| Lactobacillus plantarum is a probiotic that has the function of promoting growth and intestinal development of animal,and is widely used in food and livestock industries.However,L.plantarum has poor acid resistance and is sensitive to the environment of the digestive tract containing gastric acid,resulting in an insufficient number of live bacteria reaching the intestine to exert its probiotic effect.Therefore,in order to enhance the acid resistance of L.plantarum and improve its survival in the digestive tract.In this study,L.plantarum microcapsule was prepared by vacuum mixing method with enzymatic hydrolysate of soybean protein isolate(EHSPI)and modified phospholipid(MPC)as the main wall materials,and the physicochemical properties,in vitro digestion and storage of the microcapsule system were investigated.Next,the effects on the growth and intestinal development of chicks were further analyzed through in vivo test,and the following results were obtained:(1)The preparation of L.plantarum microcapsule was investigated and the results as follows:the microcapsule showed a regular round shape and L.plantarum was encapsulated in this system.Under neutral conditions,the particle size distribution was uniform,and the Zeta potential was negative.Microencapsulation efficiency(ME)and stability(P<0.05)of microcapsule increased with emulsification time,pre-emulsification time and MPC content.ME was significantly affected by pre-emulsification time,emulsification time,bacteria/EHSPI ratio,EHSPI content,and MPC content(P<0.05).On this basis,ME serving as the response value,the optimization test of process conditions was carried out firstly,and the optimal process conditions were obtained:pre-emulsification time 32 min and emulsification time 96 min.And then the optimization test of the system composition was carried out on the basis of these process conditions,and the optimal system compositions were obtained:bacteria/EHSPI(w/w)1:1.83,EHSPI content(w/w)4.01%,MPC content(w/w)11.41%.Under these conditions,ME was 81.31%.And the validated values were not significantly different from the predicted values(P>0.05).(2)Changes in the properties of L.plantarum microcapsule during in vitro simulated digestion(kinetic and static)were analyzed and no significant changes in Zeta potential,conductivity of microcapsule(P>0.05)during passage through simulated gastric fluid were observed.The emulsification activity index(EAI),emulsification stability index(ESI)and Z-average particle size remained stable after adaptation to the environment,and the particle size distribution was single-peaked,and the droplet size of microcapsule distribution was uniform.The internal aggregates of microcapsules continued to remain aggregated,and the number of viable bacteria in the kinetic and static digestion was 1.35×10~8CFU/m L and2.49×10~7CFU/m L,respectively,which were significantly higher than that of the control(P<0.05).Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)indicated that bothα’subunits andβsubunits were consistently present.After transferring to the simulated intestinal fluid,Zeta potential,conductivity,EAI and ESI changed significantly(P<0.05),multiple peaks appeared in the particle size distribution graph,droplet sizes varied,and the originally aggregated bacteria gradually changed to a diffuse state.All protein subunit bands disappeared at the end of the in vitro digestion.And after 120 min of digestion in the intestinal fluid,the number of viable bacteria in the dynamic and static digestion reached 5.89×10~8CFU/m L and 3.55×10~8CFU/m L,respectively,which were significantly higher than those in the control group(P<0.05),indicating that the microcapsule could protect L.plantarum to reach the intestinal fluid through the gastric fluid smoothly.(3)The stability of L.plantarum microcapsule stored at 4℃,25℃,and-20℃ for 8 w were studied,respectively,and the following results were as follows:at 4℃,the ESI and Z-average particle size of the microencapsulated system did not change significantly(P>0.05),and the EAI,Zeta potential and conductivity remained unchanged after adaptation to4℃.The particle size distribution was mostly"single peak",and the droplet distribution of microcapsule was more concentrated and uniform in size.The number of viable bacteria decreased to 1.10×10~9CFU/g at 8 w,which was significantly higher than the other temperatures(P<0.05).Theα’,αandβsubunits were consistently present in SDS-PAGE,with the same bands as in the initial EHSPI.The ESI,EAI,Zeta potential,conductivity and Z-mean particle size varied continuously at 25℃(P<0.05),with"multiple peaks"in particle size distribution and there was size coexistence of droplets of microcapsule.The number of viable bacteria continued to decrease to 1.58×10~3CFU/g at 8 w,and the bacteria showed a free state,and the subunit bands disappeared completely.At-20℃,the ESI,EAI,Zeta potential,conductivity and Z-average particle size changed significantly(P<0.05),and the particle size distribution was mostly"bimodal"and the microcapsule structure was disrupted.The viable bacterial count decreased to 7.76×10~8CFU/g at 8 w,withα’,αandβsubunits consistently present(P<0.05).(4)The effect of L.plantarum microcapsule(emulsion)on growth and intestinal development of layer chicks at 7,14 and 21 d of age was investigated.The results were as follows:the addition of microencapsulated L.plantarum to the drinking water of layer chicks significantly increased the body weight,average daily feed intake and average daily weight gain of layer chicks at 21 d of age(P<0.05),and increased the length and weight of the small intestine of layer chicks(P<0.05)and increased the capacity of the small intestine.At21 d,the number of viable bacteria of L.plantarum in the ileal contents of layer chicks was6.46×10~7CFU/g,which was significantly higher than that of the control group(P<0.05)and further increased the lactic acid content and decreased the intestinal p H(P<0.05).The addition of L.plantarum microcapsule significantly improved the villus height,crypt depth and villus height/crypt depth in different intestinal segments at different stages,especially in21 d layer chicks(P<0.05),indicating that the effect of improving intestinal development was better with longer feeding time.In summary,L.plantarum microcapsule with EHSPI-MPC as the wall materials has excellent acid resistance,which can improve their acid tolerance and survival in gastric juice and allow L.plantarum to pass through gastric juice and release in the intestine smoothly.It also has a higher number of viable bacteria stored at 4℃ and is more stable.Animal experiments have shown that L.plantarum in microencapsulation can increase the survival rate of L.plantarum in the intestine,further promote intestinal growth and development and improve the performance of chicks and regulate intestinal health. |
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