| Digestive tract living epithelial cells of weaned piglets were extracted to explore the effects of SBA on digestive tract epithelial cell metabolism of weaned piglets by metabonomics,and further reveal the mechanism of anti-nutrition effects of SBA on weaned piglets.This research is divided into three parts,and the main research results and conclusions are as follows:(1)Optimization of batch extraction method and process of SBAIn order to meet the needs of animal experiments and realize batch extraction of SBA,this study optimized the extraction conditions and purification conditions of SBA on the basis of the original experimental conditions.The extraction solution(0.9% Na Cl solution and PBS buffer(p H 7.4)),temperature(4℃ and room temperature)and time(6,12 and 24h)were optimized by a three-factor 2*2*3factorial design.The results showed that there was no significant difference in protein concentration at different time under the same temperature and extraction solution(P>0.05);At the same time and extraction solution,the protein concentration at room temperature was significantly higher than that at 4℃(P < 0.05);Under the same temperature and time conditions,the extraction efficiency of PBS(p H 7.4)solution was significantly higher than that of 0.9% Na Cl(P < 0.05).The optimization of the purification method was based on the original volume of isopropyl alcohol,according to the ratio of guar gum to sodium hydroxide and 3-chloropropylene oxide,the weight of guar gum powder was increased appropriately to improve the efficiency of the preparation of cross-linked Guar polysaccharide gel.After stirring the SBA crude extract with Guar gum,the affinity chromatography was carried out using large and small chromatographic columns respectively.In the experiment with optimized purification conditions,the yield of SBA obtained by 1 L 20% isopropyl alcohol,8.4g sodium hydroxide,60 g guar powder and 18 g 3-chloropropylene oxide in different chromatography columns was significantly higher than that obtained by other schemes(P < 0.05),the yield of SBA obtained by large column was significantly higher than that by small column(P < 0.05).The yield of SBA prepared by large chromatographic column was 542.72±16.97 mg/100 g defatted soybean powder.The purity of SBA was about 93.4%,and the activity was consistent with that of standard substance SBA.Therefore,6h of PBS(p H 7.4)solution was selected as the extraction condition at room temperature,and the efficiency of protein extraction by large chromatographic column combined with the optimized gluemaking scheme was improved by about 4 times compared with previous experiments.This process can provide technical support for SBA mass production and meet the feeding requirements in subsequent animal experiments.(2)Effects of soybean agglutinin on nutrient digestion and intestinal microstructure of weaned pigletsTwelve healthy weaner piglets(Landrace × Yorkshire,7.60±0.99kg)born on the same day were randomly divided into two groups,the blank group(fed soybean meal free diet)and the experimental group(fed 0.2% diet supplemented with laboratory self-purification SBA)with 6 replicates per group and 1 pig per replicate.The apparent digestibility of nutrients was detected.The results showed that the average daily gain of weaned piglets in SBA group was significantly decreased compared with blank control group(P < 0.05);Dry matter digestibility,ether extract digestibility and protein digestibility were also significantly decreased(P < 0.05).The results showed that the activities of trypsin,chymotrypsin,aminopeptidase and sucrase in duodenum,jejunum and ileum contents in SBA treatment group were significantly lower than those in blank treatment group(P < 0.05).The histochemical results of duodenum,jejunum and ileum(HE staining and PAS staining)showed that compared with blank control group,the villus height of weaned piglets in SBA group was significantly decreased(P < 0.05),the crypt depth was significantly increased(P < 0.05),the chorionic gland ratio was significantly decreased(P < 0.05),and the number of goblet cells was significantly increased(P < 0.05).The organelle structure and microvillus morphology of jejunum were observed by electron microscopy.The results showed that the microvilli were reduced,with varying lengths and brown particles in jejunum of weaned piglets after SBA treatment.The tight connection is incomplete and broken;The nuclei of the cells have pyretosis and irregular shape.Mitochondria cavitation phenomenon;Lipid droplets were found in the cytoplasm.These results indicate that SBA can damage intestinal morphology and microstructure of weaned piglets,reduce digestive enzyme activities,and have certain influence on nutrient digestion and absorption.(3)To explore the effects of SBA on intestinal epithelial cell metabolism of weaned piglets based on non-targeted metabolomicsSmall intestine tissues were obtained from animal experiments,epithelial cells were extracted according to Rachel Fellows et al.(2018),and the effects of SBA on intestinal epithelial cell metabolism of weaned piglets were investigated using UHPLC-Q-TOF MS analysis platform.Among them,656 kinds of metabolites were detected in positive ion mode,337 kinds were detected in negative ion mode,and a total of 993 kinds of metabolites were detected.The detected metabolites were classified and summarized according to the chemical classification information,among which lipids and lipid molecules accounted for the highest proportion,about36.052%;Organic acids and their derivatives were followed by 20.040%.Moreover,the content of organic heterocyclic compounds was 8.056%.According to the least square discriminant analysis,compared with the SBA treatment group,30 kinds of differential metabolites were up-regulated and 2 kinds were down-regulated in the positive ion mode,33 kinds were up-regulated and 4 kinds were down-regulated in the negative ion mode,a total of 69 kinds of significant differential metabolites were detected in the blank treatment group.It is speculated that SBA mainly affects the lipid metabolism function of small intestinal epithelial cells of weaned piglets,causes changes in the number of different metabolites in small intestinal epithelial cells,and significantly affects the metabolic activities of weaned piglets.With palmitic acid,cis-9-palmitoleic acid,linolenic acid,L-palmitoylcarnitine,cis-4,7,10,13,16,19-docosahexaenoic acid,12s-hydroxy-5z,8z,10 e,The metabolites of 14 z eicosaptetraenoic acid,1-stearoyl-2-oleyl-Sn-glycero-3-phosphate choline(SOPC),phosphoylcholine and leucine were mainly enriched in the blank control group,while the metabolites of acetoacetic acid were mainly enriched in the SBA treatment group.These metabolites were mainly fatty acids,choline and amino acids.The KEGG pathway of significantly different metabolites is mainly enriched in the biosynthesis signal pathway of unsaturated fatty acids,the arachidonic acid metabolic signal pathway and the degradation pathway of valine,leucine and isoleucine.Decreased oxidative metabolism of fatty acids leads to mitochondrial hollowing,and decreased phospholipid metabolism leads to intestinal epithelial damage and microvilli shedding.The decrease of arachidonic acid metabolism led to the slow proliferation and apoptosis of cells.In addition,α-linolenic acid metabolism,glycerol phospholipid metabolism signaling pathway and fatty acid degradation pathway are also significantly enriched.SBA can affect the metabolism of fatty acids,phospholipids and amino acids in small intestinal epithelial cells of weaned piglets,reduce energy release,damage cell membrane structure and thus induce intestinal inflammation,affect the proliferation of epithelial cells,and even lead to apoptosis,resulting in the decrease of nutrient digestion and absorption ability of weaned piglets and the slowing down of growth and development.In conclusion,SBA can affect intestinal morphology and microstructure,reduce metabolic capacity(fatty acids,phospholipids and amino acids)and biological function(proliferation and apoptosis),affect digestive enzyme activity,reduce intestinal nutrient digestion and absorption capacity,and affect growth performance of weaned piglets. |
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