Study On The Preparation Technology,Antibacterial Properties Of Copper-Loaded Montmorillonite And Its Application In Weaned Pigs

Long-term use of high copper and antibiotics will cause problems such as bacterial resistance and environmental pollution.At present,the use of antibiotics in feed has been banned,so finding corresponding alternatives is one of the research hotspots.In addition,montmorillonite（MMT）is an excellent mycotoxin adsorbent and metal ion negative carrier.Therefore,the purpose of this study is to prepare copper-loaded montmorillonite（Cu-MMT）with copper sulfate and montmorillonite as the main raw materials.The structural characterization,mycotoxin adsorption rate,antibacterial properties and antibacterial mechanism of copper-loaded montmorillonite were evaluated in vitro.Piglet feeding experiment was conducted to evaluate the effects of copper-loaded montmorillonite as feed additive on growth performance,immune function and antioxidant capacity of weaned piglets,so as to provide scientific basis for the application of copper-loaded montmorillonite in pig production.The main findings are as follows:Study on the preparation process,antibacterial properties and mechanism of copper-loaded montmorillonite:1)Orthogonal test showed that the order of influence of various factors on the amount of copper was:reaction temperature>material ratio>reaction PH>reaction time,and the optimum process for preparing Cu-MMT was material ratio 1,reaction time 150 min,reaction temperature 40°C,reaction PH5.1,and the maximum amount of copper downloaded under this condition was 3.76%.2)The structural characterization results of Cu-MMT show that it is still a stable layered structure.Cu²⁺mainly enters the interlayer of montmorillonite in the form of hydrated cations,resulting in an increase in interlayer spacing.The content of Cu²⁺is 4.9%.3)The adsorption capacity of Cu-MMT on aflatoxin（AFB1）,zearalenone（ZEN）and deoxynivalenol（DON）was evaluated.The results showed that compared with MMT group,the adsorption rate of Cu-MMT on AFB1 was significantly increased by 20.64%（P<0.05）,and the adsorption rate of DON was significantly increased by 33.07%（P<0.05）.Cu-MMT has stronger adsorption capacity and is a good mycotoxin absorbent.4)The antibacterial ability of Cu-MMT was evaluated by inhibition zone,MIC,MBC and growth curve.The results showed that the antibacterial effect of Cu-MMT on the three pathogenic bacteria was in the order of Salmonella>Escherichia coli>Staphylococcus aureus.Cu-MMT had better killing effect on Escherichia coli and Salmonella than copper sulfate pentahydrate.Cu-MMT with 1.0×MBC could completely kill bacteria and had strong antibacterial effect.5)The antibacterial mechanism of Cu-MMT was explored by studying the effects of Cu-MMT on cell membrane permeability,cell wall permeability,reactive oxygen species and DNA.The results of cell membrane permeability showed that compared with the control group,the OD₅₉₅ value of the three bacteria and Cu-MMT co-cultured for 6h increased significantly（P<0.05）,and the protein in the bacterial membrane leaked.The results of cell wall permeability showed that compared with the control group,the AKP content was significantly increased（P<0.05）and the cell wall permeability was increased when the three bacteria were co-cultured with Cu-MMT for 6h.The results of the effect on the production of reactive oxygen species showed that the green fluorescence intensity and number increased with the increase of Cu-MMT addition,and the reactive oxygen species produced in the three bacteria increased.The results of the effect on DNA showed that the length of DNA bands in the Cu-MMT group was the same,the brightness was reduced,and no other fragments appeared.Therefore,it can be concluded that the antibacterial mechanism of Cu-MMT is not to destroy the DNA of bacteria,but to produce reactive oxygen species,resulting in the destruction of bacterial cell membrane and the increase of cell wall permeability,affecting bacterial reproduction and reducing the number of bacteria.Effects of copper-loaded montmorillonite on growth performance,immune function and antioxidant capacity of weaned piglets:480 weaned piglets（Duroc×Landrace×Yorkshire）with an average body weight of 6.83±0.16 kg at 23±1 days of age were randomly divided into 4 groups:The control group（CON group）,Cu-MMT I group（Cu-MMT I group）,Cu-MMT II group（Cu-MMT II group）,Cu-MMT III group（Cu-MMT III group）,6 replicates in each group,20 piglets in each replicate.The CON group was fed a basal diet,the Cu-MMT I group was fed the basal diet supplemented with 1 g/kg copper-loaded montmorillonite,the Cu-MMT II group was fed the basal diet supplemented with 2 g/kg copper-loaded montmorillonite,and the Cu-MMT III group was fed the basal diet supplemented with 3 g/kg copper-loaded montmorillonite.The test period was 22 days.The results show that:1)Compared with CON group,the final weight,ADFI and ADG of Cu-MMT I group,II group and III group were significantly increased,and the diarrhea rate was significantly decreased（P<0.05）.Its supplemental level was quadratically correlated with final weight and ADG（P<0.05）,and linear and quadratically correlated with ADFI and diarrhea rate（P<0.05）.2)Compared with CON group,the content of Ig A and IL-10 in Cu-MMT I group,II group and III group increased significantly（P<0.05）,and the addition level was linearly and quadratically correlated with the content of Ig A and IL-10（P<0.05）.3)Compared with CON group,the activity of SOD in Cu-MMT I group,II group and III group was significantly increased（P<0.05）,and the content of MDA was significantly decreased（P<0.05）.The test results show that:Cu-MMT can effectively improve the growth performance,immune and antioxidant capacity of piglets and reduce the incidence of diarrhea in piglets.Based on the experimental results and economic factors,the addition of1g/kg Cu-MMT can be fed to piglets.