mg/m L.The CSNPs-Phy prepared under optimal conditions has a particle size of 249.73 nm,a Zeta potential of 38.73 mv,a PDI of 0.196,an embedding rate of 69.23%,and a drug loading of 18.04%.Chitosan nanoparticles were characterized by FTIR and DSC,and the physical encapsulation of phytase protein by chitosan nanoparticles was confirmed.2.Research on the in vitro characteristics of CSNPs-Phy found that: CSNPs-Phy has a wider temperature and p H range than free phytase,and has better resistance to high temperature,strong acid and alkali,and is more stable to trypsin degradation.The determination of the kinetic parameters of the enzymatic reaction showed that the phytase weakened the affinity between the phytase and the substrate after the chitosan nanoparticles were encapsulated.Simultaneously,chitosan nanoparticles have a certain slow-release ability for phytase protein,and a slow-release medium with high ionic strength can accelerate the release of phytase protein The model of protein release kinetics also depends on the type of slow-release medium in which chitosan nanoparticles are located.The addition of 1.0%sucrose as a freeze-dried protective agent of CSNPs-Phy can effectively prevent particle aggregation during storage and greatly improve the stability of CSNPs-Phy during storage.3.A two-step method was used to simulate in vitro feed digestion of broilers,and it was found that encapsulated phytase could better promote the release of phosphorus and the digestibility of dry matter in broilers feed than free phytase because of its better stability.In this paper,phytase nanoparticles embedded in chitosan were studied for the first time,which was different from the traditional feed enzyme preparation.It was verified that coated phytase has stronger stability than free phytase in vitro,can better promote the release of nutrient elements and the digestibility of dry matter in feed,and is expected to be a new feed additive for broilers. |
